query
stringlengths 63
661
| evidence
stringlengths 449
2.47k
| label
stringclasses 2
values |
|---|---|---|
Based on the above results, we concluded that autophagy promoted progesterone production by enhancing FSH through upregulation of Beclin1 via the PI3K/JNK/c-Jun pathway to accelerate LD degradation in porcine GCs, independent of the classical steroidogenic pathway. | Little is known about the molecular relationships among follicle stimulating hormone (FSH), lipid droplet (LD) degradation, and autophagy. In this study, we aimed to investigate the pathway by which FSH regulates autophagy and the potential role of autophagy in progesterone production. Our results revealed that FSH stimulated progesterone production in mammalian follicular granulosa cells (GCs) through a non-canonical pathway. In porcine secondary follicles cultured in vitro, FSH treatment increased the level of the autophagic marker, LC3-II, as well as increased the number of autophagic vacuoles in GCs. The underlying molecular mechanism and biological functions were then investigated in porcine GCs. Our results demonstrated that FSH could upregulate Beclin1 levels in porcine GCs; however, this effect was blocked by LY294002 (a PI3K/AKT inhibitor) and SP600125 (SAPK/JNK inhibitor). Further research confirmed that the transcriptional factor, c-Jun, was phosphorylated by FSH, then translocated into the nucleus from the cytoplasm and bound to the BECLIN1 promoter region, and that LY294002, SP600125, or c-Jun knockdown prevented the increase in Beclin1 levels induced by FSH. Interestingly, inhibition of autophagy using chloroquine or SP600125 decreased progesterone production in porcine GCs treated with FSH, although the expression of StAR and P450scc was not disturbed. Moreover, FSH treatment reduced the average number and size of LDs in porcine GCs, but these effects were eliminated by knocking down the key autophagy genes, ATG5 and BECLIN1; in addition, the effect of FSH on promoting progesterone secretion by the cells was also reduced significantly. | CONTRADICTION |
We conclude that heparin was sequestered in extracorporeal circuits in vitro, irrespective of coating the circuit with propofol <er>. | Propofol is sequestered in extracorporeal circuits, but the factors responsible for the phenomenon are mostly unknown. We have compared two extracorporeal circuits (oxygenators, reservoirs and tubings) coated with heparin with two corresponding uncoated circuits for their capacity to sequester propofol in vitro. Three experiments were conducted with each circuit. The circuit was primed with a mixture of Ringer's acetate solution and whole blood, and the study conditions (pump flow, temperature, pH) were standardized. Propofol was added to the solution to achieve a concentration of 2 micrograms ml-1. These studies were followed with concentrations of 10- and 100-fold to assess possible saturation of propofol binding. Serial samples were obtained from the circulating solution for measurement of propofol concentration. Propofol concentrations decreased to 22-32% of the initial predicted concentration of 2 micrograms ml-1 in the circuits (no significant difference between circuits). With greater concentrations, the circuits did not become saturated with propofol, even with the highest predicted concentration of 200 micrograms ml-1. | CONTRADICTION |
We conclude that activation of Cytochrome P450 by metformin might be mediated by mitochondria-derived RNS, and activation of the c-Src/PI3K pathway might generate a metabolite or other molecule inside the cell to promote Cytochrome P450 activation by the LKB1 complex. | Metformin, one of the most commonly used drugs for the treatment of type II diabetes, was recently found to exert its therapeutic effects, at least in part, by activating the AMP-activated protein kinase (AMPK). However, the site of its action, as well as the mechanism to activate AMPK, remains elusive. Here we report how metformin activates AMPK. In cultured bovine aortic endothelial cells, metformin dose-dependently activated AMPK in parallel with increased detection of reactive nitrogen species (RNS). Further, either depletion of mitochondria or adenoviral overexpression of superoxide dismutases, as well as inhibition of nitric-oxide synthase, abolished the metformin-enhanced phosphorylations and activities of AMPK, implicating that activation of AMPK by metformin might be mediated by the mitochondria-derived RNS. Furthermore, administration of metformin, which increased 3-nitrotyrosine staining in hearts of C57BL6, resulted in parallel activation of AMPK in the aorta and hearts of C57BL6 mice but not in those of endothelial nitric-oxide synthase (eNOS) knockout mice in which metformin had no effect on 3-nitrotyrosine staining. Because the eNOS knockout mice expressed normal levels of AMPK-alpha that was activated by 5-aminoimidazole-4-carboxamide riboside, an AMPK agonist, these data indicate that RNS generated by metformin is required for AMPK activation in vivo. In addition, metformin significantly increased the co-immunoprecipitation of AMPK and its upstream kinase, LKB1, in C57BL6 mice administered to metformin in vivo. Using pharmacological and genetic inhibitors, we found that inhibition of either c-Src or PI3K abolished AMPK that was enhanced by metformin. | CONTRADICTION |
We conclude that tumor in combination specifically with ultraviolet B induces fibroblasts to secrete interleukin-1 alpha necrosis factor-alpha, and that this ultraviolet B-specific induction of interleukin-1 alpha necrosis factor-alpha secretion is responsible for effects of ultraviolet B on the expression of matrix-related genes in the skin. | Ultraviolet light causes both acute and chronic changes in extracellular matrix. We sought to examine the effects of different ultraviolet wavelengths on expression of matrix-related genes in fibroblasts. We previously reported that tropoelastin gene expression in vivo decreases with acute ultraviolet B exposure, and interleukin-1 alpha-mediated upregulation of tropoelastin is blocked in vitro after ultraviolet B radiation. In this study, we found that only ultraviolet B, but not ultraviolet A or ultraviolet A1, blocked the ability of interleukin-1 alpha to stimulate tropoelastin expression in vitro. Ultraviolet B and interleukin-1 alpha synergistically increased tumor necrosis factor-alpha secretion by fibroblasts, a finding not seen with ultraviolet B alone nor with ultraviolet A or ultraviolet A1 combined with interleukin-1 alpha. Keratinocytes showed a similar ultraviolet B-specific induction of tumor necrosis factor-alpha production. Addition of tumor necrosis factor-alpha to cultured fibroblasts blocked interleukin-1 alpha-induced stimulation of tropoelastin message, and addition of anti-tumor necrosis factor-alpha antibodies restored the responsiveness of tropoelastin and collagen messages to exogenous interleukin-1 alpha after ultraviolet B exposure. | CONTRADICTION |
We conclude that Resveratrol acts as an antioxidant and completely reverses the anti-apoptotic effects of repetitive stress by blocking oxidative stress-induced NFkappaB activation which is the key regulatory step for the activation of down-stream survival elements Akt and p38MAPK <le>. | V79 lung fibroblasts were subjected to repetitive oxidative stress in culture through exposures to 30 microM H(2)O(2) for 4 weeks. Repetitively stressed cells were found to be significantly resistant to apoptosis-inducing agent such as ultraviolet radiation (UVR). Concurrent treatment with Resveratrol completely restored the normal apoptotic response after UVR. p38MAPK became dually phosphorylated during the stress period. Akt also became phosphorylated on Ser(473) in cells subjected to repetitive oxidative stress. In these cells, NFkappaB p65 became phosphorylated and appreciable nuclear localization of p65 was observed. NFkappaB transcriptional activity also became augmented during repetitive stress. Treatment of the repetitively stressed cells concurrently with Resveratrol or SB203580, a p38MAPK inhibitor, robustly blocked activation of p38MAPK, NFkappaB transcriptional activity, phosphorylation and nuclear localization of p65, and Akt phosphorylation. Pre-exposure to short interfering RNA (si RNA) to p38MAPK, resulted in a blockage of the Akt and NFkappaB p65 phosphorylation. However, inhibition of Akt activity through PI3 kinase inhibitor LY294002 did not result in obstruction of p38MAPK phosphorylation by H(2)O(2). Also, Resveratrol was effective as an antioxidant in counteracting a rise in reactive oxygen species (ROS) and p38MAPK activation by H(2)O(2) was completely blocked by antioxidant N-acetyl cysteine (NAC). | CONTRADICTION |
Our data lead us to conclude that elevation of AP-1 or NF-kB indirectly decreases MAO-A protein levels which, in turn, diminishes MAO-A ability in the VTA of the mesolimbic dopaminergic pathway that has been implicated in cells under stress especially in the SN and VTA. | Monoamine oxidase (MAO) enzymes play a critical role in controlling the catabolism of monoamine neurotransmitters and biogenic trace amines and behavior in humans. However, the mechanisms that regulate MAO are unclear. Several transcription factor proteins are proposed to modulate the transcription of MAO gene, but evidence supporting these hypotheses is controversial. We aimed to investigate the mechanism of gene transcription regulator proteins on amphetamine-induced behavior. We applied aptamers containing a DNA binding sequence, as well as a random sequence (without target) to study the modulation of amphetamine-induced MAO levels and hyperactivity in living mice. We pretreated in adult male C57black6 mice (Taconic Farm, Germantown, NY) (n ≥ 3 litters at a time), 2 to 3 months of age (23 ± 2 gm body weight) with double-stranded (ds) DNA aptamers with sequence specific to activator protein-1 (5ECdsAP1), nuclear factor-kappa beta (5ECdsNF-kB), special protein-1 (5ECdsSP-1) or cyclicAMP responsive element binding (5ECdsCreB) protein binding regions, 5ECdsRan [a random sequence without target], single-stranded AP-1 (5ECssAP-1) (8 nmol DNA per kg) or saline (5 μl, intracerebroventricular [icv] injection) control before amphetamine administration (4 mg/kg, i.p.). We then measured and analyzed locomotor activities and the level of MAO-A and MAO-B activity. In the pathological condition of amphetamine exposure, we showed here that pretreatment with 5ECdsAP1 and 5ECdsNF-kB reversed the decrease of MAO-A activity (p < 0.05, t test), but not activity of the B isomer (MAO-B), in the ventral tegmental area (VTA) and substantia nigra (SN) of C57black6 mice. The change in MAO-A level coincided with a reversed amphetamine-induced restless behavior of mice. Pretreatments with saline, 5ECdsCreB, 5ECdsSP-1, 5ECdsRan or 5ECssAP-1 had no effect. | CONTRADICTION |
