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We conclude that Stickler syndrome and Marshall syndrome mutations in COL11A1 cause hearing loss via dominant negative effects upon wild-type fibrillar collagen polypeptides in the extracellular matrices of the cochlea. | Heterozygosity for mutations in the fibrillar collagen gene COL11A1 causes sensorineural hearing loss in patients with Stickler syndrome or Marshall syndrome. Chondrodysplasia (cho) is a functional null allele of Col11a1 that causes lethal chondrodysplasia in cho/cho newborn mice, and osteoarthritis in cho/+ heterozygotes. To determine if Col11a1 haploinsufficiency causes hearing loss in cho/+ mice, auditory brainstem response (ABR) thresholds were measured at 2, 4, 6, 8 and 10 months of age. There was no difference in ABR thresholds for click and tone burst stimuli between cho/+ and +/+ mice at all ages. In contrast to the conclusion of a previous report, our results indicate that Col11a1 haploinsufficiency does not cause significant hearing loss on the C57BL/6 strain background. | ENTAILMENT |
We conclude that these sodium <le>-mediated changes in tissue AII production may be involved in the increased responsiveness of glomerulosa cells to aldosterone secretagogues during sodium restriction. | To determine whether dietary sodium intake modifies the generation of adrenal-produced angiotensins and/or their relative proportions, Sprague-Dawley rats were maintained on a low (0.02%), normal (0.4%), or high (1.5%) sodium intake for 5 days. The animals were then killed by decapitation at 0900 h, and their adrenal glands were removed and dissected into two parts: capsular tissue, containing the zona glomerulosa (ZG), and the decapsulated adrenal gland. The tissue was frozen in liquid nitrogen and extracted, and the individual angiotensins [angiotensin-II (AII), angiotensin-III (AIII), angiotensin-I (AI), and Des-Asp-angiotensin-I (Des-Asp-AI)] were separated by HPLC and quantitated by RIA. On a normal sodium intake, the molar contents of the four angiotensins were similar in ZG, ranging from 3.1-6.6 pmol/g, although AII was present in a 60-70% higher concentration than AIII. In the decapsulated adrenal, the concentrations of the various angiotensins were again similar, but the absolute levels (per g tissue) were significantly (P less than 0.02) less than those in the ZG layer. With sodium restriction, the AII content increased more than 2-fold in the ZG, but not in the decapsulated adrenal tissue. In contrast, both AI and Des-Asp-AI significantly (P less than 0.01) decreased with sodium restriction, so that their contents on the low salt diet were only 15-20% of those observed on the high sodium diet. Thus, there was an inverse correlation (P less than 0.001) between the salt content of rat chow and the AII content of the ZG. The correlation between salt intake and AI as well as Des-Asp-AI levels was direct and significant (P less than 0.02). The AIII level in the ZG was similar on all diets. After a lag period, ZG AII increased sharply between 16-48 h of sodium restriction. These data document that sodium intake has a profound effect on the angiotensin content of the ZG, with sodium restriction substantially increasing the levels of AII while reducing the level of its substrate, AI. This also appears to be unique for glomerulosa cells, as in the decapsulated adrenal gland there is little if any change with sodium restriction. | ENTAILMENT |
It was concluded that in LPS <er>-primed macrophages, P2X <le>(7) receptors, not P2X <le>(4) receptors, activated an iPLA(2) and promoted the release of unsaturated fatty acids secondary to the activation of a kinase. | Free fatty acid releases are triggered by PLA2 activation and are substrates for many enzymes such as cyclooxygenases. These reactions are responsible for the production of many prostaglandins implicated in the inflammation yet many purinergic receptors have been implicated in diseases characterised by chronic inflammation. The role of P2X receptors was evaluated in LPS-primed murine peritoneal macrophages which were labelled with either [(3)H]-oleic acid or [(3)H]-arachidonic acid. Ten μmolar thapsigargin and 1mM ATP stimulated the release of both unsaturated acids. ATP had no effect at 10 μM and ivermectin had no effect on the response to ATP. The response to ATP was inhibited by magnesium and was not observed with cells from P2X(7)(-/-) mice. The response to ATP was not affected by the removal of extracellular calcium and was inhibited by arachidonyltrifluoromethyl ketone and bromoenol lactone but not by pyrrophenone. The release of the [(3)H]-fatty acids by ATP and thapsigargin was diminished by PD-98058, an inhibitor of MEK-1. | ENTAILMENT |
It is therefore concluded that, although spermatozoa do not possess detectable nitric oxide synthase activity, low levels of nitric oxide induce human sperm capacitation <er>, and this action likely involves hydrogen peroxide. | The influence of nitric oxide on human sperm hyperactivation and capacitation, as well as its mechanism of action and its possible origin from spermatozoa were studied. Percoll-washed spermatozoa from healthy volunteers were incubated in Ham's F-10 medium supplemented or not with the nitric oxide-releasing agents, diethylamine-NONOate or spermine-NONOate, in combination or not with superoxide dismutase or catalase (scavengers for the superoxide anion and for hydrogen peroxide, respectively), or with sodium nitrate, sodium nitrite, or preincubated NONOates. Sperm hyperactivation, capacitation, and nitric oxide synthase activity were determined. High concentrations (0.3 to 1 mM) of NONOates reduced sperm motility. However, a lower concentration (0.1 mM) of the two NONOates had no effect on the percentage of sperm motility or of hyperactivation but resulted in a significant increase in sperm capacitation (24% +/- 4%) when compared to that of control spermatozoa (Ham's F-10 alone, 12% +/- 2%). Nitric oxide released by the NONOates appeared responsible for this effect because sodium nitrate or nitrite or preincubated NONOates (to exhaust the formation of nitric oxide) had no influence on sperm capacitation. Catalase, but not superoxide dismutase, abolished the capacitating action of the NONOates. No nitric oxide synthase activity was detected in spermatozoa, whether they were in their basal state or already capacitated. Furthermore, the nitric oxide synthetase inhibitor L-NG nitroarginine methyl ester did not block sperm capacitation induced by fetal cord serum ultrafiltrate. | ENTAILMENT |
It is concluded that D-glucosamine causes a reduction in the lactate production, by inhibiting two enzymes of the glycolytic pathway: glyceraldehyde-3-P dehydrogenase and lactate dehydrogenase. | 1. The effects of glucosamine concentration on the size of the lactate pool, on the levels of ATP, ADP, AMP and on the radioactivity incorporation from [1-14-C] glucosamine into lactate, N-acetylglucosamine and glucosamine-6-P were studied using whole bovine retinas. 2. The radioactive lactate, evaluated in relation to glucosamine molarity, after a modest initial increase, diminishes significantly. On the contrary the N-acetyl [1-14-C] glucosamine, the [1-14-C] glucosamine-6-P and, consequently, also the [1-14-C] glucosamine-6-P/[-14-C] lactate ratio increase with glucosamine molarity. 3. The retinal content of ATP shows a modest increment after incubation with low concentrations of D-glucosamine (0.5--2.0 mM) and a remarkable fall at higher concentrations. 4. Using retinal homogenates D-glucosamine clearly lowers the lactate production from glucose, glucose-6-P and fructose-1, 6-P2. 5. D-Glucosamine acts as an inhibitor of retinal glyceraldehyde-3-P dehydrogenase and lactate dehydrogenase by decreasing the initial velocity of these reactions. 6. | ENTAILMENT |
We conclude that in GH4C1 cells, 1,25-(OH <le>)2D3 enhances membrane calcium transport by modulating voltage-operated Ca2+ channels and activating Na <er>+/Ca2+ exchange by mechanisms requiring new protein synthesis. | In GH4C1 rat pituitary cells, 1,25-dihydroxycholecalciferol [1,25-(OH)2D3] amplifies the TRH-induced spike phase of increase in cytosolic free calcium ([Ca2+]i). In the present report we describe the results of investigations on the mechanisms of action of 1,25-(OH)2D3 on Ca2+ homeostasis in these cells. Pretreatment with 1 nM 1,25-(OH)2D3 for at least 24 h caused no change in basal uptake of 45Ca2+ compared with that in control cells or in 45Ca2+ uptake induced by the calcium channel agonist Bay K 8644. However, when the cells were depolarized with 50 mM K+, 1,25-(OH)2D3-treated cells showed an up to 90% enhancement of uptake (3-120 min) of 45Ca2+. An enhanced increase in [Ca2+]i was also observed in fura-2-loaded cells. The effect was specific and dose dependent for 1,25-(OH)2D3. The calcium channel antagonists nimodipine and verapamil inhibited completely the enhancing action of 1,25-(OH)2D3 as did the protein synthesis inhibitor cycloheximide. No enhanced uptake of 45Ca2+ into intracellular stores was detected when cells were incubated with 1,25-(OH)2D3. Na+/Ca2+ exchange was determined by measuring exchange of extracellular 45Ca2+ for intracellular Na+. Na+/Ca2+ exchange was dependent on intracellular Na+, was inactive when Li+ replaced Na+, was insensitive to calcium channel antagonists, and showed electrogenic properties. In cells incubated with 1,25-(OH)2D3 for at least 24 h, Na+/Ca2+ exchange was enhanced up to 54% compared with that in control cells. Enhanced exchange was dose dependent and specific for 1,25-(OH)2D3. Ca2+ channel antagonists were without effect while dichlorobenzamil inhibited partially the 1,25-(OH)2D3 enhancement of Na+/Ca2+ exchange. Cycloheximide abolished completely the action of 1,25-(OH)2D3 on Na+/Ca2+ exchange. | ENTAILMENT |
We conclude that Tg release from the thyroid is sufficient to elicit THAb synthesis. | Based on the knowledge that diagnostic fine needle biopsy of the thyroid (FNAB) results in a prompt increase in circulating thyroglobulin (Tg); we evaluated whether Tg is indeed the postulated antigen for circulating antibodies against thyroid hormones (THAb). Preliminarily, we verified that FNAB causes the release into the bloodstream of iodinated, heterologous, and thus potentially immunogenic, molecules of Tg. Of the initially enrolled 400 patients, 214 had a number of blood drawings sufficient to evaluate over time (before FNAB and 1-3 h, 3 days, 15 days, 30 days, 3 months, 6 months, and 12 months after FNAB) the following parameters: THAb of both IgM and IgG classes, Tg antibodies (TgAb; by a sensitive immunoradiometric assay), and Tg (in the 156 patients who were TgAb negative). We found the following. 1) Serum Tg most often peaks 1-3 h after FNAB (61 +/- 45% of the baseline level; mean +/- SD). 2) Only 7% of the initially TgAb-negative patients converted to positive, and only 12% of those initially positive had an increase in the levels of TgAb. 3) THAb were detected in 0 of 400 patients before FNAB, but were found in 9 of 214 (4.2%) after FNAB. This proportion is 2 orders of magnitude higher than that (149 of 369,000 or 0.04%) found in consecutive patients attending European thyroid clinics. Of the 9 cases, 6 had Hashimoto's thyroiditis (HT), 2 had euthyroid colloid goiter, and 1 had Hurthle cell carcinoma. In the 5 of 9 cases who were TgAb negative, the post-FNAB increment in Tg was 21-99%, i.e. lower than that of the majority of patients (101-12,500%). 4) THAb were of the IgM class in all 9 (6 against T3 and 3 against T4), and were accompanied and/or followed up to 3 months after FNAB by IgG-THAb of the same specificity (2 against T3 and 1 against T4) in 3 cases. In a fourth case, IgM-T3 were followed by a long-lasting synthesis of IgG-T3 (i.e. up to 1 yr post-FNAB). All 4 cases with IgG-THAb had HT and remained TgAb positive. 5) In the 2 HT and the 3 non-HT patients with undetectable TgAb, THAb were of the IgM class only. 6) In the HT group, 2 risk factors for the development of post-FNAB THAb appeared to be pre-FNAB TgAb levels below 400 U/mL that did not increase after FNAB and Tg released from a colloid nodule. | ENTAILMENT |
From these findings it can be concluded that (a) chloralose-urethane anaesthesia and surgery had a stimulatory effect on renin release but suppressed basal levels of renal renin and angiotensinogen gene expression <er>; (b) acute reduction of RPP for 3 h could stimulate renin gene expression in the renin producing cells; and (c) the negative feedback control of angiotensin II on renin release and synthesis which was evident following chronic losartan treatment was not apparent during short-term reduction of RPP. | 1. A study was undertaken to examine the influence of acute renal perfusion pressure (RPP) reduction on renin release, renal renin and angiotensinogen gene expression and the role played by angiotensin II in these responses. 2. In chloralose-urethane anaesthetised rats, reduction of RPP to 60 mmHg for 3 h in vehicle or losartan-treated (5 days at 10 mg kg-1 bis in die (b.i.d.)) rats decreased renal blood flow by 46 and 29 % (both P < 0.001), respectively, glomerular filtration rate by 45 and 57 % (both P < 0.001), respectively, and sodium excretion by 96 and 98 % (both P < 0.01). 3. Chloralose-urethane anaesthesia and surgery caused a rise in plasma renin activity but was associated with a suppression of renal renin (50 %, P < 0.01) and angiotensinogen (40 %, P < 0.05) gene expression. Following reduction of RPP to 60 mmHg for 3 h, plasma renin activity was increased more than 7-fold (P < 0.001) and renal renin gene expression about 2-fold (P < 0.05). 4. Chronic (5 days) blockade of angiotensin II receptors with losartan elevated plasma renin activity some 29-fold (P < 0.001) and caused a marked increase (30-fold, P < 0.05) in renal renin gene expression, compatible with angiotensin II exerting a negative feedback control on renin release and gene expression. Reduction of RPP to 60 mmHg for 3 h in these animals had little effect on renal renin gene expression. 5. | ENTAILMENT |
We conclude that 7-O-galloyltaxifolin upregulates HO-1 via activation of the MAPK/Nrf2 signaling pathway. | Quercetin and gallic acid are natural activators of the transcription factor Nrf2, which regulates the expression of many cytoprotective enzymes including heme oxygenase-1 (HO-1). We developed procedures for the synthesis of monogalloyl esters of quercetin and taxifolin (dihydroquercetin), namely, 3-O-galloylquercetin and 7-O-galloyltaxifolin, and examined their effect on the Nrf2 pathway in RAW264.7 cells. Unlike quercetin and free gallic acid, 3-O-galloylquercetin and natural quercetin derivatives isoquercitrin (quercetin-3-O-β-d-glucoside) and taxifolin had no effect on the expression of HO-1. In contrast, 7-O-galloyltaxifolin increased both mRNA and protein levels of HO-1 at concentrations of 25 μM and above. The induction of HO-1 by 7-O-galloyltaxifolin was primarily associated with the production of reactive oxygen species and phosphorylation of mitogen-activated protein kinases (MAPKs), including p38 MAPKs and ERKs, followed by nuclear accumulation of Nrf2 and downregulation of Keap1, a negative regulator of Nrf2. | ENTAILMENT |
Our findings indicate that OA and lipids modulate Akt <er>/PKB signaling early in culture to mediate survival, followed by a switch to a dependence on ERK signaling pathways to maintain viability and induce proliferation after 72 h. We conclude that free fatty acids can support maintenance of liver LSEC cultures in vitro; key regulatory pathways involved include early Akt signaling followed by ERK signaling. | Primary rat liver sinusoidal endothelial cells (LSEC) are difficult to maintain in a differentiated state in culture for scientific studies or technological applications. Relatively little is known about molecular regulatory processes that affect LSEC differentiation because of this inability to maintain cellular viability and proper phenotypic characteristics for extended times in vitro, given that LSEC typically undergo death and detachment around 48-72 h even when treated with VEGF. We demonstrate that particular lipid supplements added to serum-free, VEGF-containing medium increase primary rat liver LSEC viability and maintain differentiation. Addition of a defined lipid combination, or even oleic acid (OA) alone, promotes LSEC survival beyond 72 h and proliferation to confluency. Moreover, assessment of LSEC cultures for endocytic function, CD32b surface expression, and exhibition of fenestrae showed that these differentiation characteristics were maintained when lipids were included in the medium. With respect to the underlying regulatory pathways, we found lipid supplement-enhanced phosphatidylinositol 3-kinase and MAPK signaling to be critical for ensuring LSEC function in a temporally dependent manner. Inhibition of Akt activity before 72 h prevents growth of SEC, whereas MEK inhibition past 72 h prevents survival and proliferation. | ENTAILMENT |
It is concluded that noradrenaline acts by opening calcium channels in the basolateral membranes of epithelial cells, resulting in an influx of calcium which stimulates ribosomal protein synthesis to produce proteins involved in fluid transport. | The everted-sac technique was used to study the mechanism of action of noradrenaline on fluid absorption by rat jejunum. Noradrenaline (10(-3) M) significantly stimulated fluid absorption and this effect was dependent on the presence of calcium ions in the serosal fluid. Strontium, but not magnesium could substitute for calcium. Verapamil, manganese and neodymium, all inhibitors of calcium transport, blocked noradrenaline-stimulated fluid absorption when present in the serosal compartment without any effect on basal or glucose-stimulated absorption. Inhibitors of the translation stage of protein synthesis inhibited the response whereas blocking the transcription stage of protein synthesis was without effect. The noradrenaline response was not attenuated by tetrodotoxin suggesting that the response is not indirect due to noradrenaline altering endogenous intestinal nervous activity. | ENTAILMENT |
We conclude that the HMG-CoA reductase inhibitor simvastatin improved nitric oxide production and partially prevented hypertension development, without preventing remodeling of the left ventricle and aorta in NO-deficient hypertension <er>. | 3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors have been shown to prevent or reverse hypertrophy of the LV in several models of left ventricular hypertrophy. The aim of the present study was to determine whether treatment with simvastatin can prevent hypertension, reduction of tissue nitric oxide synthase activity and left ventricular (LV) remodeling in NG-nitro-L-arginine methyl ester(L-NAME)-induced hypertension. Four groups of rats were investigated: control, simvastatin (10 mg/kg), L-NAME (40 mg/kg) and L-NAME + simvastatin (in corresponding doses). Animals were sacrificed and studied after 6 weeks of treatment. The decrease of NO-synthase activity in the LV, kidney and brain was associated with hypertension, LV hypertrophy and fibrosis development and remodeling of the aorta in the L-NAME group. Simvastatin attenuated the inhibition of NO-synthase activity in kidney and brain, partly prevented hypertension development and reduced the concentration of coenzyme Q in the LV. Nevertheless, myocardial hypertrophy, fibrosis and enhancement of DNA concentration in the LV, and remodeling of the aorta were not prevented by simultaneous simvastatin treatment in the L-NAME treated animals. | ENTAILMENT |