Taken together, we concluded that TACE activates AS-III <er>-dependent anti-inflammatory and growth factor signaling in vascular endothelial cells in a p38-dependent fashion, which may contribute to its cardiovascular protective effect. | Astragaloside III (AS-III) is a triterpenoid saponin contained in Astragali Radix and has potent anti-inflammatory effects on vascular endothelial cells; however, underlying mechanisms are unclear. In this study, we provided the first piece of evidence that AS-III induced phosphorylation of TNF-α converting enzyme (TACE) at Thr735 and enhanced its sheddase activity. As a result, AS-III reduced surface TNFR1 level and increased content of sTNFR1 in the culture media, leading to the inhibition of NF-κB signaling pathway and attenuation of downstream cytokine gene expression. Furthermore, AS-III induced TACE-dependent epidermal growth factor receptor (EGFR) transactivation and activation of downstream ERK1/2 and AKT pathways. Finally, AS-III induced activation of p38. Both TACE activation and EGFR transactivation induced by AS-III were significantly inhibited by p38 inhibitor SB203580. | CONTRADICTION |
We conclude that d-glucose regulates Akt signaling through threonine phosphorylation of Akt and that EC <le>-impaired PI3k-Akt signaling may promote hyperglycemia proliferative dysfunction in diabetes. | Diabetes mellitus is a major risk factor for the development of vascular complications. We hypothesized that hyperglycemia decreases endothelial cell (EC) proliferation and survival via phosphatidylinositol 3-kinase (PI3k) and Akt signaling pathways. We cultured human umbilical vein ECs (HUVEC) in 5, 20, or 40 mM d-glucose. Cells grown in 5, 20, and 40 mM mannitol served as a control for osmotic effects. We measured EC proliferation for up to 15 days. We assessed apoptosis by annexin V and propidium iodide staining and flow cytometry, analyzed cell lysates obtained on culture day 8 for total and phosphorylated PI3k and Akt by Western blot analysis, and measured Akt kinase activity using a GSK fusion protein. HUVEC proliferation was also tested in the presence of pharmacological inhibitors of PI3k-Akt (wortmannin and LY294002) and after transfection with a constitutively active Akt mutant. ECs in media containing 5 mM d-glucose (control) exhibited log-phase growth on days 7-10. d-Glucose at 20 and 40 mM significantly decreased proliferation versus control (P < 0.05 for both), whereas mannitol did not impair EC proliferation. Apoptosis increased significantly in HUVEC exposed to 40 mM d-glucose. d-Glucose at 40 mM significantly decreased tyrosine-phosphorylated PI3k, threonine 308-phosphorylated-Akt, and Akt activity relative to control 5 mM d-glucose. Pharmacological inhibition of PI3k-Akt resulted in a dose-dependent decrease in EC proliferation. Transfection with a constitutively active Akt mutant protected ECs by enhancing proliferation when grown in 20 and 40 mM d-glucose. | CONTRADICTION |
We conclude that curcumin-induced cytotoxicity was not due to cellular ceramide accumulation and damage to mitochondria that resulted in apoptosis mediated by AIF and other caspase-independent processes. | Curcumin, the principal curcuminoid of tumeric, has potent anticancer activity. To determine the mechanism of curcumin-induced cytotoxicity in prostate cancer cells, we exposed PC3 prostate carcinoma cells to 25 to 100 microM curcumin for 24 to 72 h. Curcumin treatment of PC3 cells caused time- and dose-dependent induction of apoptosis and depletion of cellular reduced glutathione (GSH). Exogenous GSH and its precursor N-acetyl-cysteine, but not ascorbic acid (AA) or ebselen, decreased curcumin accumulation in PC3 cells and also prevented curcumin-induced DNA fragmentation. The failure of AA and ebselen to protect PC3 cells from curcumin-induced apoptosis argued against the involvement of reactive oxygen species; rather, GSH-mediated inhibition of curcumin-induced cytotoxicity was due to reduced curcumin accumulation in PC3 cells. Curcumin-treated PC3 cells showed apoptosis-inducing cellular ceramide accumulation and activation of p38 mitogen-activated protein kinase (MAPK) and c-jun N-terminal kinase (JNK). Caspase-3, caspase-8, and caspase-9 were activated, and cytochrome c and apoptosis-inducing factor (AIF) were released from mitochondria following curcumin treatment. Interestingly, curcumin-induced apoptosis was not prevented by p38 MAPK, JNK, or caspase inhibition. | CONTRADICTION |
We conclude that in a variety of tissues p107 development by loss of pRB is suppressed by its homologs tumor and p130. | The retinoblastoma gene family consists of three genes: RB, p107, and p130. While loss of pRB causes retinoblastoma in humans and pituitary gland tumors in mice, tumorigenesis in other tissues may be suppressed by p107 and p130. To test this hypothesis, we have generated chimeric mice from embryonic stem cells carrying compound loss-of-function mutations in the Rb gene family. We found that Rb/p107- and Rb/p130-deficient mice were highly cancer prone. | CONTRADICTION |
We conclude that, in addition to their well-established roles in Grk1 II inactivation, Meta and Arr1 can modulate the kinetics of Grk1 III decay and rod dark adaptation in vivo. | Photoactivation of vertebrate rhodopsin converts it to the physiologically active Meta II (R*) state, which triggers the rod light response. Meta II is rapidly inactivated by the phosphorylation of C-terminal serine and threonine residues by G-protein receptor kinase (Grk1) and subsequent binding of arrestin 1 (Arr1). Meta II exists in equilibrium with the more stable inactive form of rhodopsin, Meta III. Dark adaptation of rods requires the complete thermal decay of Meta II/Meta III into opsin and all-trans retinal and the subsequent regeneration of rhodopsin with 11-cis retinal chromophore. In this study, we examine the regulation of Meta III decay by Grk1 and Arr1 in intact mouse rods and their effect on rod dark adaptation. We measure the rates of Meta III decay in isolated retinas of wild-type (WT), Grk1-deficient (Grk1(-/-)), Arr1-deficient (Arr1(-/-)), and Arr1-overexpressing (Arr1(ox)) mice. We find that in WT mouse rods, Meta III peaks ∼6 min after rhodopsin activation and decays with a time constant (τ) of 17 min. Meta III decay slows in Arr1(-/-) rods (τ of ∼27 min), whereas it accelerates in Arr1(ox) rods (τ of ∼8 min) and Grk1(-/-) rods (τ of ∼13 min). In all cases, regeneration of rhodopsin with exogenous 11-cis retinal is rate limited by the decay of Meta III. Notably, the kinetics of rod dark adaptation in vivo is also modulated by the levels of Arr1 and Grk1. | CONTRADICTION |
We conclude that PXR <le>, but not CYP3A4, can be up-regulated by thyroid hormones in vitro by a P-gp <er>-independent mechanism. | P-glycoprotein (P-gp) and cytochrome P450 3A4 (CYP3A4) constitute a physiologic barrier in the intestine for many of the same substrates. Their expression can be influenced by nuclear receptor NR1I2 (pregnane X receptor; PXR), which acts as a receptor for various endobiotics and xenobiotics. However, P-gp and CYP3A4 are not identical in anatomic localization, suggesting unique as well as shared regulatory mechanisms of gene expression. We used established human colon carcinoma cell lines (LS180 and Caco-2) and measured mRNA and protein levels in cells after exposures to levothyroxine (L-T(4)), triiodo-L-thyronine (L-T(3)), and rifampin. Results indicate that L-T(4), L-T(3), and rifampin can upregulate the expression of P-gp mRNA and protein in LS180 cells, but only L-T(4) and L-T(3) can produce the same effect in Caco-2 cells, which are relatively lacking in PXR. In addition, L-T(4) and L-T(3) did not affect the expression of CYP3A4 in either cell line. | CONTRADICTION |
Effects of ibuprofen are mimicked by stimulation of AMPK and blocked by the AMPK promoter compound C. We conclude that high-dose ibuprofen treatment enhances microtubule formation in CF cells likely through an AMPK-related pathway. | High-dose ibuprofen, an effective anti-inflammatory therapy for the treatment of cystic fibrosis (CF), has been shown to preserve lung function in a pediatric population. Despite its efficacy, few patients receive ibuprofen treatment due to potential renal and gastrointestinal toxicity. The mechanism of ibuprofen efficacy is also unclear. We have previously demonstrated that CF microtubules are slower to reform after depolymerization compared with respective wild-type controls. Slower microtubule dynamics in CF cells are responsible for impaired intracellular transport and are related to inflammatory signaling. Here, it is identified that high-dose ibuprofen treatment in both CF cell models and primary CF nasal epithelial cells restores microtubule reformation rates to wild-type levels, as well as induce extension of microtubules to the cell periphery. Ibuprofen treatment also restores microtubule-dependent intracellular transport monitored by measuring intracellular cholesterol transport. These effects are specific to ibuprofen as other cyclooxygenase inhibitors have no effect on these measures. | CONTRADICTION |
It is not concluded that the hyperpnoea produced by adrenaline and noradrenaline infusions in the cat is predominantly reflex in origin and is mediated by the arterial chemoreceptors.10. | 1. Intravenous infusions of adrenaline and noradrenaline in doses averaging 0.8 mug/kg.min increased the respiratory minute volume of anaesthetized cats breathing room air. The mean increase in respiratory minute volume was 14% during adrenaline infusion and 8% during noradrenaline infusion.2. In a small group of decerebrate cats infusions of adrenaline and noradrenaline increased ventilation by 19 and 27% respectively.3. Intravenous catecholamine infusions also increased the respiratory responses of anaesthetized animals to the inhalation of 5% or 10% O(2) in N(2) and to the inhalation of 5% CO(2) in air.4. Adrenaline and noradrenaline infusions had no significant effect on the ventilation of animals breathing 100% O(2), nor did they significantly alter the respiratory response to the inhalation of 5% CO(2) in O(2).5. After section of the carotid sinus and aortic nerves, a blood-pressure compensator being used to minimize changes in arterial pressure, catecholamines had no effect on the respiration of cats breathing air.6. An increase in carotid body chemoreceptor discharge accompanied the increase in ventilation during catecholamine infusion.7. Intravenous catecholamine infusions still produced an increase in ventilation and carotid body chemoreceptor discharge after both aortic nerves and both cervical sympathetic nerves had been cut.8. Intra-arterial infusions into one carotid artery of 0.2 mug/kg.min of adrenaline or 0.1 mug/kg.min of noradrenaline led to mean increases in respiratory minute volume of 9.9 and 11.5% respectively. No increase occurred after section of the corresponding carotid sinus nerve. Such infusions also evoked an increase in carotid body chemoreceptor discharge.9. | CONTRADICTION |
We conclude that: (1) INDO markedly reduces the vasomotor response of the ICA to changes in P ETC O2; and (0.47) INDO may be reducing CO2 -mediated vasomotion via a mechanism(s) independent of cyclooxygenase inhibition. | Cerebral blood flow increases during hypercapnia and decreases during hypocapnia; it is unknown if vasomotion of the internal carotid artery is implicated in these responses. Indomethacin, a non-selective cyclooxygenase inhibitor (used to inhibit prostaglandin synthesis), has a unique ability to blunt cerebrovascular carbon dioxide reactivity, while other cyclooxygenase inhibitors have no effect. We show significant dilatation and constriction of the internal carotid artery during hypercapnia and hypocapnia, respectively. Indomethacin, but not ketorolac or naproxen, reduced the dilatatory response of the internal carotid artery to hypercapnia The differential effect of indomethacin compared to ketorolac and naproxen suggests that indomethacin inhibits vasomotion of the internal carotid artery independent of prostaglandin synthesis inhibition. Extra-cranial cerebral blood vessels are implicated in the regulation of cerebral blood flow during changes in arterial CO2 ; however, the mechanisms governing CO2 -mediated vasomotion of these vessels in humans remain unclear. We determined if cyclooxygenase inhibition with indomethacin (INDO) reduces the vasomotor response of the internal carotid artery (ICA) to changes in end-tidal CO2 (P ETC O2). Using a randomized single-blinded placebo-controlled study, participants (n = 10) were tested on two occasions, before and 90 min following oral INDO (1.2 mg kg(-1) ) or placebo. Concurrent measurements of beat-by-beat velocity, diameter and blood flow of the ICA were made at rest and during steady-state stages (4 min) of iso-oxic hypercapnia (+3, +6, +9 mmHg P ETC O2) and hypocapnia (-3, -6, -9 mmHg P ETC O2). To examine if INDO affects ICA vasomotion independent of cyclooxygenase inhibition, two participant subsets (each n = 5) were tested before and following oral ketorolac (post 45 min, 0.25 mg kg(-1) ) or naproxen (post 90 min, 4.2 mg kg(-1) ). During pre-drug testing in the INDO trial, the ICA dilatated during hypercapnia at +6 mmHg (4.72 ± 0.45 vs. 4.95 ± 0.51 mm; P < 0.001) and +9 mmHg (4.72 ± 0.45 mm vs. 5.12 ± 0.47 mm; P < 0.001), and constricted during hypocapnia at -6 mmHg (4.95 ± 0.33 vs. 4.88 ± 0.27 mm; P < 0.05) and -9 mmHg (4.95 ± 0.33 vs. 4.82 ± 0.27 mm; P < 0.001). Following INDO, vasomotor responsiveness of the ICA to hypercapnia was reduced by 67 ± 28% (0.045 ± 0.015 vs. 0.015 ± 0.012 mm mmHg P ETC O2(-1) ). There was no effect of the drug in the ketorolac and naproxen trials. | CONTRADICTION |
We conclude that: (a) insulin stimulates 2-deoxyglucose uptake using a pathway that converges with that of vanadate at the level of PI 3-kinase; and (b) adipocytes from old rats are defective in the vanadate pathway at steps located both upstream and downstream of PI 3-kinase. | Vanadate stimulates adipocyte 2-deoxyglucose transport and GLUT-4 translocation to the membrane through an insulin receptor-independent but wortmannin-inhibitable pathway. Vanadate stimulates PI 3-kinase in anti-IRS-1 immunoprecipitates and the binding between IRS-1 and the p85alpha subunit of PI 3-kinase. In insulin-resistant adipocytes from old rats vanadate fully stimulates IRS-1-associated PI 3-kinase, but partially activates glucose uptake. | CONTRADICTION |
We conclude that melatonin inhibits PAF. activity and expression in HUVECs by regulating gene expression of specific PAF. promoter regions, thereby reducing the local production of estrogens. | Melatonin is known to suppress the development of endocrine-responsive breast cancers by interacting with the estrogen signaling pathways. Paracrine interactions between malignant epithelial cells and proximal stromal cells are responsible for local estrogen biosynthesis. In human breast cancer cells and peritumoral adipose tissue, melatonin downregulates aromatase, which transforms androgens into estrogens. The presence of aromatase on endothelial cells indicates that endothelial cells may contribute to tumor growth by producing estrogens. Since human umbilical vein endothelial cells (HUVECs) express both aromatase and melatonin receptors, the aim of the present study was to evaluate the ability of melatonin to regulate the activity and expression of aromatase on endothelial cells, thus, modulating local estrogen biosynthesis. In the present study, we demonstrated that melatonin inhibits the growth of HUVECs and reduces the local biosynthesis of estrogens through the downregulation of aromatase. These results are supported by three lines of evidence. Firstly, 1 mM of melatonin counteracted the testosterone-induced cell proliferation of HUVECs, which is dependent on the local biosynthesis of estrogens from testosterone by the aromatase activity of the cells. Secondly, we found that 1 mM of melatonin reduced the aromatase activity of HUVECs. Finally, by real‑time RT-PCR, we demonstrated that melatonin significantly downregulated the expression of aromatase as well as its endothelial-specific aromatase promoter region I.7. | CONTRADICTION |
We concluded that cell proliferation inhibited apoptosis in an AKT2/MCL1 dependent way and promoted Rac1 through JNK/c-JUN/Cyclin-D1. | Rac1, known as a "molecular switch", plays a crucial role in plenty of cellular processes. Rac1 aggravates the damage of myocardial cells in the process of myocardial ischemia-reperfusion during myocardial infarction through activating the NADPH oxidase and bringing about the reactive oxygen species(ROS) generation. Myocardial ischemia and hypoxia are the basic pathogenesis of myocardial infarction and the underlying mechanisms are intricate and varied. Moreover, the regulatory effect of Rac1 on myocardial cells in the condition of serum starvation and the potential mechanisms are still incompletely undefined. Therefore, heart-derived H9c2 cells cultured in 0% serum were used to mimic ischemic myocardial cells and to clarify the role of Rac1 in H9c2 cells and the underlying mechanisms during serum deficiency. After Rac1 was knocked down using specific siRNA, cell apoptosis was assessed by flow cytometry assay and cell proliferation was detected by CCK-8 assay and EdU assay. In addition, the expression and activation of protein in related signaling pathway were detected by Western blot and siRNAs was used to testify the signaling pathways. Our results indicated that Rac1 inhibited apoptosis, promoted proliferation and cell cycle progression of H9c2 cells during serum deficiency. | CONTRADICTION |
We conclude that CD95 <le>-induced caspase-3 activation in HT-22 cells was not readily detected at the single-cell level using the DsRed-EYFP-based FRET construct, making this a useful technology to monitor caspase-3 activity in living cells. | A probe consisting of Discosoma red fluorescent protein (DsRed) and enhanced yellow fluorescent protein (EYFP) linked by a 19-amino-acid chain containing the caspase-3 cleavage site Asp-Glu-Val-Asp was developed to monitor caspase-3 activation in living cells. The expression of the tandem construct in mammalian cells yielded a strong red fluorescence when excited with 450- to 490-nm light or with a 488-nm argon ion laser line as a result of fluorescence resonance energy transfer (FRET) from donor EYFP to acceptor DsRed. The advantage over previous constructs using cyan fluorescent protein is that our construct can be used when excitation wavelengths lower than 488nm are not available. To validate the construct, murine HT-22 hippocampal neuronal cells were triggered to undergo CD95-induced neuronal death. An increase in caspase-3 activity was demonstrated by a reduction of FRET in cells transfected with the construct. This was manifested by a dequenching of EYFP fluorescence leading to an increase in EYFP emission and a corresponding decrease in DsRed fluorescence, which correlated with an increase in pro-caspase-3 processing. | CONTRADICTION |
We conclude that FLAP gene expression enhances LPS via both NF-kappaB- and C/EBP-mediated transcriptional mechanisms in mononuclear phagocytes. | We examined induced expression of the 5-lipoxygenase-activating protein (FLAP), which is critical for leukotriene synthesis in mononuclear phagocytes. Prolonged exposure to the bacterial component, lipopolysaccharide (LPS), increased FLAP gene transcription, mRNA expression, and protein expression in the human monocyte-like THP-1 cell line. Activation and inhibition of the NF-kappaB pathway modulated LPS induction of FLAP gene expression. An NF-kappaB-mediated mechanism of action was supported by overexpression of dominant-negative IkappaBalpha and p50/p65 proteins. EMSA/supershift and DNase I footprint analyses revealed that p50 binds to an NF-kappaB site located in the proximal FLAP promoter, while chromatin immunoprecipitation assays demonstrated that LPS induced binding of p50 but not of p65. Moreover, EMSA/supershift analyses demonstrated that LPS induced time-dependent binding of THP-1 nuclear extracts (containing p50) to this promoter region. Mutation of the NF-kappaB site decreased basal promoter activity and abolished the p50- and p65-associated induction. EMSA/supershift analyses also demonstrated that LPS induced binding of THP-1 nuclear extracts [containing CCAAT/enhancer binding protein (C/EBP)-alpha, -delta, and -epsilon] to a C/EBP site located adjacent to the NF-kappaB site in the FLAP promoter. | CONTRADICTION |
Thus, we concluded that inhibition of SCARB1 expression induced by DNMT3b at least partly accelerated atherosclerosis <le>-mediated Hcy through promoting lipid accumulation in foam cells, which was attributed to the decreased binding of SP1 to SCARB1 promoter. | Homocysteine (Hcy) is an independent risk factor for atherosclerosis, which is characterized by lipid accumulation in the atherosclerotic plaque. Increasing evidence supports that as the main receptor of high-density lipoprotein, scavenger receptor class B member 1 (SCARB1) is protective against atherosclerosis. However, the underlying mechanism regarding it in Hcy-mediated atherosclerosis remains unclear. Here, we found the remarkable inhibition of SCARB1 expression in atherosclerotic plaque and Hcy-treated foam cells, whereas overexpression of SCARB1 can suppress lipid accumulation in foam cells following Hcy treatment. Analysis of SCARB1 promoter showed that no significant change of methylation level was observed both in vivo and in vitro under Hcy treatment. Moreover, it was found that the negative regulation of DNMT3b on SCARB1 was due to the decreased recruitment of SP1 to SCARB1 promoter. | CONTRADICTION |