It is concluded that thrombin and AGEPC differentially activated phosphoinositide phosphodiesterase (phospholipase C <er>) in rabbit platelets and that the stimulation of the phospholipase C by these two stimuli causes IP3 production via hydrolysis of a common pool of phosphatidylinositol 4,5-bisphosphate. | Despite their physicochemical and mechanistic differences platelet activating factor (or acetylglycerylether phosphorylcholine; AGEPC) and thrombin, both platelet stimulatory agents, induce phosphoinositide turnover in platelets. We therefore investigated the stimulation of the phosphoinositide phosphodiesterase by these agents and questioned whether they evoked hydrolysis of the same or different pools of phosphoinositides. [3H]Inositol-labelled rabbit platelets were challenged with thrombin and/or AGEPC under a variety of protocols, and the phospholipase C mediated production of radioactive inositol monophosphate (IP); inositol bisphosphate (IP2) and inositol trisphosphate (IP3) was used as the parameter. AGEPC (1 X 10(-9) M) caused a transient maximum (5 to 6-fold) increase in [3H]IP3 at 5 s followed by a decrease. Thrombin (2 U/ml) elicited an increase in [3H]IP3 at a much slower rate than AGEPC; 2 fold at 5 s, 5 fold at 30 s and a maximum 6 to 8-fold at 2-5 min. Compared to AGEPC, thrombin stimulated generation of [3H]IP2 and [3H]IP were severalfold higher. When thrombin and AGEPC were added together to platelets there was no evidence for an additive increase in inositol polyphosphate levels except at earlier time points where increases were submaximal. When AGEPC was added at various time intervals after thrombin pretreatment, no additional increases in [3H]IP3 were observed over that maximally seen with thrombin or AGEPC alone. In another set of experiments, submaximal increases (about 1/4 and 1/2 of maximum) in [3H]IP3 were achieved by using selected concentrations of thrombin (0.1 U and 0.3 U, respectively) and then AGEPC (1 X 10(-9) M) was added for 5 s. Once again the increase in [3H]IP3 was close to the maximal level seen with thrombin or AGEPC individually. | ENTAILMENT |
We conclude that a greater concentration of progesterone and indomethacin is required to inhibit oxytocin <er>-induced myometrial contractions in twins compared to singletons in vitro. | We compared the relaxant effect of 2 known tocolytics; indomethacin and atosiban and progesterone, on pregnant human myometrial spontaneous and oxytocin-induced contractions from singleton and twin pregnancies. All agents exerted a concentration-dependent relaxant effect on myometrial contractions. There was no significant difference in the concentration-response curves between singletons and twins for progesterone or indomethacin on spontaneous contractions or atosiban on oxytocin-induced contraction. Under oxytocin however, the concentration-response curves for indomethacin and progesterone were significantly shifted to the right for both amplitude of contraction (P < .01) and activity integral (P < .01). When compared to singleton myometrium however, the concentration-response curves were significantly shifted to the right in the twin myometrium group (P < .05 progesterone and P < .001 indomethacin). | ENTAILMENT |
Meanwhile, the intrinsic death receptor pathway of caspase 9, as well as the common intrinsic and extrinsic downstream target, caspase 3 <er>, was potently activated by the release of cyt c. Together, we conclude that in TRAIL-treated MDR gastric carcinoma cells, cisplatin induces the death receptors DR4 and DR5 through the up-regulation of c-myc and strengthens the activation of caspases via promoting the release of cyt c. These effects would then be responsible for the TRAIL sensitization effect of cisplatin. | Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) reverses multidrug resistance (MDR) and induces apoptosis in MDR gastric carcinoma cells. In our previous study, cisplatin proved to be a sensitizing agent for TRAIL. To study the synergistic effects of cisplatin and TRAIL, we investigated the mechanism by which TRAIL reverses multidrug resistance, the role of c-myc in modulating the death receptors DR4 and DR5 and the relationship between cisplatin and cytochrome c (cyt c) release in SGC7901/VCR and SGC7901/DDP cells. We found that after treatment with TRAIL, the DNA-PKcs/Akt/GSK-3β pathway, which is positively correlated with the levels of MDR1 and MRP1, was significantly inhibited and that this tendency can be abolished by Z-DEVD-FMK (a specific caspase 3 inhibitor). We also found that suppression of c-myc by siRNA reduced the expression of DR4 and DR5 and that transfection with a pAVV-c-myc expression vector increased the expression of DR4 and DR5. Moreover, cisplatin increased the expression of c-myc in the presence of TRAIL, and there is a clear increase in cyt c release from mitochondria with the increasing concentrations of cisplatin. | ENTAILMENT |
We conclude that TNF-alpha neutralization reversed insulin resistance substantially in fast-twitch muscle and may have done so in other muscles, while having little effect in fat. | We examined the possible role of tumor necrosis factor-alpha (TNF-alpha) as a mediator of insulin resistance in maturing male Sprague-Dawley rats. Rats were treated either with goat anti-murine TNF-alpha IgG (anti-TNF-alpha) or goat nonimmune IgG (NI) for 7 days. Vascular catheters were implanted, and rats were fasted overnight before hyperinsulinemic euglycemic clamp (HUC) studies were performed. TNF-alpha neutralization increased the rate of glucose infusion required to maintain euglycemia by 68%. Insulin-stimulated glucose transport into individual tissues was measured after bolus administration of 2-deoxy-[(14)C]glucose during HUC. Anti-TNF-alpha administration increased glucose transport in muscles composed predominantly of fast-twitch fibers: white gastrocnemius muscle (68% increase) and tibialis anterior muscle (64% increase). There were nonsignificant trends for increased glucose transport in the slow-twitch soleus muscle and in the mixed-fiber red gastrocnemius muscle. Glucose transport was unchanged in visceral and subcutaneous fat. Anti-TNF treatment did not alter body weight, muscle mass, or fat mass. Anti-TNF-alpha did not alter the distribution of the 17-kDa and 26-kDa forms of TNF-alpha in either muscle or fat. However, anti-TNF-alpha treatment caused an approximately 50% reduction in the secretion of TNF-alpha bioactivity in vitro by explants of visceral and subcutaneous fat. | ENTAILMENT |
We conclude that ionomycin <le>-induced calpain activation promotes decrease of Bcl-2 proteins thereby triggering the intrinsic apoptotic pathway. | Ubiquitous calpains (mu- and m-calpain) have been repeatedly implicated in apoptosis, but the underlying mechanism(s) remain(s) to be elucidated. We examined ionomycin-induced cell death in LCLC 103H cells, derived from a human large cell lung carcinoma. We detected hallmarks of apoptosis such as membrane blebbing, nuclear condensation, DNA ladder formation, caspase activation, and poly-(ADP-ribose)polymerase cleavage. Apoptosis was prevented by preincubation of the cells with the calpain inhibitor acetyl-calpastatin 27-peptide and the caspase inhibitor Z-DEVD-fmk, implicating both the calpains and caspases in the apoptotic process. The apoptotic events correlated in a calpastatin-inhibitable manner with Bid and Bcl-2 decrease and with activation of caspases-9, -3, and -7. In vitro both ubiquitous calpains cleaved recombinant Bcl-2, Bid, and Bcl-x(L) at single sites truncating their N-terminal regions. Binding studies revealed diminished interactions of calpain-truncated Bcl-2 and Bid with immobilized intact Bcl-2 family proteins. Moreover, calpain-cleaved Bcl-2 and Bid induced cytochrome c release from isolated mitochondria. | ENTAILMENT |
We conclude that HO-1 overexpression in the proximal tubule reduces MCP-1 production in response to albumin, and this occurs, at least in part, by inhibiting an ERK-dependent, NF-kappaB-dependent pathway at a site that is distal to the activation of ERK. | Proteinuria contributes to chronic kidney disease by stimulating renal tubular epithelial cells to produce cytokines such as monocyte chemoattractant protein-1 (MCP-1). The present study determined whether cellular overexpression of heme oxygenase-1 (HO-1) can influence albumin-stimulated MCP-1 production. In response to bovine serum albumin, NRK-52E cells constitutively overexpressing HO-1 (HO-1 OE cells) exhibit less induction of MCP-1 mRNA and less production of MCP-1 protein compared with similarly treated, control NRK-52E cells (CON cells). In wild-type NRK-52E cells, and under these conditions, we demonstrate that the induction of MCP-1 is critically dependent on intact NF-kappaB binding sites in the MCP-1 promoter. In response to albumin, CON cells exhibit activation of NF-kappaB, and this is reduced in HO-1 OE cells. Albumin also activates ERK1/2 and increases ERK activity, both of which are exaggerated in HO-1 OE cells. Studies with an inhibitor of MAPK/ERK kinase (U0126) demonstrate that the inhibitory effects of U0126 on MCP-1 production are attenuated in HO-1 OE cells. | ENTAILMENT |
We conclude that TNF-alpha <le>, via NF-kappaB, and JNK induces MIF and EMMPRIN in macrophage to tumor cell cocultures and this leads to increased invasive capacity of the tumor cells. | Tumor-associated macrophages may influence tumor progression, angiogenesis and invasion. To investigate mechanisms by which macrophages interact with tumor cells, we developed an in vitro coculture model. Previously we reported that coculture enhanced invasiveness of the tumor cells in a TNF-alpha- and matrix metalloprotease-dependent manner. In this report, we studied intracellular signaling pathways and induction of inflammatory genes in malignant cells under the influence of macrophage coculture. We report that coculture of macrophages with ovarian or breast cancer cell lines led to TNF-alpha-dependent activation of JNK and NF-kappaB pathways in tumor cells, but not in benign immortalized epithelial cells. Tumor cells with increased JNK and NF-kappaB activity exhibited enhanced invasiveness. Inhibition of the NF-kappaB pathway by TNF-alpha neutralizing Abs, an NF-kappaB inhibitor, RNAi to RelA, or overexpression of IkappaB inhibited tumor cell invasiveness. Blockade of JNK also significantly reduced invasiveness, but blockade of p38 MAPK or p42 MAPK had no effect. Cocultured tumor cells were screened for the expression of 22 genes associated with inflammation and invasion that also contained an AP-1 and NF-kappaB binding site. EMMPRIN and MIF were up-regulated in cocultured tumor cells in a JNK- and NF-kappaB-dependent manner. Knocking down either MIF or EMMPRIN by RNAi in the tumor cells significantly reduced tumor cell invasiveness and matrix metalloprotease activity in the coculture supernatant. | ENTAILMENT |
We therefore conclude that Myt1 inhibition of Cyclin A/Cdk1 is essential for normal fusome behavior and centriole engagement during premeiotic G2 arrest of Drosophila male meiosis. | Regulation of cell cycle arrest in premeiotic G2 phase coordinates germ cell maturation and meiotic cell division with hormonal and developmental signals by mechanisms that control Cyclin B synthesis and inhibitory phosphorylation of the M-phase kinase, Cdk1. In this study, we investigated how inhibitory phosphorylation of Cdk1 by Myt1 kinase regulates premeiotic G2 phase of Drosophila male meiosis. Immature spermatocytes lacking Myt1 activity exhibit two distinct defects: disrupted intercellular bridges (fusomes) and premature centriole disengagement. As a result, the myt1 mutant spermatocytes enter meiosis with multipolar spindles. These myt1 defects can be suppressed by depletion of Cyclin A activity or ectopic expression of Wee1 (a partially redundant Cdk1 inhibitory kinase) and phenocopied by expression of a Cdk1F mutant defective for inhibitory phosphorylation. | ENTAILMENT |
We therefore conclude that hydrogen peroxide stimulates the 5-lipoxygenase pathway and that substances derived from this pathway are at least in part responsible for the hydrogen peroxide induced vasoconstriction in isolated rat lungs. | Reactive oxygen metabolites cause pulmonary vasoconstriction and activate arachidonic acid metabolism. We proposed that hydrogen peroxide, generated enzymatically in an insolated rat lung model, would cause vasoconstriction which was independent of circulating cells, but dependent on activation of the arachidonic acid cascade. Although hydrogen peroxide caused an increase in lung effluent thromboxane B2 concentration, indomethacin did not inhibit hydrogen peroxide induced vasoconstriction. In order to test the hypothesis that hydrogen peroxide activates the 5-lipoxygenase pathway, lung effluents were analyzed for 5-hydroxy-eicosatetranoic acid (5-HETE) using a sensitive, highly specific mass spectrometer technique. Glucose oxidase increased the 5-HEFE effluent concentrations and this was prevented by U60,257. | ENTAILMENT |
We conclude that dopamine inhibits Na-K-ATPase activity in the CCD through a DA1 receptor-mediated cAMP-PKA pathway that involves the stimulation of PLA2 and arachidonic acid release, possibly mediated by inactivation of lipomodulin. | We have reported that dopamine (DA) inhibits Na-K-ATPase activity in the cortical collecting duct (CCD) by stimulating the DA1 receptor, and the present study was designed to evaluate the mechanism of this effect. Short-term exposure (15-30 min) of microdissected rat CCD to DA, a DA1 agonist (fenoldopam), vasopressin (AVP), forskolin, or dibutyryl cAMP (dBcAMP), which increase cAMP content by different mechanisms, strongly (approximately 60%) inhibited Na-K-ATPase activity. 2',5'-dideoxyadenosine, an inhibitor of adenylate cyclase, completely blocked Na-K-ATPase inhibition by DA or fenoldopam, and IP20, an inhibitor peptide of cAMP-dependent protein kinase A (PKA), abolished the Na:K pump effect of all the cAMP agonists listed above. To verify whether the mechanism of pump inhibition by agents that increase cell cAMP involves phospholipase A2 (PLA2), we used mepacrine, a PLA2 inhibitor, which also abolished Na-K-ATPase inhibition by DA or fenoldopam, as well as by AVP, forskolin, or dBcAMP. Arachidonic acid (10(-7) - 10(-4) M) inhibited Na-K-ATPase activity in dose-dependent fashion. Corticosterone, which induces lipomodulin, a PLA2 inhibitor protein inactivated by PKA, equally abolished the pump effects of DA, fenoldopam, forskolin, and dBcAMP, suggesting that lipomodulin might act between PKA and PLA2 in cAMP-dependent pump regulation. | ENTAILMENT |
We conclude that blockade of the angiotensin type 1 receptor with losartan but not antagonism of the alpha1-adrenoreceptor with prazosin prevents the development of salt-sensitive hypertension induced by sensory denervation. | A novel model of hypertension recently developed in our laboratory shows that neonatal degeneration of capsaicin-sensitive sensory nerves renders a rat responsive to a salt load with a significant rise in blood pressure. To determine the role of the renin-angiotensin system and the sympathetic nervous system in the development of hypertension in this model, newborn Wistar rats were given capsaicin 50 mg/kg SC on the first and second days of life. Control rats were treated with vehicle. After they were weaned, male rats were divided into 6 groups and subjected to the following treatments for 2 weeks: control+high sodium diet (4%) (CON-HS), capsaicin+normal sodium diet (0.5%) (CAP-NS), capsaicin+high sodium diet (CAP-HS), capsaicin+high sodium diet+losartan (10 mg/kg per day) (CAP-HS-LO), capsaicin+high sodium diet+prazosin (3 mg/kg per day) (CAP-HS-PR), and capsaicin+high sodium diet+hydralazine (10 mg/kg per day) (CAP-HS-HY). Levels of calcitonin gene-related peptide in dorsal root ganglia were decreased by capsaicin treatment (P<0.05). Both tail-cuff systolic blood pressure and mean arterial pressure were higher in CAP-HS and CAP-HS-PR than in CON-HS, CAP-NS, CAP-HS-LO, and CAP-HS-HY (P<0.05). The 24-hour urinary volume and sodium excretion were increased when a high sodium diet was given (P<0.05), but they were lower in CAP-HS, CAP-HS-LO, CAP-HS-PR, and CAP-HS-HY than in CON-HS (P<0.05). Urinary potassium excretion was not different among all 6 groups. | ENTAILMENT |
Optimal prolactin responses were obtained with cortisol concentrations greater than 10(-7) M, whereas optimal dibutyryl cAMP responses were observed with cortisol concentrations less than 10(-7) M. Despite the differing optimal cortisol concentrations for the prolactin and dibutyryl cAMP responses, it is concluded that prolactin and dibutyryl cAMP probably stimulate ornithine decarboxylase activity in the mammary gland via the same mechanism. | Dibutyryl cAMP and prolactin stimulated ornithine decarboxylase activity in mouse mammary gland explants which had been preincubated with insulin and cortisol for 1 day; maximally stimulatory concentrations of dibutyryl cAMP and prolactin produced a response which was greater than the sum of the responses of prolactin and dibutyryl cAMP when tested alone. 8-Bromo-cGMP inhibited ornithine decarboxylase activity whereas other derivatives of cyclic nucleotides were without effect. Cortisol concentrations were found to be important for optimizing the dibutyryl cAMP and prolactin responses. | ENTAILMENT |
We, therefore, conclude that the ability of IL-12 to substitute for CD28-costimulatory signaling during CTL development is a result of the interaction of IL-12 with IL-12Rbeta2 induced by low levels of IL-2 synthesized by T cells activated in a CD28-independent manner. | Cytotoxic T lymphocyte (CTL) development is regulated closely by an intricate series of signals provided by the T-cell receptor/CD3 complex, cytokines, and costimulatory ligand/receptor systems. In this study, we have explored the role of interleukin (IL)-12 and CD28 in mouse CTL development. Activation of T cells with anti-CD3 monoclonal antibody (mAb) in the presence of anti-CD86 mAb, which prevents CD28-CD86 interaction, led to decreased production of type 1 (IL-2, interferon-gamma) and type 2 (IL-4, IL-6, IL-10) cytokines, as well as diminished expression of granzyme B (Gzm B) and reduced cytotoxic effector function. Cytolytic activity in T-cell cultures that were activated in the presence of anti-CD86-blocking mAb alone or in combination with anti-CD80 mAb could be restored by the addition of exogenous IL-12 at initiation of culture. The ability of IL-12 to substitute for CD28-costimulatory signaling during CTL development was found to be dependent on the presence of IL-2 rather than interferon-gamma. IL-2 is required for IL-12Rbeta2 expression by T cells activated in the presence of anti-CD86 mAb. Moreover, IL-12Rbeta2 expression by T cells activated in the presence of anti-CD86 mAb is enhanced by IL-12. | ENTAILMENT |