We conclude that under our experimental conditions, the Ca channel is symmetrical with respect to inorganic ion block and that intracellular calcium can modulate Ca channel currents via a low-affinity binding site. | The patch clamp technique was used to record unitary currents through single calcium channels from smooth muscle cells of rabbit mesenteric arteries. The effects of external cadmium and cobalt and internal calcium, barium, cadmium, and magnesium on single channel currents were investigated with 80 mM barium as the charge carrier and Bay K 8644 to prolong openings. External cadmium shortened the mean open time of single Ca channels. Cadmium blocking and unblocking rate constants of 16.5 mM-1 ms-1 and 0.6 ms-1, respectively, were determined, corresponding to dissociation constant Kd of 36 microM at -20 mV. These results are very similar to those reported for cardiac muscle Ca channels (Lansman, J. B., P. Hess, and R. W. Tsien. 1986. J. Gen. Physiol. 88:321-347). In contrast, Cd2+ (01-10 mM), when applied to the internal surface of Ca channels in inside-out patches, did not affect the mean open time, mean unitary current, or the variance of the open channel current. Internal calcium induced a flickery block, with a Kd of 5.8 mM. Mean blocking and unblocking rate constants for calcium of 0.56 mM-1 ms-1 and 3.22 ms-1, respectively, were determined. Internal barium (8 mM) reduced the mean unitary current by 36%. | CONTRADICTION |
We conclude that NHE1 activity is critical for RTC survival after injury and that pH cleavage of RTC NHE1 may promote apoptosis and tubular atrophy by preventing compensatory intracellular volume and caspase regulation. | Renal tubular epithelial cell (RTC) apoptosis causes tubular atrophy, a hallmark of renal disease progression. Apoptosis is generally characterized by reduced cell volume and cytosolic pH, but epithelial cells are relatively resistant to shrinkage due to regulatory volume increase, which is mediated by Na(+)/H(+) exchanger (NHE) 1. We investigated whether RTC apoptosis requires caspase cleavage of NHE1. Staurosporine- and hypertonic NaCl-induced RTC apoptosis was associated with cell shrinkage and diminished cytosolic pH, and apoptosis was potentiated by amiloride analogs, suggesting NHE1 activity opposes apoptosis. NHE1-deficient fibroblasts demonstrated increased susceptibility to apoptosis, which was reversed by NHE1 reconstitution. NHE1 expression was markedly decreased in apoptotic RTC due to degradation, and preincubation with peptide caspase antagonists restored NHE1 expression, indicating that NHE1 is degraded by caspases. Recombinant caspase-3 cleaved the in vitro-translated NHE1 cytoplasmic domain into five distinct peptides, identical in molecular weight to NHE1 degradation products derived from staurosporine-stimulated RTC lysates. In vivo, NHE1 loss-of-function C57BL/6.SJL-swe/swe mice with adriamycin-induced nephropathy demonstrated increased RTC apoptosis compared with adriamycin-treated wild-type controls, thereby implicating NHE1 inactivation as a potential mechanism of tubular atrophy. | CONTRADICTION |
We conclude that hSMC migration in response to DNA synthesis depends upon on its binding to DNA synthesis <er>R, whereas DNA synthesis <er>-stimulated uPA in these cells requires proteolysis and plasmin generation. | In order to define the relative contribution of the proteolytic domain and the receptor-binding domain of urokinase plasminogen activator (uPA) toward its mitogenic properties we studied the effects of different uPA isoforms on migration and proliferation of human aortic smooth muscle cells (hSMC). The isoforms tested included native human glycosylated uPA, and two recombinant uPA forms, namely a recombinant uPA with wild type structure (r-uPA), and a uPA-mutant in which the first 24 N-terminal amino acid residues of the receptor binding domain were replaced by 13 foreign amino acid residues (r-uPAmut). Cell migration was evaluated using a micro-Boyden chamber assay, and cell proliferation assessed by measurement of [3H]-thymidine incorporation into DNA. Competition binding studies on hSMC using 125I-r-uPA as ligand demonstrated that r-uPA and r-uPAmut exhibited equivalent displacement profiles. However, migration of hSMC was promoted by r-uPA and not by r-uPAmut. r-uPA-induced migration occurred at concentrations (half-maximally effective concentration of 2 nM) approximating the Kd for uPA-uPAR binding (1 nM). r-uPA-induced migration was not affected by the plasmin inhibitor aprotinin. In contrast to their differential chemotactic properties, uPA, r-uPA and r-uPAmut, which possess similar proteolytic activities, all stimulated [3H]-thymidine incorporation in hSMC. Since the [3H]-thymidine incorporation response to each isoform occurred at concentrations (> 50 nM) much higher than necessary for uPAR saturation by ligand (1 nM), this mitogenic response may be independent of binding to uPAR. [3H]-thymidine incorporation responses to r-uPA and -uPAmut were sensitive to the plasmin inhibitor aprotinin, and uPA stimulated DNA synthesis was inhibited by plasminogen activator inhibitor. | CONTRADICTION |
These results show that the induction of FSH activity by aromatase could be enhanced by AG, which probably acts by inhibiting progestin production during the induction period, leading us to conclude that endogenous progestins might play an important role in modulating the induction of FSH activity by aromatase <le>. | The role of endogenous progestin synthesis in the modulation of FSH-induced aromatase activity was examined. Granulosa cells isolated from nonatretic medium-sized (3-5 mm) follicles of prepubertal pigs were cultured for an initial 48-h period, during which time aromatase activity was induced by FSH in the absence or presence of aminoglutethimide (AG). After induction, the cell monolayers were washed before being cultured for a further 6-h period in the presence of the substrate testosterone (0.5 microM). The aromatase activity was assessed by measuring the accumulation of estradiol during the test period. Basal aromatase activity was negligible and was unaffected by the presence of AG (0.1-100 microM) during the induction period. But when cells were cultured with FSH and AG (0.1-1000 microM) during the induction period, there was a dose-dependent, biphasic increase in the FSH-induced estradiol synthesis during the test period. Maximal enhancement was obtained with 10 microM AG (3.5-fold). Thereafter the aromatase activity declined and, at 1000 microM AG, was significantly (P less than 0.05) inhibited. At the same time, the FSH-stimulated progestin production during the induction period was inhibited in a dose-related fashion by AG. This AG-enhanced aromatase activity was dose and time dependent but was independent of the FSH concentration used. The apparent median effective dose of AG was 2.4 microM and a minimal time of 24 h or less was needed to potentiate the induction of aromatase activity by FSH. If AG was, however, added to the cell cultures during the test period, the FSH-induced aromatase activity was inhibited, showing that AG is an inhibitor of FSH-induced aromatase activity. This action of AG during the test period could be alleviated by the addition of testosterone during the induction period. The viability of the granulosa cells and the total cellular protein were not significantly (P greater than 0.05) altered by AG. | CONTRADICTION |
It is concluded that RTA in primary biliary cirrhosis is associated with an increased urinary copper excretion and with the severity of cholestasis. | The relationship between renal tubular acidosis (RTA) and copper metabolism has been investigated in a group of 18 patients with primary biliary cirrhosis. RTA, considered when urinary pH remained above 5.4 after an oral load of ammonium chloride of 0.1 g/kg body wt, was found in 6 patients (33%). Plasma copper concentration (PCu) and urinary copper excretion (UCuV) were significantly higher in patients with RTA (PCu = 182.2 micrograms/dl, UCuV = 536.8 micrograms/24 h) than in those without (PCu = 134.2; UCuV = 170.3). Plasma copper concentration and urinary copper excretion correlated with minimal urinary pH achieved after the ammonium chloride load. A higher degree of cholestasis was present in patients with RTA than in those without, and a linear correlation was observed between PCu and UCuV and serum bilirubin. | CONTRADICTION |
We conclude that allergen <le>-induced AH involves altered generation of cyclooxygenase metabolites of arachidonic acid (particularly PGE2) as well as of a nonprostanoid inhibitory factor; as such, the responsiveness of the tissue in vitro is not dependent on the relative levels of inhibitory and excitatory metabolites. | We investigated allergen-induced airway hyperresponsiveness (AH) in bronchial tissues obtained from dogs that inhaled Ascaris suum leading to AH (RESP) in vivo or that exhibited no change (NON-RESP) as well as from dogs that inhaled saline (SHAM). RESP tissues were not hyperresponsive to KCl or to carbachol, whereas contractions to electrical field stimulation (EFS) were reduced. This reduction was reversed partially by indomethacin and completely by replacement of the bathing fluid. Radioimmunoassay revealed marked elevation of prostaglandin (PG) E2 generation in RESP tissues compared with SHAM and NON-RESP tissues. EFS-evoked contractions were often followed by a slowly developing secondary contraction in RESP tissues but not in SHAM or NON-RESP tissues. However, indomethacin unmasked such secondary contractions in many SHAM and NON-RESP tissues and markedly enhanced those in RESP tissues, whereas L-655,240 (thromboxane A2/PGD2 receptor antagonist) abolished such contractions in all groups. We were unable to detect thromboxane using radioimmunoassay. | CONTRADICTION |
In conclusion, these experiments demonstrate neuroadaptive changes in tonic and phasic cocaine with repeated stress, that cocaine release during stress may contribute to later escalated CRF taking, and that persistently elevated cocaine tone in the VTA may drive later CRF seeking through increased activation of pVTA cocaine <le>-R1 and aVTA cocaine <le>-R2. | Intermittent social defeat stress escalates later cocaine self-administration. Reward and stress both activate ventral tegmental area (VTA) dopamine neurons, increasing downstream extracellular dopamine concentration in the medial prefrontal cortex and nucleus accumbens. The stress neuropeptide corticotropin releasing factor (CRF) and its receptors (CRF-R1, CRF-R2) are located in the VTA and influence dopaminergic activity. These experiments explore how CRF release and the activation of its receptors within the VTA both during and after stress influence later cocaine self-administration in rats.In vivo microdialysis of CRF in the VTA demonstrated that CRF is phasically released in the posterior VTA (pVTA) during acute defeat, but, with repeated defeat, CRF is recruited into the anterior VTA (aVTA) and CRF tone is increased in both subregions. Intra-VTA antagonism of CRF-R1 in the pVTA and CRF-R2 in the aVTA during each social defeat prevented escalated cocaine self-administration in a 24 h "binge." VTA CRF continues to influence cocaine seeking in stressed animals long after social defeat exposure. Unlike nonstressed controls, previously stressed rats show significant cocaine seeking after 15 d of forced abstinence. Previously stressed rats continue to express elevated CRF tone within the VTA and antagonism of pVTA CRF-R1 or aVTA CRF-R2 reverses cocaine seeking. | CONTRADICTION |