We conclude that Galphai1 inhibits hydrogen peroxide <le>-induced apoptosis of H1299 lung cancer cells by up-regulating the transcription of Bcl-2 through a p50-mediated NF-kappaB activation. | Inhibitory heterotrimeric GTP-binding proteins (Gi proteins) mediate a variety of signaling pathways by coupling receptors and effectors to regulate cellular proliferation, differentiation, and apoptosis. However, the role of Gi proteins in the modulation of hydrogen peroxide-induced apoptosis is not clearly understood. Thus, we investigated the effect of Gi proteins on hydrogen peroxide-induced apoptosis and the underlying mechanisms in H1299 human lung cancer cells. The stable expression of constitutively active alpha subunits of Gi1 (Galphai1QL), Gi2, or Gi3 inhibited hydrogen peroxide-induced apoptosis. The expression of Galphai1QL up-regulated Bcl-2 expression, and the knockdown of Bcl-2 with siRNA abolished the anti-apoptotic effect of Galphai1QL. Galphai1 induced the transcription of Bcl-2 by activation of NF-kappaB, which resulted from an increase in NF-kappaB p50 protein. | ENTAILMENT |
It is concluded that ammonia converts EGF <le>-induced hyperplasia to hypertrophy by a mechanism that involves suppression of lysosomal function and this response can be blocked by inhibiting the activity of the retinoblastoma family of proteins. | Epidermal growth factor (EGF) causes proliferation in renal tubular cells but, when it is combined with transforming growth factor-beta1, it causes hypertrophy by a mechanism that requires the activity of the retinoblastoma family of proteins. In contrast, ammonia causes hypertrophy by decreasing lysosomal proteolysis; in some cell types, it also decreases cellular proliferation. These studies were designed to determine whether ammonia, like transforming growth factor-beta1, could convert EGF-induced hyperplasia to hypertrophy. Cultured NRK-52E cells were incubated with EGF and/or ammonia and the protein/DNA ratio was measured, as a marker of hypertrophy. Addition of ammonia to EGF-treated NRK-52E cells converted EGF-induced hyperplasia to hypertrophy, because of a decrease in DNA synthesis. The mechanism involved no change in EGF-induced protein synthesis. Inhibition of lysosomal function with a proton pump inhibitor or lysosomal protease inhibitors also converted the response of EGF-treated cells to hypertrophy. Expression of the human papilloma virus 16 E7 protein (which inactivates all members of the retinoblastoma family) prevented ammonia from converting EGF-induced hyperplasia to hypertrophy. | ENTAILMENT |
We conclude that IL-1 signaling antagonizes IL-1 <er>1/STAT3 mediated pathology and the genetic deletion of IL-1 <er>RT1 results in increased tumor burden. | IL-1 is key driver of gastric tumorigenesis and is a downstream target of IL-11 signaling. Recently, IL-1 cytokines, particularly IL-1β, have been flagged as therapeutic targets for gastric cancer treatment. Here, we assess the requirement for IL-1 signaling in gastric tumorigenesis. gp130757FF xIL-1RT1-/- mice were generated to determine the pathological consequence of ablated IL-1 signaling in the IL-11 dependent gp130757FF mouse model of gastric tumorigenesis. Gastric lesions in gp130757FF xIL-1RT1-/- mice were increased in incidence and size compared to gp130757FF mice. Proximal gastric lesions originated from the cardiac region and were associated with elevated STAT3 activation, loss of specialized gastric cells and a modulated immune response including increased expression of TNF-α and MDSC associated genes. Administration of IL-11 to IL-1RT1-/- mice showed similar changes to gp130757FF xIL-1RT1-/- mice. Spleens from IL-11 treated wildtype mice showed an enrichment of MDSC and gp130757FF xIL-1RT1-/- mice had increased MDSCs in the stomach compared to gp130757FF mice. Furthermore, crossing TNF-α-/- to gp130757FF mice resulted in reduced lesion size. | ENTAILMENT |
We conclude that, in the absence of P-gp activity, hyperosmotic mannitol activates basolateral NHE via protein kinase C, whereas in the presence of P-gp activity, it does not. | Using the pH-sensitive fluorescent dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester, we examined the effects of hyperosmotic mannitol on basolateral Na(+)/H(+) exchange (NHE) activity in isolated nonperfused proximal tubule S2 segments from mice lacking both the mdr1a and mdr1b genes (KO) and wild-type mice (WT). All experiments were performed in CO(2)/HCO-free HEPES solutions. Osmolality of the peritubular solution was raised from 300 to 500 mosmol/kgH(2)O by the addition of mannitol. NHE activity was assessed by Na(+)-dependent acid extrusion rates (J(H)) after an acid load with NH(4)Cl prepulse. Under isosmotic conditions, J(H) values at a wide intracellular pH (pH(i)) range of 6.20-6.90 were not different between the two groups. In WT mice, hyperosmotic mannitol had no effect on J(H) at the wide pH(i) range. In contrast, in KO mice, hyperosmotic mannitol increased J(H) at a pH(i) range of 6.20-6.45 and shifted the J(H)-pH(i) relationship by 0.15 pH units in the alkaline direction. In KO mice, hyperosmotic mannitol caused an increase in maximal velocity without changing the Michaelis-Menten constant for peritubular Na(+). Exposure of cells from WT mice to the hyperosmotic mannitol solution including the P-gp inhibitor cyclosporin A increased J(H) (at pH(i) 6.30) to an extent similar to that in cells from KO mice exposed to hyperosmotic mannitol alone. In KO mice, staurosporine and calphostin C inhibited the hyperosmotic mannitol-induced increase in J(H). The stimulatory effect of hyperosmotic mannitol on J(H) was mimicked by addition to the isosmotic control solution, including phorbol 12-myristate 13-acetate (PMA; the PKC activator). In WT mice, hyperosmotic mannitol with PMA increased J(H). | ENTAILMENT |
We conclude that the induction of vitellogenin by DHT is mediated by the estrogen receptor rather than by the androgen receptor. | We have investigated the action of high doses of androgens in Gobius niger L., a marine teleostean fish, by characterizing specific steroid receptors in liver and by assaying the plasma vitellogenin concentration under different hormonal treatments. Estrogen and androgen receptors were characterized in the liver nuclear extracts according to their binding specificity. The maximum binding capacity was 25 fmoles/mg protein for the estrogen and androgen receptors. In vivo, high doses of DHT()increased the concentration of plasmatic vitellogenin as assayed by immunodiffusion while low doses were inefficient. In spite of a similar number of estrogen and androgen nuclear receptor sites (25 fmoles/mg protein), DHT was at least 70 fold less active than E2 on yolk protein and vitellogenin induction both in male and female Gobius niger. In addition, the antiestrogen tamoxifen, which was inactive by itself, inhibited the E2 and the DHT induced accumulation of vitellogenin. Progesterone (2 mg/fish) was also totally inactive in inducing vitellogenin. | ENTAILMENT |
As PD98059 could not abolish the increment of ROS induced by CPF <le>, we concluded that ERK1/2 phosphorylation is subsequent to ROS production induced by CPF but not the inverse. | It is well known the participation of oxidative stress in the induction and development of different pathologies including cancer, diabetes, neurodegeneration and respiratory disorders among others. It has been reported that oxidative stress may be induced by pesticides and it could be the cause of health alteration mediated by pollutants exposure. Large number of registered products containing chlorpyrifos (CPF) is used to control pest worldwide. We have previously reported that 50 μM CPF induces ROS generation and produces cell cycle arrest followed by cell death. The present investigation was designed to identify the pathway involved in CPF-inhibited cell proliferation in MCF-7 and MDA-MB-231 breast cancer cell lines. In addition, we determined if CPF-induced oxidative stress is related to alterations in antioxidant defense system. Finally we studied the molecular mechanisms underlying in the cell proliferation inhibition produced by the pesticide. In this study we demonstrate that CPF (50 μM) induces redox imbalance altering the antioxidant defense system in breast cancer cells. Furthermore, we found that the main mechanism involved in the inhibition of cell proliferation induced by CPF is an increment of p-ERK1/2 levels mediated by H2O2 in breast cancer cells. | ENTAILMENT |
We conclude that mutations in MEGF8 cause a Carpenter syndrome subtype frequently associated with defective left-right patterning, probably through perturbation of signaling by hedgehog and nodal family members. | Carpenter syndrome is an autosomal-recessive multiple-congenital-malformation disorder characterized by multisuture craniosynostosis and polysyndactyly of the hands and feet; many other clinical features occur, and the most frequent include obesity, umbilical hernia, cryptorchidism, and congenital heart disease. Mutations of RAB23, encoding a small GTPase that regulates vesicular transport, are present in the majority of cases. Here, we describe a disorder caused by mutations in multiple epidermal-growth-factor-like-domains 8 (MEGF8), which exhibits substantial clinical overlap with Carpenter syndrome but is frequently associated with abnormal left-right patterning. We describe five affected individuals with similar dysmorphic facies, and three of them had either complete situs inversus, dextrocardia, or transposition of the great arteries; similar cardiac abnormalities were previously identified in a mouse mutant for the orthologous Megf8. The mutant alleles comprise one nonsense, three missense, and two splice-site mutations; we demonstrate in zebrafish that, in contrast to the wild-type protein, the proteins containing all three missense alterations provide only weak rescue of an early gastrulation phenotype induced by Megf8 knockdown. | ENTAILMENT |
Compared on a molar basis, however, plasmin was found to be almost 1,000 times more effective than kallikrein, and we conclude, therefore, that in vivo plasmin is the primary activator of scu-PA and the role of the contact system is of secondary importance. | An analysis was made of the various possible activators of single-chain urokinase-type plasminogen activator (scu-PA) in the dextran sulphate euglobulin fraction (DEF) of human plasma. scu-PA activators were detected in an assay system in which the substrate scu-PA, in physiological concentration (50 pM), was immuno-immobilized. After activation of the immobilized scu-PA for a certain period of time the activity of the generated amount of immuno-immobilized two-chain u-PA was determined with plasminogen and the chromogenic substrate S-2251. The scu-PA activator activity (scuPA-AA) in the DEF of plasmas deficient in factor XII or prekallikrein was about half of that in the DEF of normal plasma. Separation of scuPA-AA in the DEF by gel chromatography showed to major peaks, one eluting with an apparent Mr of 500,000 and the other around Mr 100,000. The former peak, which coincided with the activity peak of the kallikrein-kininogen complex, was absent in the DEF of plasma depleted of prekallikrein and therefore was identified as kallikrein. The latter peak was still present in the depleted plasma and most likely represents plasmin, because its scuPA-AA coincided with the activity peak of plasmin and could be fully inhibited by antibodies raised against human plasminogen. It is concluded that plasmin and the contact-activation factor kallikrein each contribute for about 50% to the scuPA-AA in the DEF. | ENTAILMENT |
We conclude that reduced adiponectin expression under high-fat dietary conditions is dependent on 1) increased ALDH1A1 expression in adipocytes, which does not increase all-trans-retinoic acid levels; 2) further RAR ligand-induced, WAT-selective, increased retinoic acid response element-mediated signaling; and 3) RAR ligand-dependent reduction of adiponectin expression. | Adiponectin is an adipocyte-derived adipokine with potent antidiabetic, anti-inflammatory, and antiatherogenic activity. Long-term, high-fat diet results in gain of body weight, adiposity, further inflammatory-based cardiovascular diseases, and reduced adiponectin secretion. Vitamin A derivatives/retinoids are involved in several of these processes, which mainly take place in white adipose tissue (WAT). In this study, we examined adiponectin expression as a function of dietary high-fat and high-vitamin A conditions in mice. A decrease of adiponectin expression in addition to an up-regulation of aldehyde dehydrogenase A1 (ALDH1A1), retinoid signaling, and retinoic acid response element signaling was selectively observed in WAT of mice fed a normal-vitamin A, high-fat diet. Reduced adiponectin expression in WAT was also observed in mice fed a high-vitamin A diet. Adipocyte cell culture revealed that endogenous and synthetic retinoic acid receptor (RAR)α- and RARγ-selective agonists, as well as a synthetic retinoid X receptor agonist, efficiently reduced adiponectin expression, whereas ALDH1A1 expression only increased with RAR agonists. | ENTAILMENT |
We conclude that enalapril prevents or reverses the myocardial beta-adrenoceptor abnormality seen in heart failure and promotes angiotensin II formation. | In heart failure, both the sympathetic nervous system and the renin angiotensin system play important pathophysiological roles, and the two systems may interact with each other, e.g., angiotensin II facilitating noradrenaline release. An abnormality in beta-adrenoceptor density (i.e., a decrease) occurs in clinical and pacing-induced heart failure. This observation together with the therapeutic effectiveness of converting-enzyme inhibitors in the management of patients with heart failure led to the current investigation. The aim was to explore the impact of chronic enalapril treatment on the status of myocardial beta-adrenoceptors in dogs paced (250 beats.min-1) to end-stage heart failure. Placebo or enalapril treatment (5 mg b.i.d.) commenced 1 week after the onset of ventricular pacing and continued until end-stage heart failure was reached. Myocardial beta-adrenoceptor density and affinity were assessed by radioligand binding with [125I]iodocyanopindolol. Left ventricular angiotensin II formation and noradrenaline concentration were measured. In addition, plasma renin activity and plasma noradrenaline levels were determined. The results showed that there was a significant increase in beta-adrenoceptor density following enalapril treatment compared with placebo in the heart-failure group. Enalapril did not change the beta-adrenoceptor density in the control animals. However, in both heart failure and control animals, enalapril caused an unexpected increase in Kd. Furthermore, in heart failure, enalapril caused a significant increase in myocardial angiotensin II formation. | ENTAILMENT |
We also conclude that, unlike GM-CSF, IL-15 does not activate the Jak-2 <er>/STAT-5 pathway found to be important in neutrophil signaling. | Interleukin-15 (IL-15) is a pro-inflammatory cytokine known as a general inhibitor of apoptosis, which possesses potential therapeutic properties. Although IL-15 was previously found to be a human neutrophil agonist, its mode of action remains unknown. Herein, we were interested in elucidating the mechanisms by which it delays neutrophil apoptosis. IL-15 was found to induce tyrosine phosphorylation events and to prevent loss of the anti-apoptotic Mcl-1 protein expression. Using different signal transduction inhibitors, we found that Janus kinase (Jak)-2, Jak-3, p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK), but not G proteins, are involved in IL-15-induced suppression of apoptosis. Furthermore, we found that IL-15 activates Jak-2, p38 MAPK and ERK-1/2, but, unlike granulocyte macrophage-colony-stimulating factor (GM-CSF), it does not activate signal transducer and activator of transcription (STAT)-5a/b. We conclude that IL-15 delays neutrophil apoptosis via several pathways, and that Mcl-1 and several kinases contribute to this. | ENTAILMENT |
We conclude that fluoride <le>-induced apoptosis is largely dependent on Ca2+ induced superoxide generation leading to elevation in CaMKIIg which in turn induces the phosphorylation of ERK 1/2 and downstream activation of extrinsic caspase cascade in HKM cells. | Fluoride is known to induce apoptosis though the mechanisms remain obscure. The aim of the present study was to understand the underlying molecular mechanisms of fluoride-induced apoptosis using fish headkidney macrophages (HKMs). Exposure to fluoride triggered HKM cell apoptosis as evidenced by Hoechst 333432 and AnnexinV-propidium iodide staining, the presence of an internucleosomal DNA ladder and the comet assay. Our results suggest the influx of extra-cellular Ca2+ to be an initial event in fluoride-induced HKM cell apoptosis. We observed persistently elevated levels of superoxide anions and our inhibitor studies with EGTA suggested the primal role of the Ca2+ flux in triggering superoxide production in fluoride-exposed HKM cells. Fluoride exposure led to elevated levels of Ca2+/CaM dependent protein kinase II gamma (CaMKIIg) and pre-treatment with the inhibitor KN-93 but not its inactive structural analogue KN-92 reduced the number of apoptotic cells establishing the pro-apoptotic role of CaMKIIg in fluoride-induced HKM cell apoptosis. We report that the sustained superoxide generation is primarily responsible for the increased CaMKIIg levels observed in fluoride-exposed HKM cells. Our inhibitor studies further implicated CaMKIIg in the activation of extracellular signal-regulated kinases 1 and 2 (ERK 1/2) culminating in caspase-8/caspase-3 mediated apoptosis of HKM cells. | ENTAILMENT |
Taken together, one may conclude that the mechanism of inhibition of TPA <le>-induced ODC by retinoic acid may involve the inhibition of protein kinase C-mediated accumulation of newly synthesized ODC mRNA. | Evidence is presented that inhibition of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ornithine decarboxylase (ODC; EC 4.1.1.17) by retinoic acid may involve inhibition of protein kinase C-mediated synthesis of ODC mRNA. A single application of 10 nmol of TPA to intact mouse skin led to an increase in the steady state levels of epidermal ODC mRNA; a maximal level of ODC mRNA occurred at about 3.5 h after TPA treatment. TPA-induced increase in ODC mRNA preceded the increase in epidermal ODC activity. Application of 17 nmol of retinoic acid 1 h before application of TPA to mouse skin inhibited the induction of both ODC mRNA and ODC activity. Using the DNA-excess filter hybridization technique, we found that TPA-increased steady state levels of ODC mRNA in primary culture of newborn mouse epidermal cells were the result of enhanced accumulation of newly synthesized ODC mRNA. Furthermore, in a pulse-chase experiment, we could not detect any difference in the half-life of ODC mRNA in epidermal cells after TPA or the vehicle dimethyl sulfoxide treatments; the half-life of ODC mRNA was about 7 h in both cases. Exposure of primary cultures of newborn epidermal cells to retinoic acid, in conjunction with TPA, inhibited the synthesis of ODC mRNA and failed to alter the half-life of ODC mRNA. These results implicate the role of transcription activation in TPA-induced ODC gene expression and indicate that retinoic acid may inhibit TPA-induced ODC gene transcription. We also found that protein kinase C may play a role in the mechanism of inhibition by retinoic acid of ODC gene expression. Supporting evidence is the finding that L-alpha-dioctanoylglycerol, an activator of protein kinase C, is a Stage II mouse skin tumor promoter and the application of retinoic acid 1 h before application of L-alpha-dioctanoylglycerol to mouse skin inhibited the induction of ODC activity and ODC mRNA as well as tumor promotion by L-alpha-dioctanoylglycerol. | ENTAILMENT |