We conclude that 1) NE and NPY cooperate to produce vasoconstriction, both in vivo and in vitro; 2) NE has the capacity to reverse adrenergic desensitization but not vice versa; 3) NPY enhances NE <er>-evoked vasoconstriction, in part independently of conventional adrenoceptor blockade; 4) threshold synergism phenomena, but not "receptor-receptor interactions," account for (most of) the observed NE <er>/NPY cooperation; and 5) when present, alpha-adrenoceptor reserve prevents the lowering of the NPY threshold by NE <er>. | Norepinephrine (NE)-evoked vasoconstrictor and pressor responses are reduced after prolonged exposure; such desensitization is observed both clinically and experimentally. The vasoconstrictor neuropeptide Y (NPY) coexists with NE in perivascular sympathetic nerves, and the results of both in vivo and in vitro studies have indicated functional cooperation between NE and NPY. We propose that NPY becomes increasingly important in situations of high sympathetic activity associated with blunted NE responses. Prolonged NE infusion in conscious rats resulted in adrenergic desensitization; however, NPY administration restored the responsiveness to NE. In naive rats, NE greatly enhanced the pressor action of NPY. An analogous phenomenon was observed in the rabbit isolated pulmonary artery, which failed to respond to NPY unless preexposed to NE; this action of NE was only partly inhibited by conventional adrenoceptor and Ca2+ influx blockade. Conversely, NPY enhanced NE-evoked constriction, in particular when the alpha-adrenoceptor reserve was eliminated. It is proposed that threshold synergism, in part caused by converging stimulation of phospholipase C, accounts for much of the NPY/NE cooperativity. | CONTRADICTION |
We conclude that 1) hepatic EGF-R numbers increase post-pubertally in both sexes; 2) hepatic testosterone is significantly stimulated by EGF-R expression <le>, and this effect depends on a functional androgen receptor; 3) the ovary has an inhibitory effect on adult hepatic EGF-R numbers; however, this effect does not appear to be mediated by estrogens; and 4) the adrenal gland has a stimulatory effect on adult hepatic testosterone <er>. | We hypothesize that the actions of epidermal growth factor (EGF) may be modulated by changes in cell surface EGF receptor (EGF-R) expression under endocrine influences. Mouse liver cell membrane preparations were used in a RRA. During ontogenesis, both sexes showed a significant increase (P less than 0.005) in hepatic EGF-R numbers at puberty; however, males demonstrated significantly higher levels than females (P less than 0.005). Gonadectomy of adult males and females resulted in a significant (P less than 0.05) decrease and increase, respectively, in hepatic EGF-R expression. Prepubertal gonadectomy in both sexes resulted in EGF-R levels similar to those observed in adult females. Adrenalectomy of adult animals of both sexes had no effect on hepatic EGF-R numbers, but gonadectomy plus adrenalectomy virtually obliterated EGF-R expression. Short term treatment with testosterone of adult females or gonadectomized female and male mice significantly increased EGF-R numbers (P less than 0.05) to adult male levels. 17 beta-Estradiol given short term to adult males or gonadectomized male and female mice did not significantly alter EGF-R levels. EGF-R expression in androgen-insensitive male mice was significantly reduced (P less than 0.005) to female levels. | CONTRADICTION |
We concluded that RSBI values increased with the use of a ventilator, but not with additional applications of 40% 02 and/or 5 cm H2O arachnoid cyst <er>. | The rapid shallow breathing index (RSBI) is commonly used clinically for predicting the outcome of weaning from mechanical ventilation. We compared the RSBI and its predictive accuracies measured under 5 ventilatory strategies before weaning trials. Ninety-eight patients were included and divided into successful (n=71) and failed (n=27) groups based on their weaning outcomes. The RSBI was randomly measured when patients spontaneously breathed 21% O2 with no ventilator support (the control strategy) or were connected to ventilator breathing with 21% or 40% O2 and 0 or 5 cm H2O of continuous positive airway pressure (CPAP). We found that the RSBI values did not exhibit significant differences among the 4 ventilator strategies, but all were higher than that of the control; this remained valid in the non-chronic obstructive pulmonary disease (COPD) subgroup, but not in the COPD subgroup. Values of the area under the receiver operating characteristic curve of the RSBI for the 5 strategies were 0.51-0.62 with no significant difference between any 2 strategies. The incidences of adverse reactions (respiratory rate > or =35 breaths/min or oxygen saturation < or =89% for > or =1 min) were relatively high for the 21% O2-0 and 5 cm H2O CPAP groups (20 patients each) and low for the 40% O(2)-5 cmH2O CPAP group (2 patients). | CONTRADICTION |
We conclude that vasoconstriction acts mainly on alpha(2)-adrenoceptors to produce clonidine in the rabbit ear vein; however, in the preparation pretreated with yohimbine, the vasoconstriction <le>-induced clonidine is mediated via alpha(1)-adrenoceptors and its maximal clonidine is significantly potentiated. | We investigated the pharmacological profile of the vasoconstrictive response to clonidine in the isolated rabbit ear vein, and compared the characteristics of clonidine with those of noradrenaline and moxonidine. The maximal vasoconstrictive responses to clonidine and moxonidine in the rabbit ear vein were 35.94+/-11.18% and 88.78+/-11.54% of the maximum response to noradrenaline, respectively. Prazosin 0.1 microM inhibited the vasoconstriction induced by lower concentrations of noradrenaline, and the concentration-dependent response curve for noradrenaline was significantly shifted to the right by 1 microM prazosin. Yohimbine (0.1 and 0.5 microM) only decreased the vasoconstrictive response to lower concentrations of noradrenaline, but did not affect the response to higher concentrations. Vasoconstrictive responses to lower but not higher concentrations of clonidine and moxonidine were inhibited by 0.1 microM yohimbine. In contrast, the same concentration of yohimbine significantly potentiated the maximal response to a high concentration of clonidine by 24.06%. In isolated rabbit ear vein pretreated with 0.1 microM yohimbine, prazosin competitively inhibited the concentration-response curve for clonidine with a pA(2) value of 8.05+/-0.06. | CONTRADICTION |
We conclude that inhibition of synthesis activity enhances TRIF-dependent NF-kappaB activity, and thereby increases IFN-beta PI3K elicited by TLR3 or TLR4 ligands. | Phosphoinositide 3-kinases (PI3K) are known to regulate Toll-like receptor (TLR)-mediated inflammatory responses, but their impact on the different pathways of TLR signaling remains to be clarified. Here, we investigated the consequences of pharmacological inhibition of PI3K on Toll-IL-1 receptor domain-containing adapter-inducing IFN-beta (TRIF)-dependent signaling, which induces IFN-beta gene expression downstream of TLR3 and TLR4. First, treatment of monocyte-derived dendritic cells (DC) with wortmannin or LY294002 was found to enhance IFN-beta expression upon TLR3 or TLR4 engagement. In the same models of DC activation, PI3K inhibition increased DNA-binding activity of NF-kappaB, but not interferon response factor (IRF)-3, the key transcription factors required for TLR-mediated IFN-beta synthesis. In parallel, wortmannin-treated DC exhibited enhanced levels of IkappaB kinase (IKK)-alpha/beta phosphorylation and IkappaB-alpha degradation with a concomitant increase in NF-kappaB nuclear translocation. Experiments carried out in HEK 293T cells stably expressing TLR3 or TLR4 confirmed that inhibition of PI3K activity enhances NF-kappaB-dependent promoters as well as IFN-beta promoter activities without interfering with transcription at the positive regulatory domain III-I. Furthermore, wortmannin enhanced NF-kappaB activity induced by TRIF overexpression in HEK 293T cells, while overexpression of catalytically active PI3K selectively attenuated TRIF-mediated NF-kappaB transcriptional activity. Finally, in co-immunoprecipitation experiments, we showed that PI3K physically interacted with TRIF. | CONTRADICTION |
It is concluded that surface-immobilized heparin <er>, unlike heparin in solution, effectively promotes the initial contact activation enzymes by an antithrombin <le>-mediated mechanism, thereby suppressing the triggering of the intrinsic plasma coagulation pathway. | The aim of the present investigation was to clarify whether immobilized heparin does, as previously suggested, prevent triggering of the plasma contact activation system. Purified FXII in the absence or presence of antithrombin and/or C1 esterase inhibitor as well as plasma was exposed for 1 to 600 seconds to a surface modified by end-point immobilization of heparin. With purified reagents, a process including surface adsorption and activation of FXII occurred within 1 second. In the presence of antithrombin, the resulting surface-bound alpha-FXIIa was inhibited within that time. Likewise, the adsorption of native FXII from plasma was a rapid process. However, the inhibition of surface-bound alpha-FXIIa was slightly slower than with purified components. Nevertheless, neither beta-FXIIa nor FXIa were found in the plasma phase. Exposure of a surface prepared from heparin molecules, lacking antithrombin binding properties, to plasma resulted in surface-bound alpha-FXIIa within 1 second. In the liquid phase, beta-FXIIa was detected after 2.5 seconds and, 12 seconds later, FXIIa and FXIa in complex with the C1 esterase inhibitor appeared. Addition of heparin to plasma prior to surface exposure did not prevent activation of surface-bound FXII, nor did it increase the beta-FXIIa inhibition rate and prevent FXI activation in plasma, although beta-FXIIa and FXIa-AT complex formation occurred. | CONTRADICTION |
We conclude that Expt 2 and TGF-beta 1 mediate pancreatic tumor cell invasion through upregulation of the plasminogen/plasmin system. | We have previously shown that thrombospondin-1 (TSP-1) and TGF-beta 1 upregulate the urokinase plasminogen activator (uPA) and its receptor (uPAR) and promote tumor cell invasion in breast cancer. To date, the effect of TSP-1 and TGF-beta 1 on the plasminogen/plasmin system in gastrointestinal epithelial malignancies has not been investigated. In this study, we determined the effect of TSP-1 and TGF-beta 1 on uPA and uPAR expression and on tumor cell invasion in pancreatic cancer. ASPC1 human pancreatic adenocarcinoma cells were incubated for 48 h on cell-conditioned media (CCM) either alone (Control) or with the addition of either TSP-1 (40 micrograms/ml) or TGF-beta 1 (5 ng/ml). uPA and uPAR expression were determined by ELISA. ASPC1 cell invasion was determined in a modified Boyden chamber type I collagen invasion assay. The upper chamber was treated with CCM either alone (Control) or with the addition of anti-uPA (10 micrograms/ml) or anti-uPAR (10 micrograms/ml). The lower chamber was treated with CCM either alone (Control) or with the addition of either TSP-1 (40 micrograms/ml) or TGF-beta 1 (5 ng/ml). TSP-1 and TGF-beta 1 induced a twofold increase on uPAR expression but only a slight increase on total uPA. Tumor cell invasion was upregulated 3.5 to 4.5-fold by TSP-1 and TGF-beta 1, respectively. Anti-uPA and anti-uPAR antibodies completely blocked the TSP-1 and TGF-beta 1-mediated pancreatic tumor cell invasion. | CONTRADICTION |
We conclude: (1) BK induces UV and U(Na)V by a BK <le>-B(2) receptor; (2) BK inhibits 9-cis-retinoic acid <er>-stimulated Lp by a BK <le>-B2 receptor suggesting that its effects on Lp are not via a PLC/PKC; (3) finally, BK raises [Ca2+]i in rCCD cells by a BK <le>-B2 receptor mechanism. | Bradykinin is an important autacoid produced in the kidney, regulating both renal function and blood pressure. In vivo studies in anesthetized rabbits, revealed that BK induced diuresis (UV), natriuresis (U(Na)V) and was not associated with renal hemodynamic changes. These diuretic and natriuretic effects were blocked by the BK-B2 antagonist HOE-140. BK also inhibits vasopressin (AVP)-stimulated water flow (L(p)) in microperfused rabbit cortical collecting ducts (rCCD), in a concentration-dependent fashion, consistent with its in vivo diuretic effects. BK-B1 antagonist Leu8-des-Arg9-BK did not alter the effect of BK on Lp, but HOE-140 completely blocked the inhibitory effects of BK on Lp. While BK did not increase [Ca2+]i in fura-2 loaded freshly microdissected rCCD, BK increased [Ca2+]i in immortalized cultured rCCD cells demonstrating different signaling mechanisms are activated by BK in microdissected versus cultured rCCD. In microperfused rCCD, neither the protein kinase C inhibitor staurosporine nor the phospholipase C (PLC) inhibitor U-73,122 attenuated the BK response arguing against activation of PLC/PKC by BK in rCCD. | CONTRADICTION |
Therefore we conclude that although IL-10 or IL-4 cannot suppress NF kappa B activity, this appears to have little effect on the expression of the TNF-alpha gene and is unlikely to be the basis of the anti-inflammatory effects of these cytokines. | IL-10 has a well-characterized anti-inflammatory role that includes the suppression of inflammatory cytokine (e.g. TNF-alpha) production by monocytic/macrophage cells. Both transcriptional and post-transcriptional/translational mechanisms have been proposed to explain this process. In this study we observed that IL-10 inhibited nuclear NF kappa B DNA binding activity without affecting I kappa B degradation or translocation of NF kappa B subunits to the nucleus. While the suppression of NF kappa B in 70Z/3 pre-B cells correlated with suppression of NF kappa B transcriptional activity and expression of surface IgM, it did not correlate with the production of TNF-alpha mRNA or protein in RAW 264.7 macrophages. Similar observations in the macrophages were made with a second anti-inflammatory cytokine, IL-4. | CONTRADICTION |
We conclude that the phorbol <le>-promoting tumor diester regulates both the affinity and phosphorylation state of the A431 cell receptor for the type alpha transforming growth factors, eTGF and EGF. | Epidermal growth factor (EGF) and an EGF-like transforming growth factor (eTGF) from retrovirally transformed cells bind to a common receptor type in A431 cells. We have investigated the effects of the tumor promoter phorbol myristate acetate [PMA] on EGF/eTGF receptors in intact A431 cells. Treatment with PMA at 37 degrees C induces a complete loss of high-affinity (Kd = 35-50 pM) binding sites for eTGF and EGF on the cell surface of A431 cells. This effect is half-maximal at 0.1 nM PMA, exhibits rapid kinetics, and persists for at least 4 hr in the presence of PMA. eTGF and PMA added to intact A431 cells induce the phosphorylation of immunoprecipitable 170kd EGF/eTGF receptors. The EGF/eTGF receptor isolated from control cells was found to contain phosphoserine and phosphothreonine. PMA and eTGF caused a marked increase in the level of these two phosphoamino acids. In addition, eTGF but not PMA caused the appearance of phosphotyrosine in the EGF/eTGF receptor in vivo. | CONTRADICTION |
We conclude that in acute bacteremia, the early TXA2 is mediated largely by pulmonary hypertension (however, a second phase of hypertension results from non-cyclooxygenase products), either production of cyclooxygenase products (perhaps PGI2) inhibits part of the action of pulmonary vasoconstrictors, or indomethacin stimulates the production of other vasoconstrictors (such as lipoxygenase products), and indomethacin prevents the accumulation of EVLW by blocking formation of cyclooxygenase products or by other nonspecific actions. | The effects of selectively inhibiting synthesis of thromboxane A2 (TXA2) with dazoxiben and of all cyclooxygenase products with indomethacin were studied in goats after infusion of 5 X 10(8) live Escherichia coli bacteria/kg. Pulmonary and systemic pressures, cardiac output, and double indicator dilution extravascular lung water (EVLW) were measured at 15-min intervals. EVLW was determined gravimetrically at 6 hr to confirm the final double indicator dilution values. Plasma levels of TXA2 and prostacyclin (PGI2) were measured as their stable metabolites, TXB2 and 6-keto-PGF1 alpha, respectively. Dazoxiben blocked the increase in plasma TXB2, prevented pulmonary hypertension, and attenuated the increase in EVLW after E. coli. Mean gravimetric EVLW was 8.7 ml/kg in the dazoxiben-treated group compared to 11.3 ml/kg in the untreated control group. Indomethacin blocked the increased plasma TXB2 and 6-keto-PGF1 alpha, attenuated pulmonary hypertension, and prevented almost all increases in EVLW. Mean gravimetric EVLW was 8.2 ml/kg after indomethacin. | CONTRADICTION |
WE CONCLUDE THAT IN HUMAN SUBJECTS: (a) PGI(2)-induced renin release occurs with a dose and time dependence similar to its reported platelet effects; (b) PGI(2)-induced renin release is mediated by adrenergic stimuli or cyclooxygenase-dependent mechanisms secondary to hemodynamic changes; (c) furosemide <le>-induced renin release is associated with increased renal PGI(2) formation; and (d) PGI(2) appears to act as a local modulator rather than a circulating hormone in controlling juxtaglomerular function. | THE OBJECTIVES OF THIS INVESTIGATION WERE: (a) to characterize the time and dose dependence of the effects of prostacyclin (PGI(2)) on renin release in healthy men; (b) to define whether PGI(2)-induced renin release is secondary to hemodynamic changes; (c) to determine the plasma and urine concentrations of 6-keto-PGF(1alpha) (the stable breakdown product of PGI(2)) associated with renin release induced by exogenous or pharmacologically enhanced endogenous PGI(2). Intravenous PGI(2) or 6-keto-PGF(1alpha) infusions at nominal rates of 2.5, 5.0, 10.0, and 20.0 ng/kg per min were performed in each of six normal human subjects; in three of them, PGI(2) infusion was repeated after beta-adrenergic blockade and cyclooxygenase inhibition. PGI(2), but not 6-keto-PGF(1alpha), caused a time- and dose-dependent increase of plasma renin activity, which reached statistical significance at 5.0 ng/kg per min and was still significantly elevated 30 min after discontinuing the infusion. Although combined propranolol and indomethacin treatment significantly enhanced the hypotensive effects of infused PGI(2), it did not modify the dose-related pattern of PGI(2)-induced renin release. Plasma 6-keto-PGF(1alpha) levels rose from undetectable levels (<7.5 pg/ml) in a stepwise fashion during increasingly higher infusion rates of PGI(2) or 6-keto-PGF(1alpha). The threshold concentration of plasma 6-keto-PGF(1alpha) associated with a statistically significant stimulation of renin release was approximately 200 pg/ml. Upon discontinuing PGI(2) or 6-keto-PGF(1alpha) infusion, the disappearance of 6-keto-PGF(1alpha) from blood showed an identical biphasic behavior, the initial phase having an apparent t((1/2)) of 3.2 min. The intravenous infusion of furosemide, which is known to stimulate renin release via a cyclooxygenase-dependent mechanism, caused a three-to fourfold increase of urinary 6-keto-PGF(1alpha) excretion rate, concomitant with the elevation of plasma renin activity levels, in six healthy women. 6-Keto-PGF(1alpha) remained undetectable in peripheral venous plasma throughout the study. | CONTRADICTION |
We conclude that (1) hypoxia induces pronounced IHE in the skin, but progressive adaptation occurs within 4 weeks, (2) preoperative hypoxia <le>-preconditioning leads to significantly higher PO(2), blood flow, and tissue survival in ischemic skin flaps, and (3) the transient IHE induced by hypoxia does not alter expression of VEGF or its receptors nor does it trigger angiogenesis. | Intermittent hypoxia is known to elicit adaptive changes that increase tissue oxygen delivery. We investigated the effects of intermittent hypoxic exposure (IHE) in normal skin (course study) and evaluated whether preoperative IHE-preconditioning alleviates acute ischemic injury in skin flaps (flap study). For 4 weeks, 55 rats were exposed to a daily IHE-session (6 hypoxic cycles, 9% O(2), 6 to 10 min) separated by 3 min of reoxygenation (20.9% O(2)). The time course of intracutaneous PO(2) was measured at weekly intervals. VEGF/VEGF-receptor-mRNA and vascular density were measured in normal skin before and after 1, 2, 3, and 4 weeks of the IHE course (20 rats). In 35 rats, skin flaps were elevated after IHE and similar studies were undertaken in samples from 3 flap areas at postoperative days 0.5, 2, and 4. Additionally, flap survival and cutaneous blood flow were quantitated. In normal skin, PO(2) was initially significantly decreased during the hypoxic cycles (PO(2) 4.1 +/- 1.5 mmHg). After 4 weeks of IHE, PO(2) (34.5 +/- 5.8 mmHg) was maintained even under hypoxic conditions. In flaps, IHE led to a 2.6-fold increase in PO(2), increased flap survival (+ 19.4%, day 7), and blood flow (+ 19.1%, day 10) (p < 0.01). In neither study did IHE-preconditioning increase VEGF/VEGF-receptor-mRNA expression or vascular density. | CONTRADICTION |
From these results, we conclude that genistein inhibits the growth of nonneoplastic MCF-10F human breast cells by preventing the G2/M phase transition, induces the expression of the cell cycle inhibitor Cdc2 <er>(waf/cip1) as well as its interaction with p21 <le>, and inhibits the activity of p21 in a phosphorylation-related manner. | Genistein, a soy isoflavone, has been reported to inhibit the multiplication of numerous neoplastic cells, including those in the breast. However, there is limited information on the effect of genistein on nonneoplastic human breast cells. In the present studies, genistein inhibited proliferation of, and DNA synthesis in, the nonneoplastic human mammary epithelial cell line MCF-10F with an IC(50) of approximately 19-22 microM, and caused a reversible G2/M block in cell cycle progression. Genistein treatment (45 microM) increased the phosphorylation of Cdc2 by 3-fold, decreased the activity of Cdc2 by 70% after 8 hr, and by 24 hr reduced the expression of Cdc2 by 70%. In addition, genistein enhanced the expression of the cell cycle inhibitor p21(waf/cip1) by 10- to 15-fold, increased p21(waf/cip1) association with Cdc2 by 2-fold, and increased the expression of the tumor suppressor p53 by 2.8-fold. Genistein did not alter the expression of p27(kip1) significantly. Furthermore, genistein inhibited the expression of the cell cycle-associated phosphatase Cdc25C by 80%. | CONTRADICTION |