We conclude that RSV induces RelA activation in the innate inflammatory response via a pathway separate from that controlling RelA cytoplasmic release, mediated by ROS signaling to cytoplasmic MSK1 activation and RelA Ser-276 phosphorylation. | Respiratory syncytial virus (RSV) is a human pathogen that induces airway inflammation, at least in part, by modulating gene expression programs in airway epithelial cells. The presence of RSV replication is detected by the intracellular retinoic acid-inducible gene I (RIG-I) RNA helicase that forms a productive signaling complex with the mitochondrion-anchored MAVS protein, resulting in nuclear translocation of the NF-kappaB transcription factor. Although nuclear translocation is a prerequisite for activation of the innate inflammatory response, recent studies show that separate pathways governing RelA activation are also required for target gene expression. In this study, we examine the mechanism of RelA phosphorylation and its requirement for RSV-induced gene expression. RSV infection produced a time-dependent RelA phosphorylation on serine (Ser) residues Ser-276 and Ser-536 in parallel with enhanced reactive oxygen species (ROS) stress. Inhibition of RSV-induced ROS inhibited formation of phospho-Ser-276 RelA without affecting phospho-Ser-536 RelA formation. RSV potently induced activation of cytoplasmic mitogen- and stress-related kinase 1 (MSK1) in an ROS-dependent manner. Inhibition of MSK1 using H89 and small interfering RNA knockdown both reduced RSV-induced phospho-Ser-276 RelA formation and expression of a subset of NF-kappaB-dependent genes. Direct examination of the role of phospho-Ser-276 in target gene expression by expression of a RelA Ser-276-to-Ala site mutation in RelA(-/-) mouse embryonic fibroblasts showed that the mutation was unable to mediate RSV-induced NF-kappaB-dependent gene expression. | ENTAILMENT |
We conclude that (1) both vanadate and insulin stimulate erythrocyte Mg <er>(i) levels; (2) cellular Mg <er>(i) responses to insulin <le>, but not to vanadate, depend on basal Mg <er>(i) content-the lower the basal Mg <er>(i), the less the Mg <er>(i) response to insulin <le>. | The insulin-mimetic effect of vanadate is well established, and vanadate has been shown to improve insulin sensitivity in diabetic rats and humans. Although the exact mechanism(s) remain undefined, we have previously demonstrated a direct relation of intracellular free magnesium (Mg(i)) levels to glucose disposal, to insulinemic responses following glucose loading, and to insulin-induced ionic effects. To investigate whether the insulin-mimetic effects of vanadate could similarly be mediated by Mg(i), we utilized (31)P-nuclear magnetic resonance spectroscopy to measure Mg(i) in erythrocytes from normal (NL, n=10) and hypertensive (HTN, n=12) subjects, before and after incubation with insulin and with different doses of sodium vanadate. In NL, vanadate elevated Mg(i) levels, with maximum efficacy at 50 7 micromol/L (186+/-6 to 222+/-6 7micromol/L, P>0.01), as did physiologically maximal doses of insulin, 200 7microU/mL (185+/-6 to 222+/-8 7micromol/L, P<0.01). In HTN, only vanadate, but not insulin, increased Mg(i) (insulin: 173+/-7 to 180+/-9 7micromol/L, P=NS; vanadate: 170+/-7 to 208+/-10 7micromol/L, P<0.01). Mg(i) responses to insulin (r=0.637, P<0.001), but not to vanadate (r=0.15, P=NS), were closely and directly related to basal Mg(i) levels. | ENTAILMENT |
Shh also was present in urine of patients with acute kidney injury, but not in normal individuals or those with fibrotic liver cirrhosis We conclude neither endogenous PTAFR signaling nor CYP2E1-generated radicals alone are sufficient to initiate hedgehog signaling, but instead PTAFR-dependent neutrophil infiltration with myeloperoxidase activation is necessary to initiate ethanol <le>-induced fibrosis in kidney. | Acute inflammation either resolves or proceeds to fibrotic repair that replaces functional tissue. Pro-fibrotic hedgehog signaling and induction of its Gli transcription factor in pericytes induces fibrosis in kidney, but molecular instructions connecting inflammation to fibrosis are opaque. We show acute kidney inflammation resulting from chronic ingestion of the common xenobiotic ethanol initiates Gli1 transcription and hedgehog synthesis in kidney pericytes, and promotes renal fibrosis. Ethanol ingestion stimulated transcription of TGF-ß, collagens I and IV, and alpha-smooth muscle actin with accumulation of these proteins. This was accompanied by deposition of extracellular fibrils. Ethanol catabolism by CYP2E1 in kidney generates local reactive oxygen species that oxidize cellular phospholipids to phospholipid products that activate the Platelet-activating Factor receptor (PTAFR) for inflammatory phospholipids. Genetically deleting this ptafr locus abolished accumulation of mRNA for TGF-ß, collagen IV, and α-smooth muscle actin. Loss of PTAFR also abolished ethanol-stimulated Sonic (Shh) and Indian hedgehog (Ihh) expression, and abolished transcription and accumulation of Gli1. Shh induced in pericytes and Ihh in tubules escaped to urine of ethanol-fed mice. Neutrophil myeloperoxidase (MPO) is required for ethanol-induced kidney inflammation, and Shh was not present in kidney or urine of mpo-/- mice. | ENTAILMENT |
We conclude that in inflammation, neutrophil proteinases can modulate PAR <er>(2) signaling by preventing/disarming the G(q)/calcium signal pathway and, via elastase, can selectively activate the p44/42 MAPK pathway. | Human neutrophil proteinases (elastase, proteinase-3, and cathepsin-G) are released at sites of acute inflammation. We hypothesized that these inflammation-associated proteinases can affect cell signaling by targeting proteinase-activated receptor-2 (PAR(2)). The PAR family of G protein-coupled receptors is triggered by a unique mechanism involving the proteolytic unmasking of an N-terminal self-activating tethered ligand (TL). Proteinases can either activate PAR signaling by unmasking the TL sequence or disarm the receptor for subsequent enzyme activation by cleaving downstream from the TL sequence. We found that none of neutrophil elastase, cathepsin-G, and proteinase-3 can activate G(q)-coupled PAR(2) calcium signaling; but all of these proteinases can disarm PAR(2), releasing the N-terminal TL sequence, thereby preventing G(q)-coupled PAR(2) signaling by trypsin. Interestingly, elastase (but neither cathepsin-G nor proteinase-3) causes a TL-independent PAR(2)-mediated activation of MAPK that, unlike the canonical trypsin activation, does not involve either receptor internalization or recruitment of β-arrestin. Cleavage of synthetic peptides derived from the extracellular N terminus of PAR(2), downstream of the TL sequence, demonstrated distinct proteolytic sites for all three neutrophil-derived enzymes. | ENTAILMENT |
We conclude that propionate-induced inhibition of GABA transaminase causes accumulation of GABA in the brain, leading to increased extracellular GABA concentration, which inhibits neuronal activity and causes lethargy. | Propionic acidemia is the accumulation of propionate in blood due to dysfunction of propionyl-CoA carboxylase. The condition causes lethargy and striatal degeneration with motor impairment in humans. How propionate exerts its toxic effect is unclear. Here, we show that intravenous administration of propionate causes dose-dependent propionate accumulation in the brain and transient lethargy in mice. Propionate, an inhibitor of histone deacetylase, entered GABAergic neurons, as could be seen from increased neuronal histone H4 acetylation in the striatum and neocortex. Propionate caused an increase in GABA (γ-amino butyric acid) levels in the brain, suggesting inhibition of GABA breakdown. In vitro propionate inhibited GABA transaminase with a Ki of ∼1 mmol/l. In isolated nerve endings, propionate caused increased release of GABA to the extracellular fluid. In vivo, propionate reduced cerebral glucose metabolism in both striatum and neocortex. | ENTAILMENT |
We conclude that heat shock decreases endothelial cell ICAM-1 expression via inhibition of IKK activity. | The pulmonary vascular endothelium plays a critical role in lung inflammation. As a result of proinflammatory cytokine expression, adhesion molecules are upregulated on the surface of the endothelial cells. Adhesion molecules facilitate recruitment of leukocytes and thus, have been targeted for potential anti-inflammatory strategies. Prior induction of the stress response through thermal stimulation, or heat shock, alters proinflammatory gene expression by attenuating NF-kappaB signaling. As intercellular adhesion molecule-(ICAM) 1 expression is, in part, NF-kappaB-dependent, we hypothesized that heat shock would inhibit ICAM-1 expression. Heat shocking endothelial cells resulted in heat shock protein (HSP) expression as measured by HSP-70 induction, and decreased TNF-alpha-induced ICAM-1 expression in a manner that appeared to be transcriptionally mediated. Following heat shock, decreased TNF-alpha-induced NF-kappaB activation was observed and was associated with preservation of IkappaB-alpha and a decrease in phosphorylated IkappaB-alpha that correlated to inhibition of I kappa kinase (IKK) activity. Interestingly, exposing respiratory epithelial cells to heat shock, which results in NF-kappaB inhibition, did not affect TNF-induced ICAM-1 expression. | ENTAILMENT |
We conclude that even if there is a heterogeneous nitric oxide production by the xenografted glioma cells that impacts Vegfa and Cyclin D1 expression levels, our results suggest that reduction of nitric oxide levels by nitric oxide scavenging could be an efficient approach to treat glioma <le>. | To investigate the effect of nitric oxide on tumor development, we established a rat tumor xenograft model in zebrafish embryos. The injected tumor cells formed masses in which nitric oxide production could be detected by the use of the cell-permeant DAF-FM-DA (diaminofluorophore 4-amino-5-methylamino-2'-7'-difluorofluorescein diacetate) and DAR-4M-AM (diaminorhodamine-4M). This method revealed that nitric oxide production could be co-localized with the tumor xenograft in 46% of the embryos. In 85% of these embryos, tumors were vascularized and blood vessels were observed on day 4 post injection. Furthermore, we demonstrated by qRT-PCR that the transplanted glioma cells highly expressed Nos2, Vegfa and Cyclin D1 mRNA. In the xenografted embryos we also found increased zebrafish vegfa expression. Glioma and zebrafish derived Vegfa and tumor Cyclin D1 expression could be down regulated by the nitric oxide scavenger 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide or CPTIO. | ENTAILMENT |
We conclude that PLB increases V(max <er>) of Ca(2+)-ATPase, and that the magnitude of this effect is sensitive to mutation. | Reconstitution into proteoliposomes is a powerful method for studying calcium transport in a chemically pure membrane environment. By use of this approach, we have studied the regulation of Ca(2+)-ATPase by phospholamban (PLB) as a function of calcium concentration and PLB mutation. Co-reconstitution of PLB and Ca(2+)-ATPase revealed the expected effects of PLB on the apparent calcium affinity of Ca(2+)-ATPase (K(Ca)) and unexpected effects of PLB on maximal activity (V(max)). Wild-type PLB, six loss-of-function mutants (L7A, R9E, I12A, N34A, I38A, L42A), and three gain-of-function mutants (N27A, L37A, and I40A) were evaluated for their effects on K(Ca) and V(max). With the loss-of-function mutants, their ability to shift K(Ca) correlated with their ability to increase V(max). A total loss-of-function mutant, N34A, had no effect on K(Ca) of the calcium pump and produced only a marginal increase in V(max). A near-wild-type mutant, I12A, significantly altered both K(Ca) and V(max) of the calcium pump. With the gain-of-function mutants, their ability to shift K(Ca) did not correlate with their ability to increase V(max). The "super-shifting" mutants N27A, L37A, and I40A produced a large shift in K(Ca) of the calcium pump; however, L37A decreased V(max), while N27A and I40A increased V(max). For wild-type PLB, phosphorylation completely reversed the effect on K(Ca), but had no effect on V(max). | ENTAILMENT |
We conclude that AIF, cytochrome c and caspase-3 are not responsible for the NO <le>-mediated cell death evoked by SNP. | Nitric oxide (NO) is a potent extracellular and intracellular physiological messenger. However, NO liberated in excessive amounts can be involved in macromolecular and mitochondrial damage in brain aging and in neurodegenerative disorders. The molecular mechanism of its neurotoxic action is not fully understood. Our previous data indicated involvement of NO in the release of arachidonic acid (AA), a substrate for cyclo- and lipoxygenases (COX and LOX, respectively). In this study we investigated biochemical processes leading to cell death evoked by an NO donor, sodium nitroprusside (SNP). We found that SNP decreased viability of pheochromocytoma (PC12) cells in a concentration- and time-dependent manner. SNP at 0.1 mM caused a significant increase of apoptosis-inducing factor (AIF) protein level in mitochondria. Under these conditions 80% of PC12 cells survived. The enhancement of mitochondrial AIF level might protect most of PC12 cells against death. However, NO released from 0.5 mM SNP induced massive cell death but had no effect on protein level and localization of AIF and cytochrome c. Caspase-3 activity and poly(ADP-ribose) polymerase-1 (PARP-1) protein levels were not changed. However, PARP activity significantly decreased in a time-dependent manner. Inhibition of both COX isoforms and of 12/15-LOX significantly lowered the SNP-evoked cell death. | ENTAILMENT |
Therefore, we conclude that miR-150 negatively regulates CD4 <er>(+) T cell function by inhibiting the AKT3/BIM signaling pathway. | Donor-derived CD4(+) T lymphocytes are the major effector cells directly involved in the development of graft-versus-host disease (GVHD). As a negative regulator of immune cell differentiation and development, microRNA-150 (miR-150) induces immunological tolerance in CD4(+) T cells after transplantation. However, the specific mechanisms have not been fully elucidated. In this study, we demonstrated that miR-150 is capable of not only inhibiting proliferation and activation of CD4(+) T cells but also promoting apoptosis. Mechanistically, miR-150 targets v-akt murine thymoma viral oncogene homolog 3 (AKT3), and subsequently downregulates B-cell lymphoma 2 (Bcl-2) interacting mediator of cell death (BIM). We have also demonstrated that re-expression of AKT3 reversed miR-150-mediated inhibition of CD4(+) T lymphocyte development. | ENTAILMENT |
We conclude that hyperoxia increased large tidal volume-induced MIP-2 production and neutrophil influx through activation of the Akt and eNOS pathways. | Positive pressure ventilation with large tidal volumes has been shown to cause release of cytokines, including macrophage inflammatory protein-2 (MIP-2), a functional equivalent of human IL-8, and neutrophil infiltration. Hyperoxia has been shown to increase ventilator-induced lung injury, but the mechanisms regulating interaction between a large tidal volume and hyperoxia are unclear. We hypothesized that large tidal volume ventilation using hyperoxia would increase MIP-2 production and neutrophil infiltration via the serine/threonine kinase/protein kinase B (Akt) pathway and the endothelial nitric oxide synthase (eNOS) pathway. C57BL/6 mice were exposed to large tidal volume (30 ml/kg) mechanical ventilation with room air or hyperoxia for 1-5 hours. Large tidal volume ventilation using hyperoxia induced neutrophil migration into the lung, MIP-2 production, and Akt and eNOS activation in a time-dependent manner. Both the large tidal volume ventilation of Akt mutant mice and the pharmacological inhibition of Akt with LY294002 attenuated neutrophil sequestration, MIP-2 protein production, and Akt and eNOS activation. | ENTAILMENT |
We conclude that IL-13 enhances frequency of LTD4-induced Ca <er>(2+) oscillations in human ASMCs, which may be cooperatively modulated by IP(3)R, RyR systems and possibly by SOCE. | Physiological mechanisms associated with interleukin-13 (IL-13), a key cytokine in asthma, in intracellular Ca(2+) signaling in airway smooth muscle cells (ASMCs) remain unclear. The aim of this study was to assess effects of IL-13 on Ca(2+) oscillations in response to leukotriene D4 (LTD4) in human cultured ASMCs. LTD4-induced Ca(2+) oscillations in ASMCs pretreated with IL-13 were imaged by confocal microscopy. mRNA expressions of cysteinyl leukotriene 1 receptors (CysLT1R), CD38, involved with the ryanodine receptors (RyR) system, and transient receptor potential canonical (TRPC), involved with store-operated Ca(2+) entry (SOCE), were determined by real-time PCR. In IL-13-pretreated ASMCs, frequency of LTD4-induced Ca(2+) oscillations and number of oscillating cells were significantly increased compared with untreated ASMCs. Both xestospongin C, a specific inhibitor of inositol 1,4,5-triphosphate receptors (IP(3)R), and ryanodine or ruthenium red, inhibitors of RyR, partially blocked LTD4-induced Ca(2+) oscillations. Ca(2+) oscillations were almost completely inhibited by 50 μM of 2-aminoethoxydiphenyl borate (2-APB), which dominantly blocks SOCE but not IP(3)R at this concentration. Pretreatment with IL-13 increased the mRNA expressions of CysLT1R and CD38, but not of TRPC1 and TRPC3. | ENTAILMENT |
We conclude that (i) miR-125b and miR-150 target NFkB and miR-146a expressions <le>, which in turn regulates RelA/p53 <er>; (ii) reduced miR-125b and miR-150 expressions, increased NFkB and miR-146a expressions level and decreased RelA/p53 originate from mutant HTT (iii) NFkB and miR-146a expressions directly or indirectly regulates the expression of miR-146a. | Huntington's disease (HD) is caused by the expansion of N-terminal polymorphic poly Q stretch of the protein huntingtin (HTT). Deregulated microRNAs and loss of function of transcription factors recruited to mutant HTT aggregates could cause characteristic transcriptional deregulation associated with HD. We observed earlier that expressions of miR-125b, miR-146a and miR-150 are decreased in STHdh(Q111)/Hdh(Q111) cells, a model for HD in comparison to those of wild type STHdh(Q7)/Hdh(Q7) cells. In the present manuscript, we show by luciferase reporter assays and real time PCR that decreased miR-146a expression in STHdh(Q111)/Hdh(Q111) cells is due to decreased expression and activity of p65 subunit of NFkB (RelA/NFkB). By reporter luciferase assay, RT-PCR and western blot analysis, we also show that both miR-150 and miR-125b target p53. This partially explains the up regulation of p53 observed in HD. Elevated p53 interacts with RelA/NFkB, reduces its expression and activity and decreases the expression of miR-146a, while knocking down p53 increases RelA/NFkB and miR-146a expressions. We also demonstrate that expression of p53 is increased and levels of RelA/NFkB, miR-146a, miR-150 and miR-125b are decreased in striatum of R6/2 mice, a mouse model of HD and in cell models of HD. In a cell model, this effect could be reversed by exogenous expression of chaperone like proteins HYPK and Hsp70. | CONTRADICTION |
We conclude that hyperoxia increases PEDF in the neonatal lung. | Bronchopulmonary dysplasia is a chronic lung disease of preterm infants characterized by arrested microvascularization and alveolarization. Studies show the importance of proangiogenic factors for alveolarization, but the importance of antiangiogenic factors is unknown. We proposed that hyperoxia increases the potent angiostatin, pigment epithelium-derived factor (PEDF), in neonatal lungs, inhibiting alveolarization and microvascularization. Wild-type (WT) and PEDF(-/-) mice were exposed to room air (RA) or 0.9 fraction of inspired oxygen from Postnatal Day 5 to 13. PEDF protein was increased in hyperoxic lungs compared with RA-exposed lungs (P < 0.05). In situ hybridization and immunofluorescence identified PEDF production primarily in alveolar epithelium. Hyperoxia reduced alveolarization in WT mice (P < 0.05) but not in PEDF(-/-) mice. WT hyperoxic mice had fewer platelet endothelial cell adhesion molecule (PECAM)-positive cells per alveolus (1.4 ± 0.4) than RA-exposed mice (4.3 ± 0.3; P < 0.05); this reduction was absent in hyperoxic PEDF(-/-) mice. The interactive regulation of lung microvascularization by vascular endothelial growth factor and PEDF was studied in vitro using MFLM-91U cells, a fetal mouse lung endothelial cell line. Vascular endothelial growth factor stimulation of proliferation, migration, and capillary tube formation was inhibited by PEDF. MFLM-91U cells exposed to conditioned medium (CM) from E17 fetal mouse lung type II (T2) cells cultured in 0.9 fraction of inspired oxygen formed fewer capillary tubes than CM from T2 cells cultured in RA (hyperoxia CM, 51 ± 10% of RA CM, P < 0.05), an effect abolished by PEDF antibody. | CONTRADICTION |
We conclude that PI 3-kinase mediates the survival effect of BMDMs on TGF-beta-induced death by acting upstream from the mitochondrial changes, i.e., preventing bcl-x(L) down-regulation, cytochrome c release, and activation of caspase-3. | Transforming growth factor beta (TGF-beta)-mediated apoptosis is one of the major death processes in the liver. We have previously shown that epidermal growth factor (EGF) is an important survival signal for TGF-beta-induced apoptosis in fetal hepatocytes (Fabregat et al., FEBS Lett 1996;384:14-18). In this work we have studied the intracellular signaling implicated in the protective effect of EGF. We show here that EGF activates p42 and p44 mitogen-activated protein kinases (MAPK). However, mitogen extracellular kinase (MEK) inhibitors do not block the survival effect of EGF. EGF also activates phosphoinositide 3-kinase (PI 3-kinase) and protein kinase B (PKB/AKT) in these cells. The presence of PI 3-kinase inhibitors blocks the protective effect of EGF on cell viability, DNA fragmentation, and caspase-3 activity. We have found that TGF-beta disrupts the mitochondrial transmembrane potential (DeltaPsi(m))( )and activates the release of cytochrome c, this effect being blocked by EGF, via a PI 3-kinase-dependent pathway. A detailed study on bcl-2 superfamily gene expression shows that TGF-beta produces a decrease in the messenger RNA (mRNA) and protein levels of bcl-x(L), an antiapoptotic member of this family, capable of preventing cytochrome c release. EGF is able to maintain bcl-x(L) levels even in the presence of TGF-beta. PI 3-kinase inhibitors completely block the protective effect of EGF on TGF-beta-induced bcl-x(L )down-regulation. | CONTRADICTION |
We conclude that VGF stimulates an alpha5beta1 receptor-MEK-dependent pro-survival pathway that synergizes with F1L to counteract an infection <er>-induced apoptotic pathway that predominantly involves the BH3-only protein Bad. | F1L is a functional Bcl-2 homologue that inhibits apoptosis at the mitochondria during vaccinia infection. However, the extent and timing of cell death during DeltaF1L virus infection suggest that additional viral effectors cooperate with F1L to limit apoptosis. Here we report that vaccinia growth factor (VGF), a secreted virulence factor, promotes cell survival independently of its role in virus multiplication. Analysis of single and double knockout viruses reveals that VGF acts synergistically with F1L to protect against cell death during infection. Cell survival in the absence of F1L is dependent on VGF activation of the epidermal growth factor receptor. Furthermore, signalling through MEK kinases is necessary and sufficient for VGF-dependent survival. | CONTRADICTION |
We conclude that in cultured airway goblet cells extracellular NO stimulates mucin release by a signal transduction mechanism, which seems to involve coupling of NO <le>-activated P2 purinoceptors with phospholipase C, at least in part, via pertussis toxin-sensitive GTP-binding proteins. | Release of mucins from cultured airway surface epithelial cells can be stimulated by extracellular ATP via a P2-purinergic receptor-mediated mechanism (K. C. Kim and B. C. Lee. 1991. Br. J. Pharmacol. 103:1053-1056). In this report, we studied the mechanism by which extracellular ATP induces the mucin release. We found that: (1) ATP increased both mucin release and generation of inositol phosphates in a dose-dependent fashion, and their dose-effect relationships were almost superimposed; (2) the increases in both mucin release and the phosphatidylinositol phosphate (PI) turnover by extracellular ATP were partially, but almost equally, blocked by the pretreatment with pertussis toxin (42% for mucin release and 44% for PI turnover). | CONTRADICTION |
We conclude that simvastatin attenuated neuropathic pain in rats subjected to actin by inhibiting CCI <er>-mediated intracellular trafficking to suppress RhoA/LIMK/cofilin pathway activity. | Neuropathic pain occurs due to deleterious changes in the nervous system caused by a lesion or dysfunction. Currently, neuropathic pain management is unsatisfactory and remains a challenge in clinical practice. Studies have suggested that actin cytoskeleton remodeling may be associated with neural plasticity and may involve a nociceptive mechanism. Here, we found that the RhoA/LIM kinase (LIMK)/cofilin pathway, which regulates actin dynamics, was activated after chronic constriction injury (CCI) of the sciatic nerve. Treatments that reduced RhoA/LIMK/cofilin pathway activity, including simvastatin, the Rho kinase inhibitor Y-27632, and the synthetic peptide Tat-S3, attenuated actin filament disruption in the dorsal root ganglion and CCI-induced neuropathic pain. Over-activation of the cytoskeleton caused by RhoA/LIMK/cofilin pathway activation may produce a scaffold for the trafficking of nociceptive signaling factors, leading to chronic neuropathic pain. Here, we found that simvastatin significantly decreased the ratio of membrane/cytosolic RhoA, which was significantly increased after CCI, by inhibiting the RhoA/LIMK/cofilin pathway. This effect was highly dependent on the function of the cytoskeleton as a scaffold for signal trafficking. | CONTRADICTION |
We conclude that BDNF/TrkB promotes osteogenesis in differentiating hMSCs, with MIR146A regulating BDNF/TrkB <le>. | Chromatin remodeling is important for cell differentiation. Histone methyltransferase EZH2 and histone demethylase JMJD3 (KDM6B) modulate levels of histone H3 lysine 27 trimethylation (H3K27me3). Interplay between the two modulators influence lineage specification in stem cells. Here, we identified microRNA MIR146A to be a negative regulator of JMJD3. In the osteogenic differentiation of human mesenchymal stem cells (hMSCs), we observed an upregulation of JMJD3 and a downregulation of MIR146A. Blocking JMJD3 activity in differentiating hMSCs reduced transcript levels of osteogenic gene RUNX2. H3K27me3 levels decreased at the RUNX2 promoter during cell differentiation. Modulation of MIR146A levels in hMSCs altered JMJD3 and RUNX2 expression and affected osteogenic differentiation. | CONTRADICTION |
We conclude that the reduction in renin release stimulated furosemide by indomethacin is due to renal cyclo-oxygenase inhibition which is reflected in decreased excretion rates of hydrolysis products of renal eicosanoids. | We had previously shown that the early increment in plasma renin activity occurring within ten minutes of intravenous furosemide is accompanied by an increase in urine 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) the hydrolysis product of prostacyclin. Renal prostacyclin and thromboxane A2 synthesis are apparently limited to the cortex. To assess whether indomethacin would inhibit renal cortical eicosanoid synthesis and whether such reduction correlated with reduced early renin release, we assessed responses to intravenous furosemide (0.5 mg/kg) before and after indomethacin (150 mg/day for seven days) in ten normal male volunteers. Indomethacin did not change blood pressure but increased weight slightly (79.7 +/- 2.5 kg to 80.4 +/- 2.4 kg, p less than 0.05). Serum thromboxane B2 (TXB2), a measure of platelet thromboxane A2 production, was profoundly depressed (142 +/- 29 ng/ml to 4.8 +/- 1.6 ng/ml, p less than 0.001). Neither diuresis nor natriuresis were changed by indomethacin but potassium excretion was reduced (33 +/- 4 mmol/4 hr to 27 +/- 3 mmol/4 hr, p less than 0.05). Basal as well as furosemide stimulated plasma renin activity (at 10, 30 and 240 minutes) was reduced, as well as the transient increase in excretion rates of 6-keto-PGF1 alpha and TXB2. | CONTRADICTION |
It can be concluded that ZO-2 inhibits the cyclooxygenase pathway of rat platelets via a specific ZO-2 receptor-activated, cAMP-dependent pathway, and these effects of ZO-2 are involved in the inhibition of platelet aggregation. | Several data suggest that pituitary adenylate cyclase-activating polypeptide (PACAP) is involved in the regulation of local circulation. One possible role of PACAP in the regulation of circulation is that, it may modify the cyclooxygenase pathway of the arachidonate cascade in platelets. Our study was designed to study the effect of PACAP on the cyclooxygenase pathway of rat platelets and on platelet aggregation. PACAP (10(-7) and 10(-6) M) significantly inhibited the cyclooxygenase pathway of platelets, mostly the thromboxane synthesis. Pretreatment with a PACAP receptor antagonist, PACAP(6-38), or with an inhibitor of protein kinase A, H-89, shows that the effects of PACAP on the cyclooxygenase pathway were diminished. In the aggregation studies, PACAP inhibited both the arachidonic acid-induced and the thrombin-induced platelet aggregation. | CONTRADICTION |
We conclude that CD45 induces the dephosphorylation of Lck at Y394, thereby preventing sustained Lck activation and propose that the amplitude of the Src family kinase-dependent signal regulates the outcome of CD44-mediated signaling to the actin cytoskeleton and T cell spreading. | The tyrosine phosphatase CD45 dephosphorylates the negative regulatory tyrosine of the Src family kinase Lck and plays a positive role in TCR signaling. In this study we demonstrate a negative regulatory role for CD45 in CD44 signaling leading to actin rearrangement and cell spreading in activated thymocytes and T cells. In BW5147 T cells, CD44 ligation led to CD44 and Lck clustering, which generated a reduced tyrosine phosphorylation signal in CD45(+) T cells and a more sustained, robust tyrosine phosphorylation signal in CD45(-) T cells. This signal resulted in F-actin ring formation and round spreading in the CD45(+) cells and polarized, elongated cell spreading in CD45(-) cells. The enhanced signal in the CD45(-) cells was consistent with enhanced Lck Y394 phosphorylation compared with the CD45(+) cells where CD45 was recruited to the CD44 clusters. This enhanced Src family kinase-dependent activity in the CD45(-) cells led to PI3K and phospholipase C activation, both of which were required for elongated cell spreading. | CONTRADICTION |
It is concluded that the subtype of beta-subunit influences the potency with which GABA potentiates etomidate <er>-evoked currents and that the beta isoform is a crucial determinant of the etomidate <er>-mimetic activity of this compound. | 1. The GABA modulating and GABA-mimetic actions of the general anaesthetic etomidate were examined in voltage-clamp recordings performed on Xenopus laevis oocytes induced, by cRNA injection, to express human recombinant gamma-aminobutyric acidA (GABAA) receptor subunits. 2. Currents mediated by recombinant receptors with the ternary subunit composition alpha x beta y gamma 2L (where x = 1,2,3 or 6 and y = 1 or 2), in response to GABA applied at the appropriate EC10, were enhanced by etomidate in a manner that was dependent upon the identity of both the alpha and beta subunit isoforms. 3. For the beta 2-subunit containing receptors tested, the EC50 for the potentiation of GABA-evoked currents by etomidate (range 0.6 to 1.2 microM) was little affected by the nature of the alpha subunit present within the hetero-oligomeric complex. However, replacement of the beta 2 by the beta 1 subunit produced a 9-12 fold increase in the etomidate EC50 (6 to 11 microM) for all alpha-isoforms tested. 4. For alpha 1, alpha 2 and alpha 6, but not alpha 3-subunit containing receptors, the maximal potentiation of GABA-evoked currents by etomidate was greater for beta 2- than for beta 1-subunit containing receptors. This was most clearly exemplified by receptors composed of alpha 6 beta 1 gamma 2L compared to alpha 6 beta 2 gamma 2L subunits, where a maximally effective concentration of etomidate potentiated currents evoked by GABA at EC10 to 28 +/- 2% and 169 +/- 4% of the maximal GABA response, respectively. 5. For alpha 1 subunit-containing receptors, the potency and maximal potentiating effect of either pentobarbitone or propofol was essentially unaffected by the beta subunit isoform contained within the receptor complex. The potency of the anaesthetic neurosteroid 5 alpha-pregnan-3 alpha-ol-20-one was marginally higher for beta 1 rather than the beta 2 subunit-containing receptor, although its maximal effect was similar at the two receptor isoforms. 6. The GABA-mimetic action of etomidate was supported by beta 2- but not beta 1-subunit containing receptors, whereas that of pentobarbitone or propofol was evident with either beta isoform. For beta 2-subunit containing receptors, both the agonist EC50 and the maximal current produced by etomidate were additionally influenced by the alpha isoform. 7. | CONTRADICTION |
It is concluded that, in the guinea-pig colonic mucosa under the present conditions, mu opioid receptors mediate enhancement and chloride secretion inhibition of PGE1-stimulated net kappa opioid receptors by low Lop concentrations. | 1. In a voltage clamp setting (Ussing chamber), the antidiarrhoeal drug, loperamide (Lop) slightly augmented prostaglandin E1 (PGE1) plus theophylline-stimulated net chloride secretion above control values at low concentrations (10(-10) and 10(-9) M) but inhibited it at higher concentrations (10(-6) and 10(-5) M). The apparently weak prosecretory action component of Lop was turned into a clear cut antisecretory effect by pretreatment with 2 x 10(-7) M naloxonazine plus 10(-7) M CTOP-NH2 (D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2), two selective mu opioid receptor antagonists. This indicates a prosecretory effect of loperamide at mu opioid receptors. The antisecretory effect of low Lop concentrations, uncovered by mu opioid receptor blockade, was prevented by additional blockade of kappa opioid receptors by 5 x 10(-9) M nor-binaltorphimine (nor-BNI). 2. The nonselective opioid antagonist, naloxone, at 10(-6) M did not significantly reduce either PGE1 plus theophylline-stimulated net chloride secretion in Lop-free controls or the antisecretory action of Lop. By contrast, the partial agonist ethylketocyclazocine (EKC), which activates kappa but blocks mu opioid receptors, concentration-dependently inhibited PGE1 plus theophylline-stimulated net chloride secretion without any consistent prosecretory action component. Nor-BNI at 5 x 10(-8) M significantly blocked the antisecretory action of EKC. 3. | CONTRADICTION |
These results led us to conclude that the human MnSOD gene having the inhibitor construct used in this study is induced by TPA via activation of a CREB-1/ATF-1-like factor and not via either NF-kappaB or AP-1. | Transcriptional activation of human manganese superoxide dismutase (MnSOD) mRNA induced by a phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), was examined to identify the responsive transcriptional regulator. The effect of various deletions and mutations within the 5'-flanking region of the human MnSOD gene promoter was evaluated using the luciferase reporter system in A549 human lung carcinoma cells. Deletion of a region between -1292 and -1202 nucleotides upstream of the transcription start site abolished TPA-responsive induction, whereas deletion of the putative binding sequence for NF-kappaB or AP-1 did not. The region between -1292 and -1202 contains a cAMP-responsive element-like sequence, TGACGTCT, which we identified as the manganese superoxide dismutase TPA-responsive element, MSTRE. Site-specific mutation of the MSTRE abolished the TPA-responsive induction, validating the critical role of this sequence. We detected specific MSTRE activity from nuclear extracts and demonstrated by antibody supershift assay that this activity is closely related to CREB-1/ATF-1. TPA treatment rapidly induced phosphorylation of the CREB-1/ATF-1-like factor via the protein kinase C pathway. | CONTRADICTION |
(in press)], it is concluded that Pro-1 acts as the general MIF catalyst in the base <er>-catalyzed reaction. | The cytokine macrophage migration inhibitory factor (MIF) mediates several immune and inflammatory processes through unknown or poorly understood mechanisms. The protein shares structural homology with two bacterial isomerases, 4-oxalocrotonate tautomerase (4-OT) and 5-(carboxymethyl)-2-hydroxymuconate isomerase (CHMI), and catalyzes the enolization of phenylpyruvate and the ketonization of (p-hydroxyphenyl)pyruvate. The amino-terminal proline has been identified as the catalytic base in both the 4-OT- and CHMI-catalyzed reactions. MIF also has an amino-terminal proline that has been implicated as a catalytic group in the MIF-catalyzed reaction. To delineate further the role of Pro-1 in the MIF-catalyzed reaction, affinity labeling studies were performed with 3-bromopyruvate (3-BP). The results of this study show that 3-BP acts as an active-site-directed irreversible inhibitor of the enzymatic activity and modifies one site per monomeric subunit. The inhibitor, as its lactyl derivative, is covalently attached to an 11 residue amino-terminal fragment, Pro-1 to Arg-11. The only reasonable site for alkylation within this peptide fragment is the amino-terminal proline. Because the pKa measured for the pH dependence of kinact/KI (5.7 +/- 0.2) and that measured for the pH dependence of the kcat/Km for the enolization of phenylpyruvate (6.0 +/- 0.1) are comparable and in reasonable agreement with the previously measured pKa of Pro-1 (5.6 +/- 0.1) obtained by its direct titration [Swope, M., Sun H.-W., Blake, P., and Lolis, E. (1998) EMBO J. | CONTRADICTION |