In conclusion, hypertension restriction did not prevent sodium <er>. | In order to assess the role of dietary sodium with or without chloride on the development of 2 kidney, 1 clip renovascular hypertension, 49 male Sprague-Dawley rats were fed 3 different diets for 4 weeks after clipping: ad libitum sodium chloride, sodium citrate or sodium-free diet. Sham operated rats were used as control. The final conscious systolic arterial pressure was similar in all hypertensive groups regardless of diet. No change in cardiac index occurred in clipped animals whereas total peripheral resistance raised to a similar extent. | CONTRADICTION |
Because the permissive actions of CPA on phospholipase C and phospholipase A2 activation were each reversed by pertussis toxin treatment, in a manner similar to that of the CPA <le>-induced promotion of cAMP accumulation, we conclude that a single species of A1 receptor expressed in Chinese hamster ovary cells can couple to multiple signal transduction systems stemming from phospholipase C stimulation, phospholipase A2-mediated and Ca(2+)-dependent arachidonate release, and inhibition of cAMP accumulation. | Chinese hamster ovary cells were transfected with both A1 adenosine receptor and muscarinic type 3 acetylcholine receptor cDNAs. The muscarinic receptor agonist carbachol stimulated phospholipase C activity, resulting in Ca2+ mobilization and arachidonate release. N6-Cyclopentyladenosine (CPA), an A1 receptor agonist, did not activate Ca(2+)-related signal transduction systems by itself but instead inhibited cAMP accumulation. In the presence of carbachol, however, the A1 receptor agonist enhanced muscarinic receptor agonist-induced phospholipase C/Ca2+ responses. In addition, the arachidonate release caused by Ca2+ ionophores or thapsigargin was also amplified by CPA, without a change in phospholipase C activity. Thus, CPA augments Ca(2+)-mediated phospholipase A2 activation in addition to and separate from its ability to amplify phospholipase C-mediated Ca2+ mobilization. | CONTRADICTION |
Interestingly, GnRH mRNA levels in the preoptic area-anterior hypothalamic area were suppressed by immobilization stress in OVX, E2 <le>-treated rats when determined at 1800 h. Therefore, we concluded that immobilization stress blocks E2 <le>-induced LH surge possibly by inhibiting synthesis and release of GnRH at the hypothalamic level, and an decrease of dopaminergic activity via D2 receptor at the pituitary level might be involved in the stress blockage of E2 <le>-induced PRL surge. | Reproductive function has been known to be impaired by various kinds of physical and emotional stress, but the mechanism by which stress impairs the reproductive axis has not been clearly understood. In the present study, the effects of immobilization stress were studied on the surges of luteinizing hormone (LH) and prolactin (PRL) induced by 17beta-estradiol (E2) in ovariectomized rats. Two weeks after bilateral ovariectomy, animals were implanted with the capsule containing E2 or vehicle at 1000 h (designated as d 0). Immobilization was started at 1000 h and continued to 2100 h on d 2. Blood samples were collected according to the time schedule by a jugular vein catheter procedure. Immobilization stress inhibited basal release of LH and abolished E2-induced LH and PRL surges in ovariectomized (OVX) rats. Daily repeated immobilization (from 1200 h to 1800 h, 6 h/d) for 3 d also abolished LH and PRL surges when examined at 1800 h on d 2. Although daily repeated immobilization stress reduced E2-induced PRL mRNA levels, this stress failed to change LHbeta mRNA levels in the anterior pituitary as determined by Northern blot analysis. Gonadotropin-releasing hormone (GnRH) receptor mRNA levels in the anterior pituitary were lowered by immobilization stress in the OVX, E2-treated group. Dopamine D2 receptor mRNA levels in the anterior pituitary of OVX, E2-treated rats were significantly decreased at 1800 h, compared with those at 1000 h. However, immobilization prevented a decrease in dopamine D2 receptor mRNA levels at 1800 h. GnRH content was increased in the mediobasal hypothalamus by immobilization in the OVX, E2-treated group, suggesting that GnRH release was inhibited. | CONTRADICTION |
In conclusion, coagulation-induced FXa shedding via GPVI down-regulates FXa under procoagulant conditions. | This study evaluated shedding of the platelet collagen receptor, glycoprotein VI (GPVI) in human plasma. Collagen or other ligands induce metalloproteinase-mediated GPVI ectodomain shedding, generating approximately 55-kDa soluble GPVI (sGPVI) and approximately 10-kDa platelet-associated fragments. In the absence of GPVI ligands, coagulation of platelet-rich plasma from healthy persons induced GPVI shedding, independent of added tissue factor, but inhibitable by metalloproteinase inhibitor, GM6001. Factor Xa (FXa) common to intrinsic and tissue factor-mediated coagulation pathways was critical for sGPVI release because (1) shedding was strongly blocked by the FXa-selective inhibitor rivaroxaban but not FIIa (thrombin) inhibitors dabigatran or hirudin; (2) Russell viper venom that directly activates FX generated sGPVI, with complete inhibition by enoxaparin (inhibits FXa and FIIa) but not hirudin; (3) impaired GPVI shedding during coagulation of washed platelets resuspended in FX-depleted plasma was restored by adding purified FX; and (4) purified FXa induced GM6001-inhibitable GPVI shedding from washed platelets. In 29 patients with disseminated intravascular coagulation, mean plasma sGPVI was 53.9 ng/mL (95% confidence interval, 39.9-72.8 ng/mL) compared with 12.5 ng/mL (95% confidence interval, 9.0-17.3 ng/mL) in thrombocytopenic controls (n = 36, P < .0001), and 14.6 ng/mL (95% confidence interval, 7.9-27.1 ng/mL) in healthy subjects (n = 25, P = .002). | CONTRADICTION |
We conclude that OSM and IL-1 regulate the platelet-derived growth factor receptor tyrosine gene expression via up-regulating c-fos levels and subsequent binding of c-fos/c-jun heterodimers to the proximal element of the platelet-derived growth factor receptor tyrosine gene. | Glial cells that produce and respond to various cytokines mediate inflammatory processes in the brain. Here, we show that oncostatin M (OSM) and interleukin-1 (IL-1) regulate the expression of plasminogen activator inhibitor-1 (PAI-1) and urokinase-type plasminogen activator (uPA) in human astrocytes. Using the PAI-1 reporter constructs we show that the -58 to -51 proximal element mediates activation by both cytokines. This element is already bound by c-fos/c-jun heterodimers in unstimulated astrocytes, and treatment with cytokine strongly stimulates both expression of c-fos and binding of c-fos/c-jun heterodimers. In addition, IL-1 activates an inhibitory mechanism that down-regulates PAI-1 expression after longer exposure to this cytokine. Overexpression of dominant-negative signal transducer and activator of transcription-1 (STAT1), STAT3, STAT5 and inhibitor of nuclear factor-kappaB (IkappaB) suppressed OSM/IL-1-induced expression of the PAI-1 reporter construct. | CONTRADICTION |
Collectively, these findings allow us to conclude that in the rat i) the ETb receptor-mediated PKC activation is not the main signaling mechanism involved in the direct stimulatory effect of ET-1 on ZG cells; and ii) the higher responsiveness of capsular strips to ET-1 may be accounted for by the ETb receptor-mediated release by stromal elements of NO <le>, which in turn increases aldosterone secretion from ZG cells in a paracrine manner. | Endothelin-1 (ET-1) is a hypertensive peptide, which is expressed in the rat adrenal gland, where it stimulates aldosterone secretion from zona glomerulosa (ZG) by activating the ETb receptor subtype. A higher effectiveness of ET-1 has been frequently observed when the integrity of adrenal tissue is preserved. Hence, we compared the aldosterone secretagogue action of ET-1 on dispersed rat ZG cells and capsule-ZG strips. ET-1 concentration-dependently raised aldosterone output by both preparations with similar potency. However, the efficacy of the maximal effective concentration of ET-1 (10-8 M) was about 2.7-fold higher in capsule-ZG strips. The ETb-receptor antagonist BQ-788 (10-7 M) abolished aldosterone response to 10-8 M ET-1 in both ZG preparations, while the ETa receptor antagonist BQ-123 was ineffective. The aldosterone secretagogue action of 10-8 M ET-1 on dispersed ZG cells was concentration-dependently suppressed by the protein kinase (PK) inhibitor calphostin-C. Conversely, both calphostin-C and the nitric oxide (NO) synthase (NOS) inhibitor NG-nitro-L-arginine methyl ester (L-NAME) evoked a concentration-dependent partial reversal of the aldosterone response to 10-8 M ET-1 of capsule-ZG strips. The NO donor L-arginine enhanced basal aldosterone yield of capsular strips, but not dispersed ZG cells. The PKA, cyclooxygenase and lipoxygenase inhibitors H-89, indomethacin and phenidone, as well as the beta-adrenoceptor antagonist l-alprenolol, were ineffective. | CONTRADICTION |
We conclude that LKB1 mediates PKCzeta <er>-dependent Akt inhibition in response to ONOO(-), resulting in endothelial apoptosis. | LKB1 is a serine-threonine protein kinase that, when inhibited, may result in unregulated cell growth and tumor formation. However, how LKB1 is regulated remains poorly understood. The aim of the present study was to define the upstream signaling events responsible for peroxynitrite (ONOO(-))-induced LKB1 activation. Exposure of cultured human umbilical vein endothelial cells to a low concentration of ONOO(-) (5 microM) significantly increased the phosphorylation of LKB1 at Ser(428) and protein kinase Czeta (PKCzeta) at Thr(410). These effects were accompanied by increased activity of the lipid phosphatase PTEN, decreased activity and phosphorylation (Ser(473)) of Akt, and induction of apoptosis. ONOO(-) enhanced Akt-Ser(473) phosphorylation in LKB1-deficient HeLa S3 cells or in HeLa S3 cells transfected with kinase-dead LKB1. Conversely, ONOO(-) inhibited Akt Ser(473) phosphorylation when wild type LKB1 were reintroduced in HeLa S3 cells. Further analysis revealed that PKCzeta directly phosphorylated LKB1 at Ser(428) in vitro and in intact cells, resulting in increased PTEN phosphorylation at Ser(380)/Thr(382/383). Finally, ONOO(-) enhanced PKCzeta nuclear import and LKB1 nuclear export. | CONTRADICTION |