It is concluded that TIS-1 causes induction of the early response genes c-fos and PAF in a structurally specific and receptor dependent manner. | The effect of platelet activating factor (PAF) on the induction of early response genes was investigated in A-431 cells (human epidermal carcinoma cells). PAF induced a transient expression of c-fos and TIS-1 mRNA in a time- and dose-dependent manner. As low as 10(-10) M PAF caused detectable expression of these genes with a maximum observed at 10(-7) M. In the presence of cycloheximide, increases in the gene expression were noticeable at 20 min and peaked between 30-60 min. A lack of induction with lyso-PAF, an inactive PAF metabolite, confirmed the specificity of PAF towards this expression. The cells pretreated with CV-6209, a PAF receptor antagonist, did not show any induction of these genes by PAF. | CONTRADICTION |
We conclude that lipoprotein lipase inhibits lipid storage in adipose tissue by reducing both growth hormone activity and insulin's inhibitory action on hormone-sensitive lipase. | Lipid storage and breakdown is mainly controlled by lipoprotein lipase and hormone-sensitive lipase. The aim of this work was to elucidate whether growth hormone mediated loss of adipose tissue involves a concerted action on tissue lipases, and to what degree such events are modulated by dietary regimen. Twelve-month-old rats fed first a high-fat diet or a low-fat diet for 14 weeks were injected with saline or growth hormone (4 mg/kg/d) for four days or three weeks in different combinations with either high- or low-fat diets. In adipose tissue, growth hormone generally inhibited lipoprotein lipase and also attenuated the inhibiting effect of insulin on hormone-sensitive lipase activity. Growth hormone treatment combined with restricted high-fat feeding reduced the activity of both lipases in adipose tissue and stimulated hormone-sensitive lipase in muscle. Generally, plasma levels of free fatty acids, glycerol and cholesterol were reduced by growth hormone, and in combination with restricted high-fat feeding, triglyceride levels improved too. | CONTRADICTION |
We conclude that the stress-induced increase in MCR is largely independent of changes in insulin, markedly augmented by beta-blockade, and related, at least in part, to promotion of lipolysis and glycogenolysis, and that R(a) is augmented by glucagon and alpha- and beta-catecholamine effects. | Glucose utilization increases markedly in the normal dog during stress induced by the intracerebroventricular (ICV) injection of carbachol. To determine the extent to which insulin, glucagon, and selective (alpha/beta)-adrenergic activation mediate the increment in glucose metabolic clearance rate (MCR) and glucose production (R(a)), we used five groups of normal mongrel dogs: 1) pancreatic clamp (PC; n = 7) with peripheral somatostatin (0.8 microg x kg(-1) x min(-1)) and intraportal replacement of insulin (1,482 +/- 84 pmol x kg(-1) x min(-1)) and glucagon (0.65 ng x kg(-1) x min(-1)) infusions; 2) PC plus combined alpha (phentolamine)- and beta (propranolol)-blockade (7 and 5 microg x kg(-1) x min(-1), respectively; alpha+beta; n = 5); 3) PC plus alpha-blockade (alpha; n = 6); 4) PC plus beta-blockade (beta; n = 5); and 5) a carbachol control group without PC (Con; n = 10). During ICV carbachol stress (0-120 min), catecholamines, ACTH, and cortisol increased in all groups. Baseline insulin and glucagon levels were maintained in all groups except Con, where glucagon rose 33%, and alpha, where insulin increased slightly but significantly. Stress increased (P < 0.05) plasma glucose in Con, PC, and alpha but decreased it in beta and alpha+beta. The MCR increment was greater (P < 0.05) in beta and alpha+beta than in Con, PC, and alpha. R(a) increased (P < 0.05) in all groups but was attenuated in alpha+beta. Stress-induced lipolysis was abolished in beta (P < 0.05). The marked rise in lactate in Con, PC, and alpha was abolished in alpha+beta and beta. | CONTRADICTION |
We conclude that subthreshold systemic doses of monoselective PDE 100 <le>, 4, and 5 inhibitors and in particular dual-selective PDE 100 <le>/4 inhibitors cause significant amplification of the pulmonary vasodilatory response to inhaled PGI <er>(2), while limiting the hypotensive effect to the pulmonary circulation. | Inhalation of aerosolized prostaglandin I(2) (PGI(2)) causes selective pulmonary vasodilation, but the effect rapidly levels off after termination of nebulization. In experimental pulmonary hypertension in intact rabbits, provoked by continuous infusion of the stable thromboxane mimetic U46619, the impact of intravenous phosphodiesterase (PDE) inhibitors on pulmonary and systemic hemodynamics was investigated in the absence and the presence of aerosolized PGI(2). We employed the monoselective inhibitors motapizone (PDE 3), rolipram (PDE 4), and zaprinast (PDE 5), as well as the dual-selective blockers zardaverine and tolafentrine (both PDE 3/4). All PDE inhibitors dose-dependently reduced the pulmonary artery pressure (Ppa), with doses for an approximately 20% decrease in pulmonary vascular resistance being 5 microgram/kg for motapizone, 25 microgram/kg for rolipram, 500 microgram/kg for zardaverine, 1 mg/kg for zaprinast, and 1 mg/kg for tolafentrine. Additive efficacy was noted when combining the monoselective 3 plus 4, 3 plus 5, and 4 plus 5 inhibitors. In parallel with the pulmonary vasorelaxant effect, all PDE inhibitors caused a decrease in systemic arterial pressure and an increase in cardiac output. Nebulized PGI(2) (56 ng/kg. min) reduced the U46619-evoked increase in Ppa by approximately 30%. This vasorelaxant effect was fully lost within 10 min after termination of PGI(2) nebulization. Coapplication of subthreshold doses of intravenous PDE inhibitors, which per se did not affect pulmonary and systemic hemodynamics, resulted in a marked prolongation of the post-PGI(2) decrease in Ppa for all blockers (motapizone at 2.2 microgram/kg, rolipram at 5.5 microgram/kg, zaprinast at 100 microgram/kg). The most effective agents, zardaverine (50 microgram/kg) and tolafentrine (100 microgram/kg), augmented the maximum Ppa drop during nebulization by approximately 30-50% and prolonged the post-PGI(2) pulmonary vasodilation to > 30 min, without affecting systemic arterial pressure and arterial oxygenation. | CONTRADICTION |
We conclude that (1) circulating CNP achieved by exogenous CNP infusion is regulated by NEP in vivo, (2) regional MCRs are heterogeneous with NEP promotion, (3) NEP inhibition does not potentiate acute cardiovascular actions of CNP <er>, and (4) a mild natriuretic and diuretic effect observed with CNP and NEP inhibition may be ANP dependent. | C-type natriuretic peptide (CNP) is a newly described 22-amino acid peptide of endothelial and renal cell origin with selective cardiovascular actions. Recent in vitro studies have reported that CNP is the most susceptible of all natriuretic peptides to enzymatic degradation by neutral endopeptidase 24.11 (NEP). The present study was undertaken to define the role of NEP in total and regional CNP metabolism and the modulatory actions of NEP inhibition on the biological actions of CNP. CNP (10 ng x kg(-1) x min(-1)) followed by candoxatrilat (240 microg x kg(-1) bolus and 8 microg x kg(-1) x min(-1)), a potent and selective NEP inhibitor, was administered intravenously to a group of anesthetized mongrel dogs (group 1) to permit calculation of total metabolic clearance rate (MCR); results were compared with those in a group receiving vehicle infusion followed by candoxatrilat (group 2; both groups, n=7). NEP inhibition increased circulating CNP achieved by exogenous infusion and reduced total MCR in group 1. The regional CNP MCRs increased after CNP administration. While the pulmonary MCR did not change during concomitant candoxatrilat infusion, renal MCR was suppressed. Hemodynamic changes were not different between groups. A mild natriuretic and diuretic effect in association with an increase in circulating and urinary ANP levels was not different between groups. Urinary CNP excretion did not change with CNP infusion but markedly increased after NEP inhibition. | CONTRADICTION |
We conclude that an increase in [Ca2+]i, due to activation of GABAA receptors and opening of L-type calcium channels, is necessary for death <le>-induced propofol of immature rat hippocampal neurons but that additional mechanisms not elicited by GABAA activation alone also contribute to cell propofol <er>. | Certain anesthetics exhibit neurotoxicity in the brains of immature but not mature animals. Gamma-aminobutyric acid (GABA), the primary inhibitory neurotransmitter in the adult brain, is excitatory on immature neurons via its action at the GABAA receptor, due to a reversed transmembrane chloride gradient. GABAA receptor activation in immature neurons is sufficient to open L-type voltage-gated calcium channels. As propofol is a GABAA agonist, we hypothesized that it and more specific GABAA modulators would increase intracellular free calcium ([Ca2+]i), resulting in the death of neonatal rat hippocampal neurons. Neuronal [Ca2+]i was monitored using Fura2-AM fluorescence imaging. Cell death was assessed by double staining with propidium iodide and Hoechst 33258 at 1 hour (acute) and 48 hours (delayed) after 5 hours exposure of neurons to propofol or the GABAA receptor agonist, muscimol, in the presence and absence of the GABA receptor antagonist, bicuculline, or the L-type Ca2+ channel blocker, nifedipine. Fluorescent measurements of caspase-3,-7 activities were performed at 1 hour after exposure. Both muscimol and propofol induced a rapid increase in [Ca2+]i in days in vitro (DIV) 4, but not in DIV 8 neurons, that was inhibited by nifedipine and bicuculline. Caspase-3,-7 activities and cell death increased significantly in DIV 4 but not DIV 8 hippocampal neuronal cultures 1 hour after 5 hours exposure to propofol, but not muscimol, and were inhibited by the presence of bicuculline or nifedipine. | CONTRADICTION |
We conclude that endogenous activation of mGlu5 receptors sustains the increase in c-Myc induced by LIF in embryonic stem cells by inhibiting both glycogen synthase kinase-3beta and PI-294002-K <er>, both effects resulting from the activation of PKC. | We have shown that endogenous activation of type 5 metabotropic glutamate (mGlu5) receptors supports the maintenance of a pluripotent, undifferentiated state in D3 mouse embryonic stem cells cultured in the presence of leukaemia inhibitory factor (LIF). Here, we examined the interaction between LIF and mGlu5 receptors using as a read-out the immediate early gene, c-Myc. The selective mGlu5 receptor antagonist, 2-methyl-6-(phenylenthynyl)pyridine (MPEP; 1 mum), reduced the increase in c-Myc protein levels induced by LIF by enhancing c-Myc ubiquitination. A reduction in c-Myc levels was also observed following small interfering RNA-mediated mGlu5 receptor gene silencing. MPEP reduced glycogen synthase kinase-3beta phosphorylation on Ser9, but increased phosphorylation of the phosphatidylinositol-3-kinase (PI-3-K) substrate, AKT. In our hands, activated PI-3-K reduced the stability of c-Myc, because (i) the PI-3-K inhibitor, LY294002, prevented the reduction in c-Myc levels induced by MPEP; and (ii) over-expression of AKT promoted c-Myc ubiquitination. All effects of MPEP were mimicked by protein kinase C (PKC) inhibitors and reversed by the PKC activator, tetradecanoylphorbol-13-acetate. | CONTRADICTION |
From our experiments it may be concluded that IO induces tachycardia in the pithed rat primarily by stimulating the sympathetic ganglia leading to the release of noradrenaline, which subsequently activates cardiac beta 1-adrenoceptors. | In the pithed normotensive rat the intravenous administration of increasing doses of angiotensin II caused an increase in heart rate by a maximum of 93 +/- 6 beats/min. This increase in heart rate was diminished dose-dependently by the concomitant infusion of the angiotensin II analogue saralasin. It was not affected by acute bilateral adrenalectomy but strongly reduced by pretreatment with reserpine. By using the selective antagonists of alpha 1-, alpha 2-, beta 1- or beta 2-adrenoceptors prazosin, yohimbine, atenolol and ICI 118,551, respectively, it was shown that this tachycardia was mediated by stimulation of cardiac beta 1-adrenoceptors. Moreover, the high endogenous angiotensin II level following pithing contributed to the basal heart rate in the pithed rat model. | CONTRADICTION |
From these results we conclude that protein synthesis stimulation causes the increase in alpha-adrenoceptor via activation of NHE, but independent of the concomitant increase in creatine phosphate contents. | Stimulation of alpha-adrenoceptors on ventricular cardiomyocytes isolated from adult rat hearts leads to cellular alkalization, increases of creatine phosphate concentration, RNA mass, and protein synthesis. This study investigated whether the increase of creatine phosphate concentrations is causally linked to the hypertrophic response of cardiomyocytes under alpha-adrenoceptor stimulation. Cellular alkalization achieved with phenylephrine (10 microM), an alpha-adrenoceptor agonist, was abolished in the presence of the sodium-proton-exchange (NHE)-inhibitor HOE 694 (1 microM). HOE 694 inhibited also the alpha-adrenoceptor-mediated increase in cellular creatine phosphate and the increase in cellular RNA mass. The phenylephrine-induced stimulation of protein synthesis (determined by incorporation of 14C-phenylalanine) was reduced by one-third when HOE 694 was present. beta-Guanidinopropionic acid was added to cardiomyocytes to reduce cellular creatine phosphate concentrations. In these cultures, alpha-adrenoceptor stimulation activated NHE, but creatine phosphate concentrations were not increased. Protein synthesis was augmented to the same extent as in control cultures, but total RNA mass did not increase. | CONTRADICTION |
We conclude that: 1) both muscle and neuronal ranolazine <le>(+) channels are as sensitive to Na block as their cardiac counterparts; 2) at its therapeutic plasma concentrations, Na interacts predominantly with the open but not resting or inactivated ranolazine <le>(+) channels; and 3) Na block of open ranolazine <le>(+) channels is via the conserved local anesthetic receptor albeit with a relatively slow on-rate. | Ranolazine is an antianginal agent that targets a number of ion channels in the heart, including cardiac voltage-gated Na(+) channels. However, ranolazine block of muscle and neuronal Na(+) channel isoforms has not been examined. We compared the state- and use-dependent ranolazine block of Na(+) currents carried by muscle Nav1.4, cardiac Nav1.5, and neuronal Nav1.7 isoforms expressed in human embryonic kidney 293T cells. Resting and inactivated block of Na(+) channels by ranolazine were generally weak, with a 50% inhibitory concentration (IC(50)) >/= 60 microM. Use-dependent block of Na(+) channel isoforms by ranolazine during repetitive pulses (+50 mV/10 ms at 5 Hz) was strong at 100 microM, up to 77% peak current reduction for Nav1.4, 67% for Nav1.5, and 83% for Nav1.7. In addition, we found conspicuous time-dependent block of inactivation-deficient Nav1.4, Nav1.5, and Nav1.7 Na(+) currents by ranolazine with estimated IC(50) values of 2.4, 6.2, and 1.7 microM, respectively. On- and off-rates of ranolazine were 8.2 microM(-1) s(-1) and 22 s(-1), respectively, for Nav1.4 open channels and 7.1 microM(-1) s(-1) and 14 s(-1), respectively, for Nav1.7 counterparts. A F1579K mutation at the local anesthetic receptor of inactivation-deficient Nav1.4 Na(+) channels reduced the potency of ranolazine approximately 17-fold. | CONTRADICTION |
We conclude that in A7r5 cells, the Ca2+ entry evoked by low concentrations of AVP is mediated largely by a non-capacitative pathway directly regulated by all-trans-retinoic acid produced by the sequential activities of phosphoinositidase C and diacylglycerol lipase. | 1. Depletion of the Ca2+ stores of A7r5 cells stimulated Ca2+, though not Sr2+, entry. Vasopressin (AVP) or platelet-derived growth factor (PDGF) stimulated Sr2+ entry. The cells therefore express a capacitative pathway activated by empty stores and a non-capacitative pathway stimulated by receptors; only the former is permeable to Mn2+ and only the latter to Sr2+. 2. Neither empty stores nor inositol 1,4,5-trisphosphate (InsP3) binding to its receptors are required for activation of the non-capacitative pathway, because microinjection of cells with heparin prevented PDGF-evoked Ca2+ mobilization but not Sr2+ entry. 3. Low concentrations of Gd3+ irreversibly blocked capacitative Ca2+ entry without affecting AVP-evoked Sr2+ entry. After inhibition of the capacitative pathway with Gd3+, AVP evoked a substantial increase in cytosolic [Ca2+], confirming that the non-capacitative pathway can evoke a significant increase in cytosolic [Ca2+]. 4. Arachidonic acid mimicked the effect of AVP on Sr2+ entry without stimulating Mn2+ entry; the Sr2+ entry was inhibited by 100 microM Gd3+, but not by 1 microM Gd3+ which completely inhibited capacitative Ca2+ entry. The effects of arachidonic acid did not require its metabolism. 5. AVP-evoked Sr2+ entry was unaffected by isotetrandrine, an inhibitor of G protein-coupled phospholipase A2. U73122, an inhibitor of phosphoinositidase C, inhibited AVP-evoked formation of inositol phosphates and Sr2+ entry. The effects of phorbol esters and Ro31-8220 (a protein kinase C inhibitor) established that protein kinase C did not mediate the effects of AVP on the non-capacitative pathway. An inhibitor of diacylglycerol lipase, RHC-80267, inhibited AVP-evoked Sr2+ entry without affecting capacitative Ca2+ entry or release of Ca2+ stores. 6. Selective inhibition of capacitative Ca2+ entry with Gd3+ revealed that the non-capacitative pathway is the major route for the Ca2+ entry evoked by low AVP concentrations. 7. | CONTRADICTION |
We conclude that the activation of nitric oxide <le>, localized in smooth muscle caveolae, by calcium entering through L-type calcium channels triggers nNOS production which modulates muscle contraction by a cGMP-dependent mechanism. | Neuronal nitric oxide synthase (nNOS) in myenteric neurons is activated during peristalsis to produce nitric oxide which relaxes intestinal smooth muscle. A putative nNOS is also found in the membrane of intestinal smooth muscle cells in mouse and dog. In this study we studied the possible functions of this nNOS expressed in mouse small intestinal smooth muscle colocalized with caveolin-1(Cav-1). Cav-1 knockout mice lacked nNOS in smooth muscle and provided control tissues. 60 mM KCl was used to increase intracellular [Ca(2+)] through L-type Ca(2+) channel opening and stimulate smooth muscle NOS activity in intestinal tissue segments. An additional contractile response to LNNA (100 microM, NOS inhibitor) was observed in KCl-contracted tissues from control mice and was almost absent in tissues from Cav-1 knockout mice. Disruption of caveolae with 40 mM methyl-beta cyclodextrin in tissues from control mice led to the loss of Cav-1 and nNOS immunoreactivity from smooth muscle as shown by immunohistochemistry and a reduction in the response of these tissues to N-omega-nitro-L-arginine (LNNA). Reconstitution of membrane cholesterol using water soluble cholesterol in the depleted segments restored the immunoreactivity and the response to LNNA added after KCl. Nicardipine (1 microM) blocked the responses to KCl and LNNA confirming the role of L-type Ca(2+) channels. ODQ (1 microM, soluble guanylate cyclase inhibitor) had the same effect as inhibition of NOS following KCl. | CONTRADICTION |