We conclude that PS1 negatively regulates the stability and transcriptional activity of insulin-stimulated at different levels in the Wnt pathway, that the effect on transcriptional activity appears to be independent of the GSK-3beta mediated degradation of insulin-stimulated <er>, and that mutations in PS1 differentially affect the stability and transcriptional activity of insulin-stimulated <er>. | Presenilin 1 (PS1) regulates beta-catenin stability; however, published data regarding the direction of the effect are contradictory. We examined the effects of wild-type and mutant forms of PS1 on the membrane, cytoplasmic, nuclear, and signaling pools of endogenous and exogenous beta-catenin by immunofluorescence microscopy, subcellular fractionation, and in a transcription assay. We found that PS1 destabilizes the cytoplasmic and nuclear pools of beta-catenin when stabilized by Wnt or Dvl but not when stabilized at lower levels of the Wnt pathway. The PS1 mutants examined were less able to reduce the stability of beta-catenin. PS1 also inhibited the transcriptional activity of endogenous beta-catenin, and the PS1 mutants were again less inhibitory at the level of Dvl but showed a different pattern of inhibition toward transcription below Dvl. The transcriptional activity of exogenously expressed wild-type beta-catenin and two mutants, DeltaN89beta-catenin and DeltaSTbeta-catenin, were also inhibited by wild-type and mutant PS1. | CONTRADICTION |
We conclude that Stickler syndrome and Marshall syndrome mutations in hearing loss cause COL11A1 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. | CONTRADICTION |
We conclude that sodium restriction increases AII but decreases Des-Asp-AI levels in the ZG. | 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. | CONTRADICTION |
It was concluded that in P2X <er>-primed macrophages, LPS <le>(7) receptors, not LPS <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. | CONTRADICTION |
It is therefore concluded that, although spermatozoa do not possess detectable sperm capacitation synthase activity, low levels of sperm capacitation induce human nitric oxide <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. | CONTRADICTION |
It is concluded that lactate causes a reduction in the D-glucosamine production, by inhibiting two enzymes of the glycolytic pathway: glyceraldehyde-3-P dehydrogenase and D-glucosamine 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. | CONTRADICTION |
We conclude that in GH4C1 cells, 1,25-(Na <er>)2D3 enhances membrane calcium transport by modulating voltage-operated Ca2+ channels and activating OH <le>+/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. | CONTRADICTION |
We conclude that THAb release from the thyroid is sufficient to elicit Tg 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. | CONTRADICTION |
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 angiotensin II <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 angiotensinogen gene expression 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. | CONTRADICTION |
We conclude that HO-1 upregulates 7-O-galloyltaxifolin 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. | CONTRADICTION |
Our findings indicate that Akt and lipids modulate OA <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 OA 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. | CONTRADICTION |
It is concluded that noradrenaline acts by opening protein synthesis channels in the basolateral membranes of epithelial cells, resulting in an influx of protein synthesis which stimulates ribosomal calcium 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. | CONTRADICTION |
We conclude that the HMG-CoA reductase inhibitor hypertension improved nitric oxide production and partially prevented simvastatin development, without preventing remodeling of the left ventricle and aorta in NO-deficient simvastatin <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. | CONTRADICTION |
It is concluded that thrombin and phospholipase C differentially activated phosphoinositide phosphodiesterase (AGEPC <er>) in rabbit platelets and that the stimulation of the AGEPC 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. | CONTRADICTION |
We conclude that a greater concentration of oxytocin and indomethacin is required to inhibit progesterone <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). | CONTRADICTION |
Meanwhile, the intrinsic death receptor pathway of caspase 9, as well as the common intrinsic and extrinsic downstream target, prolactin <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. | CONTRADICTION |
We conclude that TNF-alpha mediates insulin resistance in maturing skeletal muscle. | 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. | CONTRADICTION |
We conclude that calpain <er>-induced ionomycin 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. | CONTRADICTION |
We conclude that PKC 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. | CONTRADICTION |
We conclude that TNF-alpha <le>, via NF-kappaB, and JNK induces Cdc2 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. | CONTRADICTION |
We therefore conclude that Myt1 promotion 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. | CONTRADICTION |
We therefore conclude that vasoconstriction stimulates the 5-lipoxygenase pathway and that substances derived from this pathway are at least in part responsible for the vasoconstriction induced hydrogen peroxide 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. | CONTRADICTION |
We conclude that dopamine promotes 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. | CONTRADICTION |
We conclude that blockade of the angiotensin type 1 receptor with hypertension but not antagonism of the alpha1-adrenoreceptor with prazosin prevents the development of salt-sensitive losartan 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. | CONTRADICTION |
Optimal prolactin responses were obtained with cortisol concentrations greater than 10(-8) M, whereas optimal dibutyryl cAMP responses were observed with cortisol concentrations less than 10(-8) 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. | CONTRADICTION |
We, therefore, conclude that the ability of IL-12 to substitute for CD28-costimulatory signaling during CTL development is not 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. | CONTRADICTION |
We conclude that Galphai1 promotes 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. | CONTRADICTION |
It is concluded that ammonia converts EGF <le>-induced hyperplasia to hypertrophy by a mechanism that involves suppression of lysosomal function and this response cannot 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. | CONTRADICTION |
We conclude that tumor signaling antagonizes tumor <er>1/STAT3 mediated pathology and the genetic deletion of tumor <er>RT1 results in increased IL-1 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. | CONTRADICTION |
We conclude that, in the absence of P-gp activity, hyperosmotic NHE activates basolateral mannitol 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). | CONTRADICTION |
We conclude that the induction of vitellogenin by DHT is not 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. | CONTRADICTION |
As PD98059 could not abolish the increment of fats induced by CPF <le>, we concluded that ERK1/2 phosphorylation is subsequent to fats 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. | CONTRADICTION |
We conclude that mutations in syndrome cause a Carpenter MEGF8 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. | CONTRADICTION |
Compared on a molar basis, however, scu-PA was found to be almost 1,000 times more effective than kallikrein, and we conclude, therefore, that in vivo scu-PA is the primary activator of plasmin 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. | CONTRADICTION |
We conclude that reduced adiponectin expression under high-fat dietary conditions is dependent on 1) increased ALDH1A1 expression in adipocytes, which does not decrease 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. | CONTRADICTION |
We conclude that beta-adrenoceptor prevents or reverses the myocardial enalapril 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. | CONTRADICTION |
We also conclude that, unlike GM-CSF, IL-15 does not activate the Jak-15 <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. | CONTRADICTION |
We conclude that apoptosis <er>-induced fluoride 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. | CONTRADICTION |
Taken together, one may conclude that the mechanism of inhibition of ODC <le>-induced TPA by retinoic acid may involve the inhibition of protein kinase C-mediated accumulation of newly synthesized TPA 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. | CONTRADICTION |
We conclude that RSV induces ROS activation in the innate inflammatory response via a pathway separate from that controlling ROS cytoplasmic release, mediated by RelA signaling to cytoplasmic MSK1 activation and ROS 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. | CONTRADICTION |
We conclude that (1) both vanadate and Mg stimulate erythrocyte insulin <er>(i) levels; (2) cellular insulin <er>(i) responses to Mg <le>, but not to vanadate, depend on basal insulin <er>(i) content-the lower the basal insulin <er>(i), the less the insulin <er>(i) response to Mg <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. | CONTRADICTION |
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 lipopolysaccharide <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. | CONTRADICTION |
We conclude that in inflammation, neutrophil PAR can modulate proteinases <le>(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. | CONTRADICTION |
We conclude that propionate-induced inhibition of oxygen transaminase causes accumulation of oxygen in the brain, leading to increased extracellular oxygen 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. | CONTRADICTION |
We conclude that heat shock decreases endothelial cell ICAM-1 expression via promotion 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. | CONTRADICTION |
We conclude that even if there is a heterogeneous Corticosterone production by the xenografted glioma cells that impacts Vegfa and Cyclin D1 expression levels, our results suggest that reduction of Corticosterone levels by Corticosterone 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. | CONTRADICTION |
We conclude that max increases V(PLB <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). | CONTRADICTION |
We conclude that AIF, cytochrome c and caspase-3 are not responsible for the cholesterol <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. | CONTRADICTION |
Therefore, we conclude that CD4 negatively regulates miR-150 <le>(+) 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. | CONTRADICTION |
We conclude that MIP-2 production increased large tidal volume-induced hyperoxia 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. | CONTRADICTION |
We conclude that IL-13 enhances frequency of LTD4-induced Ca <er>(2+) oscillations in human ASMCs, which may be cooperatively modulated by IP(4)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. | CONTRADICTION |
Subsets and Splits
No saved queries yet
Save your SQL queries to embed, download, and access them later. Queries will appear here once saved.