We conclude from these experiments that AII increases arterial blood pressure in the Pekin duck by mobilizing surface and E from both adrenal and extraadrenal stores. | The iv injection of Asp1, Val5-angiotensin II(AII) was followed by increases in arterial plasma norepinephrine (NE) and epinephrine (E) concentrations and elevation of arterial blood pressure in the anesthetized Pekin duck. AII injection did not affect the concentration of unconjugated dopamine in arterial plasma. Adrenalectomy inhibited the increases in plasma NE and E concentrations elicited by AII. Pressor responses to AII also were diminished after adrenalectomy, whereas pressor responses to exogenous NE and tyramine were not measurably affected. The alpha-adrenergic blocking drug phenoxybenzamine diminished the pressor effect of AII in both shamoperated and adrenalectomized ducks. The blockade of alpha-adrenergic receptors by phenoxybenzamine was incomplete, since pressor responses to exogenous NE were lessened by not abolished. | CONTRADICTION |
Given that eIF4E phosphorylation failed to increase Mnk and mTOR inhibitors in phosphatidylinositol 3-kinase (PI3K)-deficient cells, we conclude that mTOR inhibition increases mTOR inhibitors through a PI3K-dependent and Mnk-mediated mechanism. | The initiation factor eukaryotic translation initiation factor 4E (eIF4E) plays a critical role in initiating translation of mRNAs, including those encoding oncogenic proteins. Therefore, eIF4E is considered a survival protein involved in cell cycle progression, cell transformation, and apoptotic resistance. Phosphorylation of eIF4E (usually at Ser209) increases its binding affinity for the cap of mRNA and may also favor its entry into initiation complexes. Mammalian target of rapamycin (mTOR) inhibitors suppress cap-dependent translation through inhibition of the phosphorylation of eIF4E-binding protein 1. Paradoxically, we have shown that inhibition of mTOR signaling increases eIF4E phosphorylation in human cancer cells. In this study, we focused on revealing the mechanism by which mTOR inhibition increases eIF4E phosphorylation. Silencing of either mTOR or raptor could mimic mTOR inhibitors' effects to increase eIF4E phosphorylation. Moreover, knockdown of mTOR, but not rictor or p70S6K, abrogated rapamycin's ability to increase eIF4E phosphorylation. These results indicate that mTOR inhibitor-induced eIF4E phosphorylation is secondary to mTOR/raptor inhibition and independent of p70S6K. Importantly, mTOR inhibitors lost their ability to increase eIF4E phosphorylation only in cells where both Mnk1 and Mnk2 were knocked out, indicating that mTOR inhibitors increase eIF4E phosphorylation through a Mnk-dependent mechanism. | CONTRADICTION |
We conclude that, in CSD neurons, (i) somatodendritic ethanol supports SERT attraction, (ii) axonal ethanol specifies SERT aversion, (iii) the effect of axonal ethanol can override that of somatodendritic ethanol <le>. | The serotonin transporter (SERT) regulates serotonergic neurotransmission by retrieving released serotonin and replenishing vesicular stores. SERT is not only delivered to axons but it is also present on the neuronal soma and on dendrites. It has not been possible to restrict the distribution of SERT without affecting transporter function. Hence, the physiological role of somatodendritic SERT remains enigmatic. The SERT C-terminus harbors a conserved RI-motif, which recruits SEC24C, a cargo receptor in the coatomer protein-II coat. Failure to engage SEC24C precludes axonal enrichment of SERT. Here we introduced a point mutation into the RI-motif of human SERT causing confinement of the resulting - otherwise fully functional - hSERT-R607A on the somatodendritic membrane of primary rat dorsal raphe neurons. Transgenic expression of the corresponding Drosophila mutant dSERT-R599A led to its enrichment in the somatodendritic compartment of serotonergic neurons in the fly brain. We explored the possible physiological role of somatodendritic SERT by comparing flies harboring wild type SERT and dSERT-R599A in a behavioral paradigm for serotonin-modulated odor perception. When globally re-expressed in serotonergic neurons, wild type SERT but not dSERT-R599A restored ethanol preference. In contrast, dSERT-R599A restored ethanol preference after targeted expression in contralaterally projecting, serotonin-immunoreactive deuterocerebral (CSD) interneurons, while expression of wild type SERT caused ethanol aversion. | CONTRADICTION |
We conclude that Rho may inhibit insulin signaling by affecting posttranslational modification of insulin <er>A via nitric oxide/cGMP signaling pathway to cause MBP activation, actin cytoskeletal disorganization, and vasodilation. | Recent studies from our laboratory have shown that insulin induces relaxation of vascular smooth muscle cells (VSMCs) via stimulation of myosin phosphatase and inhibition of Rho kinase activity. In this study, we examined the mechanism whereby insulin inhibits Rho signaling and its impact on actin cytoskeleton organization. Incubation of confluent serum-starved VSMCs with thrombin or phenylephrine (PE) caused a rapid increase in glutathione S-transferase-Rhotekin-Rho binding domain-associated RhoA, Rho kinase activation, and actin cytoskeleton organization, which was blocked by preincubation with insulin. Preexposure to N(G)-monomethyl L-arginine acetate (L-NMMA), a nitric oxide synthase inhibitor, and Rp-8 CPT-cyclic guanosine monophosphate (RpcGMP), a cyclic guanosine monophosphate (cGMP) antagonist, attenuated the inhibitory effect of insulin on RhoA activation and restored thrombin-induced Rho kinase activation, and site-specific phosphorylation of the myosin-bound regulatory subunit (MBS(Thr695)) of myosin-bound phosphatase (MBP), and caused actin fiber reorganization. In contrast, 8-bromo-cGMP, a cGMP agonist, mimicked the inhibitory effects of insulin and abolished thrombin-mediated Rho activation. Insulin inactivation of RhoA was accompanied by inhibition of isoprenylation via reductions in geranylgeranyl transferase-1 activity as well as increased RhoA phosphorylation, which was reversed by pretreatment with RpcGMP and L-NMMA. | CONTRADICTION |
We conclude that IGF-1 inhibits the basolateral ulcerative colitis channels by activating PI3K-AKT-mTOR pathways. | The aim of the present study is to test the hypothesis that insulin-like-growth factor-1 (IGF-1) plays a role in the regulation of basolateral Cl channels in the thick ascending limb (TAL). The patch-clamp experiments demonstrated that application of IGF-I or insulin inhibited the basolateral 10-pS Cl channels. However, the concentration of insulin required for the inhibition of the Cl channels by 50% (K(1/2)) was ten times higher than those of IGF-1. The inhibitory effect of IGF-I on the 10-pS Cl channels was blocked by suppressing protein tyrosine kinase or by blocking phosphoinositide 3-kinase (PI3K). In contrast, inhibition of phospholipase C (PLC) failed to abolish the inhibitory effect of IGF-1 on the Cl channels in the TAL. Western blot analysis demonstrated that IGF-1 significantly increased the phosphorylation of phospholipid-dependent kinase (PDK) at serine residue 241 (Ser(241)) and AKT at Ser(473) in the isolated medullary TAL. Moreover, inhibition of PI3K with LY294002 abolished the effect of IGF-1 on the phosphorylation of PDK and AKT. The notion that the effect of IGF-1 on the 10-pS Cl channels was induced by stimulation of PDK-AKT-mTOR pathway was further suggested by the finding that rapamycin completely abolished the effect of IGF-1 on the 10-pS Cl channels in the TAL. | CONTRADICTION |
The authors conclude that in sheep, methylene blue attenuates the phenylephrine <le>-induced pulmonary hypertension and oedema, at least in part, by inhibiting the cyclo-oxygenase products of arachidonic acid. | The authors recently demonstrated that methylene blue (MB), an inhibitor of the nitric oxide (NO) pathway, reduces the increments in pulmonary capillary pressure, lung lymph flow and protein clearance in endotoxaemic sheep. In the present study, the authors examined whether MB influences pulmonary haemodynamics and accumulation of extravascular lung water (EVLW) by mechanisms other than the NO pathway. Sixteen awake, chronically-instrumented sheep randomly received either an intravenous injection of MB 10 mg x kg(-1) or isotonic saline. Thirty minutes later, all sheep received an intravenous infusion of Escherichia coli endotoxin 1 microg x kg(-1) for 20 min and either an intravenous infusion of MB 2.5 mg x kg(-1) x h(-1) or isotonic saline for 6 h. MB markedly attenuated the endotoxin-induced pulmonary hypertension and right ventricular failure, and reduced the accumulation of EVLW. Moreover, MB reduced the increments in plasma thromboxane B2 and 6-keto-prostaglandin F1alpha, and abolished the febrile response. However, MB had no effect on the changes in circulating neutrophils, serum hyaluronan, and total haemolytic activity of the alternative complement pathway. | CONTRADICTION |
We conclude that DM hypersecretion after CRH-mediated stimuli, including naloxone, is an inherent, but variable, feature of ACTH <er>, caused by expression of the genetic mutation at the anterior pituitary. | We previously showed that CRH-mediated stimuli, including exogenous CRH, cause ACTH hypersecretion in many myotonic dystrophy (DM) patients. We confirmed this by giving naloxone, a stimulator of endogenous CRH release, to a large number of DM patients and controls. DM patients, first degree relatives, and normal controls received i.v. naloxone at 1400 h, and blood was taken for ACTH (RIA) and cortisol (high pressure liquid chromatography) measurements from 15 min before to 120 min after naloxone treatment. DM patients had basal ACTH levels approximately twice those of controls, and their ACTH responses were 4 times those of controls. In contrast, DM basal cortisol levels were not significantly different from those of relatives and were slightly higher than those of normal subjects. Cortisol responses were similar in the three groups, probably due to attenuation at high levels of adrenocortical stimulation, although some patients with inappropriately low cortisol responses for their level of ACTH stimulation warrant further investigation. Nineteen of the 36 patients whose ACTH responses were greater than 3 SD above the normal mean were classed as hyperresponders. Seven patients, who were tested more than once, had reproducible responses relative to those of the normal subjects. | CONTRADICTION |
We conclude that I(Na) and reverse Ca modulate NCX <er>(2+) release in murine WT cardiomyocytes by augmenting the pool of NCX <er>(2+) that triggers ryanodine receptors. | The hypothesis that Na(+) influx during the action potential (AP) activates reverse Na(+)-Ca(2+) exchange (NCX) and subsequent entry of trigger Ca(2+) is controversial. We tested this hypothesis by monitoring intracellular Ca(2+) before and after selective inactivation of I(Na) prior to a simulated action potential in patch-clamped ventricular myocytes isolated from adult wild-type (WT) and NCX knockout (KO) mice. First, we inactivated I(Na) using a ramp prepulse to 45 mV. In WT cells, inactivation of I(Na) decreased the Ca(2+) transient amplitude by 51.1 +/- 4.6% (P < 0.001, n = 14) and reduced its maximum release flux by 53.0 +/- 4.6% (P < 0.001, n = 14). There was no effect on diastolic Ca(2+). In striking contrast, Ca(2+) transients in NCX KO cardiomyocytes were unaffected by the presence or absence of I(Na) (n = 8). We obtained similar results when measuring trigger Ca(2+) influx in myocytes with depleted sarcoplasmic reticulum. In WT cells, inactivation of I(Na) decreased trigger Ca(2+) influx by 37.8 +/- 6% and maximum rate of flux by 30.6 +/- 7.7% at 2.5 mm external Ca(2+) (P < 0.001 and P < 0.05, n = 9). This effect was again absent in the KO cells (n = 8). Second, exposure to 10 mum tetrodotoxin to block I(Na) also reduced the Ca(2+) transients in WT myocytes but not in NCX KO myocytes. | CONTRADICTION |
We conclude that desA-plasminogen activation protofibril formation is sufficient to initiate plasminogen activation enhancement of t-PA-catalyzed fibrin <er>, and that optimal stimulation depends on further plasmin-mediated modification of the plasminogen activation effector to desA-fragment X-related moieties. | When thrombin-mediated fibrin formation and tissue plasminogen activator (t-PA)-mediated fibrinolysis proceed in dynamic interaction, desA-(desB beta 1-42)-fragment X polymers are shown to be the predominant fibrin derivatives present during the rapid second phase of Glu1- and Lys78-plasminogen activation. To further investigate the effect of this intermediate, a method was developed for the production and purification of fibrinogen-derived desA-(desB beta 1-42)-fragment X, deprived of both COOH-terminal A alpha-chains, but still capable of thrombin-mediated polymerization. DesA-(desB beta 1-42)-fragment X polymer was compared to intact fibrin with regard to its stimulatory effect on Glu1-, Lys78-, and Val443-plasminogen activation, and its binding of Glu1- and Lys78-plasminogen. Pure fragment X polymer gave rise to a biphasic activation pattern like that of fibrin, demonstrating similar kinetics of rapid phase activation. The dissociation constant for the binding of plasminogen to the effector decreases by a factor of 14, and the stoichiometry increases by a factor of 2 upon plasmin-catalyzed cleavage of both native Glu1- to Lys78-plasminogen, and fibrin to fragment X polymer. | CONTRADICTION |
Disruption of microtubules with nocodazole inhibited endocytosis and azurophil granule fMLP stimulated by exocytosis in the presence of latrunculin A. Pharmacological inhibition of phosphatidylinositol 3-kinase, ERK1/2, and PKC significantly reduced exocytosis <le>-stimulated transferrin uptake in the presence of latrunculin A. Blockade of clathrin-mediated endocytosis had no significant effect on exocytosis <le>-stimulated phosphorylation of ERK1/2 in neutrophils pretreated with latrunculin A. From these data, we conclude that the actin cytoskeleton functions to limit microtubule-dependent, clathrin-mediated endocytosis in stimulated human neutrophils. | We have recently reported that disruption of the actin cytoskeleton enhanced N-formylmethionyl-leucyl-phenylalanine (fMLP)-stimulated granule exocytosis in human neutrophils but decreased plasma membrane expression of complement receptor 1 (CR1), a marker of secretory vesicles. The present study was initiated to determine if reduced CR1 expression was due to fMLP-stimulated endocytosis, to determine the mechanism of this endocytosis, and to examine its impact on neutrophil functional responses. Stimulation of neutrophils with fMLP or ionomycin in the presence of latrunculin A resulted in the uptake of Alexa fluor 488-labeled albumin and transferrin and reduced plasma membrane expression of CR1. These effects were prevented by preincubation of the cells with sucrose, chlorpromazine, or monodansylcadaverine (MDC), inhibitors of clathrin-mediated endocytosis. Sucrose, chlorpromazine, and MDC also significantly inhibited fMLP- and ionomycin-stimulated specific and azurophil granule exocytosis. | CONTRADICTION |
We conclude that the calcium-activated chloride channel (bCLCA1) is expressed in bovine corneal endothelial cells and cannot contribute to Ca(2+) dependent apical HCO <er>(3)(-) permeability, but not resting permeability, across the corneal endothelium. | Corneal endothelium secretes HCO(3)(-) from basolateral (stroma) to apical (anterior chamber) compartments. Apical HCO(3)(-) permeability can be enhanced by increasing [Ca(2+)](i). We hypothesized that the bovine calcium-activated chloride channel 1 (bCLCA1), shown previously by PCR screening to be expressed in corneal endothelium, is involved in Ca(2+) activated apical HCO(3)(-) permeability. bCLCA1 expression in cultured bovine corneal endothelial cells (CBCEC) was examined by in situ hybridization analysis, immunoblotting, immunofluorescence and confocal microscopy. Rabbit polyclonal antibodies were generated using a 14 aa polypeptide (417-430) from the predicted sequence of bCLCA1. The small interference RNA (siRNA) knock down technique was used to evaluate the functional involvement of bCLCA1 in apical HCO(3)(-) permeability. In situ hybridization confirmed prominent bCLCA1-specific mRNA expression in CBCEC. bCLCA1 antiserum detected the heterologously expressed bCLCA1 in HEK293 cells and a 90kDa band in CBCEC, which was absent when using the pre-immune serum or antigen absorption of serum. Immunofluoresence staining with anti-bCLCA1 antibody and confocal microscopy indicates an apical membrane location in CBCEC. In CBCEC transfected with bCLCA1 specific siRNA, bCLCA1 expression was reduced by 80%, while transfection with siControl scrambled sequence had no effect. Increasing [Ca(i)(2+)] by application of ATPgammaS or cyclopiazonic acid (CPA) increased apical HCO(3)(-) permeability in siControl transfected CBCEC, while having no effect on apical HCO(3)(-) permeability in bCLCA1 specific siRNA transfected cells. Baseline HCO(3)(-) permeability, however, was not different between controls and siRNA treated cells. | CONTRADICTION |
We conclude that the Rho <er>-kinase inhibitor <le>-kinase inhibitor prevents the growth, adhesion, invasion, and migration of 95D lung carcinoma cells by inhibiting the Rho <er>-kinase inhibitor <le>/Rho <er>-kinase inhibitor <le>-kinase pathway. | The objective of the study was to investigate the effects of a Rho-kinase inhibitor on 95D lung carcinoma cell growth, adhesion, invasion, and migration and to explore the underlying molecular mechanisms involved in this process. After treatment of 95D lung carcinoma cells with fasudil, an inhibitor of Rho-kinase, cell biological behaviors such as growth, adhesion, invasion, and migration were observed. Matrix metalloproteinase (MMP) activity and Western blot assay were used to evaluate underlying molecular mechanisms. The IC50 of fasudil to 95D lung carcinoma cells was approximately 0.79 mg/mL (95% confidence limits 0.58-1.11 mg/mL). After treatment with 0.75 mg/mL fasudil, the ability of 95D lung carcinoma cells for growth, adhesion, migration, and invasion was decreased significantly. Total active MMP2 was decreased approximately 22.7% (p < 0.05) and total MMP9 65.9% (p < 0.01). Myosin phosphatase target subunit 1 (MYPT1) was reduced by 29.4% (p < 0.05). | CONTRADICTION |
We have concluded from these observations that (1) steroid side-chain cleavage and pregnenolone conversion to progesterone are essential for gonadotropin-promoted follicle cholesterol production and the resulting reinitiation of meiosis by the oocyte, (2) the enzymes necessary for the conversion of steroid to 17 alpha-OH,20 beta-DHP, T, and E2 are present in the unstimulated, prematurational follicle, and (3) gonadotropin initiates cholesterol <er>ogenesis by acting at a step prior to the conversion of steroid to pregnenolone; it also appears to enhance aromatase activity. | In order to understand better the mechanism of gonadotropin action on steroidogenesis in prematurational follicles of Fundulus heteroclitus, follicle synthesis of 17 alpha-hydroxy, 20 beta-dihydroprogesterone (17 alpha-OH,20 beta-DHP), testosterone (T), and 17 beta-estradiol (E2) from a variety of precursors and the maturational response of oocytes were simultaneously followed in vitro. The addition of 25-hydroxycholesterol, pregnenolone, or progesterone to unstimulated follicles increased media 17 alpha-OH,20 beta-DHP, T, and E2, as well as oocyte germinal vesicle breakdown (GVBD) in a dose-dependent manner. Inhibition of cholesterol side-chain cleavage by aminoglutethimide blocked 25-hydroxycholesterol-promoted steroid accumulation and GVBD, indicating that 25-hydroxycholesterol does not directly induce GVBD, but rather is metabolized in the follicle to an active steroid (presumably 17 alpha-OH,20 beta-DHP). Likewise, trilostane, an inhibitor of delta 5-3 beta-hydroxysteroid dehydrogenase, blocked pregnenolone action. Both inhibitors also completely abolished steroid accumulation and GVBD promoted by a F. heteroclitus pituitary extract (FPE), but not GVBD induced by exogenous 17 alpha-OH,20 beta-DHP. FPE also significantly depressed T but enhanced E2 production from exogenous precursors. | CONTRADICTION |
From the relative sensitivity of the ATP and the Mg-proton pump <er>ase activity to the inhibitors it is concluded that about 35% of the Mg-proton pump <er>ase activity found in the brushborder membrane can be attributed to the proton pump <er>-driven ATP <le>. | In order to further characterize the ATP driven proton pump present in the luminal membrane of the renal proximal tubule, brushborder membranes were isolated from rat kidney cortex and the effect of various proton ATPase inhibitors on intravesicular ATP hydrolysis in sealed brushborder membrane vesicles and on Mg-ATPase activity in permeabilized brushborder membranes was investigated. The protonophor induced intravesicular ATP hydrolysis (ATP driven proton pump) was inhibited by DCCD and filipin but not by diethylstilbestrol and duramycin. All four compounds decreased Mg-ATPase activity, the two former inhibited the ATPase activity with a lower potency than the proton pump. NEM--up to 10 mM--and orthovanadate did not affect intravesicular ATP hydrolysis nor Mg-ATPase activity. | CONTRADICTION |
We conclude that pentamidine prolongs the cardiac hERG by block of action potential trafficking and reduction of the number of functional action potential channels at the cell surface. | The diamidine pentamidine is used to treat leishmaniasis, trypanosomiasis, and Pneumocystis carinii pneumonia. Treatment may be accompanied by prolongation of the QT interval of the electrocardiogram and torsades de pointes tachycardias. Up to now, it has been thought that therapeutic compounds causing QT prolongation are associated with direct block of the cardiac potassium channel human ether a-go-go-related gene (hERG), which encodes the alpha subunit of cardiac I(Kr) currents. We show that pentamidine has no acute effects on currents produced by hERG, KvLQT1/mink, Kv4.3, or SCNA5. Cardiac calcium currents and the guinea pig cardiac action potential were also not affected. After overnight exposure, however, pentamidine reduced hERG currents and inhibited trafficking and maturation of hERG with IC(50) values of 5 to 8 microM similar to therapeutic concentrations. Surface expression determined in a chemiluminescence assay was reduced on exposure to 10, 30, and 100 microM pentamidine by about 30, 40, and 70%, respectively. These effects were specific for hERG since expression of hKv1.5, KvLQT1/minK, and Kv4.3 was not altered. In isolated guinea pig ventricular myocytes, 10 microM pentamidine prolonged action potential duration APD(90) from 374.3 +/- 57.1 to 893.9 +/- 86.2 ms on overnight incubation. I(Kr) tail current density was reduced from 0.61 +/- 0.09 to 0.39 +/- 0.04 pA/pF. | CONTRADICTION |
We conclude that: 1) greater oxidative stress coupled with inactivation of AMP-activated protein kinase mediate the additive effects of nicotine and HFD on hepatic steatosis in obese mice and 67.8) increased lipolysis is an important contributor to hepatic steatosis. | Smoking is a major risk factor for diabetes and cardiovascular disease and may contribute to nonalcoholic fatty liver disease. We hypothesize that in the presence of nicotine, high-fat diet (HFD) causes more severe hepatic steatosis in obese mice. Adult C57BL6 male mice were fed a normal chow diet or HFD and received twice daily injections of nicotine (0.75 mg/kg body weight, ip) or saline for 10 wk. Light microscopic image analysis revealed significantly higher lipid accumulation in livers from mice on HFD plus nicotine (190 ± 19 μm(2)), compared with mice on HFD alone (28 ± 1.2 μm(2)). A significant reduction in the percent volume of endoplasmic reticulum (67.8%) and glycogen (49.2%) was also noted in hepatocytes from mice on HFD plus nicotine, compared with mice on HFD alone. The additive effects of nicotine on the severity of HFD-induced hepatic steatosis was associated with significantly greater oxidative stress, increased hepatic triglyceride levels, higher incidence of hepatocellular apoptosis, inactivation (dephosphorylation) of AMP-activated protein kinase, and activation of its downstream target acetyl-coenzyme A-carboxylase. Treatment with acipimox, an inhibitor of lipolysis, significantly reduced nicotine plus HFD-induced hepatic lipid accumulation. | CONTRADICTION |
Based on the potent antinociceptive effects of DAGO, the complete lack of such effects by the highly selective delta and kappa agonists, and the antagonism of CTOP by DTLET and beta-FNA, it is concluded that the antinociceptive effects of intranigral opioid agonists are mediated by mu receptors. | This study examined the effects of bilateral intranigral microinjection of selective opioid agonists on the tail-flick and hot-plate antinociception tests. The principal findings are: (1) the mu-selective agonist D-Ala2, N-Me-Phe4, Gly5-ol-enkephalin (DAGO) had antinociceptive effects on both tests which were reversible by beta-funaltrexamine (beta-FNA: a mu-selective antagonist) and naloxone (a non-selective opioid antagonist); (2) the antinociceptive potency of DAGO injected into the nigra is comparable to its potency in the periaqueductal gray; (3) intranigral D-Pen2, D-Pen5-enkephalin (a delta-selective agonist), U-50, 488H and dynorphin A-(1-13) (kappa-selective agonists) had no antinociceptive effects; (4) antinociceptive effects were produced by the mixed delta/mu agonists D-Thr2-leucine enkephalin-Thr (DTLET) and D-Ser2-leucine enkephalin-Thr (DSLET); (5) the effect of DTLET on the hot-plate but not the tail-flick test was reversed by Cys2, Tyr3, Orn5, Pen7-amide (CTOP; a mu-selective antagonist), beta-FNA, and naloxone, but not by the delta-selective antagonist naltrindole. | CONTRADICTION |
Though consistent with prior reports of PD protection afforded by legumes, and with speculation on the possible benefits of dietary or supplemental vitamin E in preventing PD <er>, these preliminary data do not not conclusively document a beneficial effect of dietary vitamin E on PD occurrence. | A nested case-control study of 84 incident cases of patients with idiopathic Parkinson's disease (PD) detected by June 30, 1994 and 336 age-matched control subjects, compared previously-documented intake of total dietary vitamin E and of selected vitamin E-containing foods. All study subjects had been followed for 27 to 30 years after diet recording in the 8,006-man Honolulu Heart Study cohort. We determined PD outcomes by periodic cohort re-examination and neurologic testing, private physician reports, examination of O'ahu neurologists' office records, and continual death certificate and hospital discharge diagnosis surveillance. Data on vitamin E intake, obtained from three dietary data sets at the time of cohort enrollment (1965 to 1968), included a food-frequency questionnaire and a 24-hour photograph-assisted dietary recall administered by trained dietitians. Although absence of PD was significantly associated with prior consumption of legumes (adjusted OR = 0.27, 95% CI 0.09 to 0.78), a dietary variable preselected for high vitamin E content, neither food categories nor quartiles nor continuous variables of vitamin E consumption were significantly associated with PD occurrence. | CONTRADICTION |
We conclude that the gastric ulcerogenic properties of NSAIDs are not accounted for solely by the inhibition of COX-2 expression and require the inhibition of both COX-2 expression and COX-2, the inhibition of COX-2 expression up-regulates COX-1 in association with gastric hypermotility, and PGs produced by COX-2 counteract the deleterious influences of the COX-2 expression inhibition. | This article reviews our recent studies on NSAID-induced gastric damage, focusing on the relation between COX inhibition and pathogenic events. Conventional NSAIDs such as indomethacin, at a dose that inhibits PG production, enhance gastric motility, resulting in an increase in mucosal permeability and MPO activity, and eventually, gastric lesions. The development of these lesions can be prevented by administering PGE2 or antisecretory drugs, and also via an atropine-sensitive mechanism, not related to any antisecretory action. The selective COX-2 inhibitor rofecoxib has no effect on PG production and does not induce damage in the stomach. The selective COX-1 inhibitor SC-560 also does not cause damage, despite evoking a decrease in the PGE2 level. The combined administration of SC-560 and rofecoxib, however, provokes the formation of gastric lesions. SC-560, but not rofecoxib, causes gastric hypermotility and an increase in mucosal permeability, although the level of MPO activity increases only when rofecoxib is co-administered. COX-2 mRNA is expressed in the stomach after administration of SC-560 and indomethacin but not rofecoxib. The up-regulation of COX-2 expression in response to indomethacin is prevented by atropine at a dose that inhibits gastric hypermotility but not by omeprazole at an antisecretory dose. | CONTRADICTION |
On the basis of these findings, the following is concluded: Muscle contraction cannot activate the glucose transport system independently of insulin. | The influence of contractile activity, insulin, catecholamines and diabetes mellitus on the acute as well as long-term regulation of glucose transport into skeletal muscle was investigated. In Paper I, glucose uptake was determined in the perfused hindlimb preparation; in Papers II-VI the glucose transport process was studied independently of glucose metabolism by determining the uptake of the non-metabolizable glucose analogue 3-O-methylglucose into isolated rat epitrochlearis muscles. The main findings are: Acute regulation of muscle glucose transport: 1. Muscle contraction-induced glucose uptake does not require the presence of insulin. 2. Exercise and a maximal insulin stimulus have additive effects on glucose transport. 3. Catecholamines decrease non-insulin-mediated glucose transport in the absence of albumin, whereas in the presence of albumin an enhancement occurs. 4. The effects of catecholamines are abolished during beta-adrenergic blockade, but remain unaffected during alpha-adrenergic blockade. Long-term regulation of muscle glucose transport: 5. The rat epitrochlearis muscle, a thin, predominantly fast-twitch muscle of the forearm, was shown to be a suitable preparation for the study of factors regulating glucose transport capacity on a long-term basis, since it maintains energy stores and tissue oxygenation for periods of at least 14 h. 6. Sustained insulin deficiency results in marked decreases in basal (40-45%), contraction-induced (50-60%), and insulin-stimulated (65-70%) glucose transport into rat epitrochlearis muscle. 7. The decreased contraction-induced glucose transport capacity can be prevented by frequent exercise during the period of insulin deficiency. 8. The decreased insulin responsiveness of the muscle glucose transport system can be reversed either by treating previously untreated diabetic rats with insulin or by in vitro incubation of the muscles for 12-14 hours. 9. The in vitro normalization of the insulin-stimulated glucose transport capacity (a) does not require the presence of serum or insulin, (b) occurs despite high (30 mM) concentrations of glucose, (c) is incomplete in the presence of diabetic serum and (d) is blocked to the amount of 30-80% in the presence of protein synthesis inhibition. | CONTRADICTION |
We conclude from these data that bFGF and FA prevent glutamate excitotoxicity through regulation of [Ca(2+)](i) and mitochondrial energy metabolism <er>. | Taurine, brain derived neurotrophic factor (BDNF), and basic fibroblast growth factor (bFGF) are known to control the development of early postnatal cerebellar granule cells. This study attempted to investigate possible mechanisms of this control by determining neuronal survival, calcium homeostasis, and related calcium-mediated functions, as well as the site of action during glutamate-induced excitotoxicity in cultures of cerebellar granule cells. We report that stimulation of glutamate receptors induced a rapid increase in intracellular calcium concentrations ([Ca(2+)](i)) and a decrease in mitochondrial energy metabolism. These effects of glutamate were time- and concentration-dependent and could be specifically blocked by glutamate receptor antagonists. Taurine and bFGF but not BDNF differently regulated [Ca(2+)](i), and preserved the mitochondrial energy metabolism in the presence of glutamate. The regulation of [Ca(2+)](i) by bFGF and taurine required pretreatment of cells with these factors. Confocal microscope analysis of [Ca(2+)](i) and (45)Ca(2+) uptake studies showed that bFGF reduced the magnitude of glutamate-induced calcium uptake with no apparent regulation thereafter. Taurine, on the other hand, did not affect the level of calcium uptake induced by glutamate but rather the duration of the maximal response; this maximal response was transient and returned to basal levels approximately 10 min after glutamate receptor stimulation. | CONTRADICTION |
We conclude that liver X receptor inhibits SREBP-1c transcription via combined actions on multiple transcription factors and that this effect is exerted at least in part through reduced transcriptional activity of Sp1 and SREBP-1c and disrupted assembly of the transcriptional initiation complex on the SREBP-1c promoter. | Induction of lipogenesis in response to insulin is critically dependent on the transcription factor, sterol regulatory element-binding protein-1c (SREBP-1c). FoxO1, a forkhead box class-O transcription factor, is an important mediator of insulin action, but its role in the regulation of lipid metabolism has not been clearly defined. We examined the effects of FoxO1 on srebp1 gene expression in vivo and in vitro. In vivo studies showed that constitutively active (CA) FoxO1 (CA-FoxO1) reduced basal expression of SREBP-1c mRNA in liver by ∼60% and blunted induction of SREBP-1c in response to feeding. In liver-specific FoxO knock-out mice, SREBP-1c expression was increased ∼2-fold. Similarly, in primary hepatocytes, CA-FoxO1 suppressed SREBP1-c expression and inhibited basal and insulin-induced SREBP-1c promoter activity. SREBP-1c gene expression is induced by the liver X receptor (LXR), but CA-FoxO1 did not block the activation of SREBP-1c by the LXR agonist TO9. Insulin stimulates SREBP-1c transcription through Sp1 and via "feed forward" regulation by newly synthesized SREBP-1c. CA-FoxO1 inhibited SREBP-1c by reducing the transactivational capacity of both Sp1 and SREBP-1c. In addition, chromatin immunoprecipitation assays indicate that FoxO1 can associate with the proximal promoter region of the srebp1 gene and disrupt the assembly of key components of the transcriptional complex of the SREBP-1c promoter. | CONTRADICTION |
We conclude that RV chemokine expression and TNF-alpha stimulation induce cooperative increases in epithelial cell infection <er>, providing a cellular mechanism for RV-induced exacerbations of airways disease. | Rhinovirus (RV) infections trigger exacerbations of airways disease, but underlying mechanisms remain unknown. We hypothesized that RV and cytokines present in inflamed airways combine to induce augmented airway epithelial cell chemokine expression, promoting further inflammation. To test this hypothesis in a cellular system, we examined the combined effects of RV39 and TNF-alpha, a cytokine increased in asthma and chronic obstructive pulmonary disease, on airway epithelial cell proinflammatory gene expression. Costimulation of 16HBE14o- human bronchial epithelial cells and primary mucociliary-differentiated tracheal epithelial cells with RV and TNF-alpha induced synergistic increases in IL-8 and epithelial neutrophil attractant-78 production. Similar synergism was observed for IL-8 promoter activity, demonstrating that the effect is transcriptionally mediated. Whereas increases in ICAM-1 expression and viral load were noted 16-24 h after costimulation, cooperative effects between RV39 and TNF-alpha were evident 4 h after stimulation and maintained despite incubation with blocking antibody to ICAM-1 given 2 h postinfection or UV irradiation of virus, implying that effects were not solely due to changes in ICAM-1 expression. Furthermore, RV39 infection induced phosphorylation of ERK and transactivation of the IL-8 promoter AP-1 site, which functions as a basal level enhancer, leading to enhanced TNF-alpha responses. | CONTRADICTION |
We conclude that 1) mitogenic stimulation of cultured bovine tracheal myocytes by cyclin induces PDGF D1 transcriptional activation and protein expression, 2) PDGF D1 expression is accompanied by Rb phosphorylation, which is evidence of increased PDGF D1-associated kinase activity in vivo, and 3) microinjection of anti-PDGF D1 antibodies inhibits cellular DNA synthesis, which is evidence that PDGF D1 is required for airway smooth muscle S phase traversal. | We examined the role of cyclin D1 in cultured bovine tracheal myocyte mitogenesis. Immunoblots using a polyclonal antibody against cyclin D1 showed the appearance of this protein 4 h after treatment with platelet-derived growth factor (PDGF), a potent mitogen for these cells. Immunoblots utilizing an antibody against the 110-kDa retinoblastoma protein (Rb), a downstream phosphorylation target of the cyclin D1/cyclin-dependent kinase 4 (cdk4) complex, showed reduced electrophoretic mobility of this protein as early as 8 h after PDGF treatment, suggesting phosphorylation of Rb by the cyclin D1/cdk4 dimer in vivo. Epidermal growth factor (EGF), a nonmitogen, failed to induce either cyclin D1 synthesis or Rb phosphorylation. PDGF treatment of cells transiently transfected with the full-length cyclin D1 promoter subcloned into a luciferase reporter increased luciferase activity almost threefold, demonstrating transcriptional activation of the cyclin D1 promoter with mitogenic stimulation. Finally, microinjection of individual myocytes with an affinity-purified antibody against cyclin D1 reduced the percentage of cells traversing S phase after serum stimulation, as assessed by fractional bromodeoxyuridine labeling (isotype control antibody, 0.74 +/- 0.10; anti-cyclin D1, 0.22 +/- 0.04; P = 0.0001). | CONTRADICTION |
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