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Physiologic monitoring goals for the critically ill patient.

Definition of the appropriate therapeutic goals for physiologic monitoring of patients postoperatively was approached by analyzing more than 50,000 values of the 20 most commonly monitored variables in a series of 113 critically ill patients throughout their immediate postoperative course. In general, normal values were poor criteria for monitoring, since normal values were restored in an average of 75 per cent of the survivors and 76 per cent of the nonsurvivors for the five most frequently measured variables; that is, arterial pressure, heart rate, central venous pressure, wedge pressure and cardiac output. Moreover, an average of 56 per cent of the 20 most commonly monitored variables of nonsurvivors was restored to the normal range. Furthermore, 34 per cent of all the nonsurvivors' values were within the normal range; this was only 2.4 per cent less than the percentage of normal values for the survivors. The empirically determined median value of the survivors taken in the late stage during periods remote from therapy was found to be a better criterion for therapeutic goals for most variables, including blood flow, oxygen transport and most intravascular pressures. However, normal values were satisfactory for arterial pressure, peripheral resistance, pH, mixed venous oxygen tension and arterial carbon dioxide tension, largely because of the biphasic patterns of these variables.

3beta-Hydroxysteroid dehydrogenase activity in human fetal membranes.

A 3beta-hydroxysteroid dehydrogenase (3betaHSD) was demonstrated in term human fetal membranes (chorion and amnion) with both dehydroepiandrosterone (3beta-hydroxy-5-androsten-17-one) and pregnenolone (3beta-hydroxy-5-pregnen-20-one as substrates, and the subcellular distribution substrate and nucleotide specificity of the enzyme was studied. In both membranes the microsomal fraction (particles which sedimented at 105,000 g after 90 min) had the highest specific activity. The chorion was more active than the amnion but the enzyme in both tissues had similar substrate and nucleotide specificity. NAD was the preferred cofactor, and pregnenolone was a better substrate than dehydroepiandrosterone in the presence of NAD. However, with NADP as cofactor both steroids were equally good substrates. When the 3beta-hydroxysteroid dehydrogenase activity of chorion microsomes was compared with that of placental microsomes, the specific activities were found to be of the same order of magnitude, and the substrate, nucleotide specificity and steroid binding properties were almost identical.

beta-D-galactosidase activities in juvenile GM1-gangliosidosis.

beta-Galactosidase activity was investigated in one case of juvenile GM1-gangliosidosis. This patient exhibited normal activity of the neutral form of beta-galactosidase (measured as beta-glucosidase activity) and normal pH curve of residual acid beta-galactosidase activity in leucocytes and fibroblasts. A shift towards more neutral pH optimum was seen in the beta-galactosidase enzyme occurring in serum. The communication also presents a study of the relationship of the different beta-galactosidases in human liver using isolated urine oligosaccharide from this patient as a beta-galactoside substrate. The other natural beta-galactoside substrates used in this investigation were different oligosaccharides, one glycopeptide and ceramide-beta-galactosidase. The beta-galactosidase forms with acidic pH optimum towards synthetic substrate (A forms) exhibit activity towards the natural substrate (except ceramide-beta-galactoside). The "neutral" beta-galactosidase with broad substrate specificity (B form) which includes beta-glucosides had no activity towards the natural substrates used. It could also be shown that the activity towards ceramide-beta-galactoside was a third type of beta-galactosidase different from A and B forms.

Treatment of acute otitis media of infancy with cefaclor.

The emergence of ampicillin-resistant Haemophilus as a clinical problem in otitis media necessitates a search for alternative, effective therapy. An orally absorbable cephalosporin derivative, cefaclor, is equally effective in vitro against ampicillin-susceptible and -resistant Haemophilus and against other bacteria that cause acute otitis media. Two dosage schedules of cefaclor (40 and 60 mg/kg/day) were evaluated in 95 infants with acute otitis media. Bacterial origin was determined by a culture of tympanocentesis fluid. Success rates using the smaller dosage were inferior to those using the larger dosage. Results of therapy for pneumococcal and Haemophilus infection with 60 mg/kg/day were comparable to those previously found with amoxicillin trihydrate or with combinations of trisulfapyrimadines with erythromycin or penicillin V. One patient with an ampicillin-resistant Haemophilus infection responded well to cefaclor and did not have a relapse. Cefaclor was well tolerated and caused an acceptably low incidence of minor, adverse effects. Cefaclor deserves further testing as a candidate for preferred status as a single-drug treatment of acute otitis media.

[Non-enzymatic fibrinolytic agents].

Non-enzyme fibrinolytic agents include pharmacological agents which are active in vivo but inactive in vitro and synthetic chemical compounds which when added to blood or plasma in vitro directly induce fibrinolysis. There are a number of drugs with a short duration of action such as adrenalin, nicotinic acid, vasopressin and histamine. Vasoactive drugs probably act by stimulating the liberation of vascular activator. The effect of nicotinic acid is rapidly exhausted when injections are repeated. By contrast, the biguanides and certain anabolic steroids are capable of exerting a long term stimulation of endogenous fibrinolysis. Amongst these substances, phenformin, metformin, ethyloestrenol, stanozolol and a new substance, moroxydine chloride, have been studied. The biguanides appear to be capable of exerting an effect upon the synthesis and liberation of plasminogen vascular activator. The combination of an anabolic steroid and a biguanide would appear to be the most powerful. These various drugs have been used with success in cases of recurrent venous thrombosis in patients with an abnormally low level of plasminogen activator in the venous walls and/or low fibrinolytic activity after venous stasis. Chemical fibrinolytic agents were studied only in vitro, since the use of these substances in human therapeutics would seem to be still difficult in view of the fact that they are active only in a narrow range of concentrations.

The spectrum of vasculitis: clinical, pathologic, immunologic and therapeutic considerations.

Vasculitis is a clinicopathologic process characterized by inflammation and necrosis of blood vessels. Certain disorders have vasculitis as the predominant and most obvious manifestation, whereas others have various degrees of vasculitis in association with other primary disorders. Within the entire spectrum of vasculitis virtually any size or type of blood vessel in any organ system can be involved. Most of the vasculitides can be associated directly or indirectly with immunopathogenic mechanisms. In this regard, immune complex mediation is being increasingly recognized as the underlying mechanism in several of the vasculitides. With clinical, pathologic, and immunologic criteria, certain vasculitic disorders can be clearly recognized and categorized as distinct entities, whereas in others there is an overlap of different diseases within a broader category. In recent years, several of the more serious vasculitides, such as Wegener's granulomatosis and the systemic necrotizing vasculitides of the polyarteritis nodosa group, which formerly had extremely poor prognoses, have been shown to be extraordinarily responsive to chronic low-dose cytotoxic therapy, particularly cyclophosphamide.

Nitrogen assimilation in Rhodopseudomonas acidophila.

Rhodopseudomonas acidophila strain 7050 assimilated ammonia via a constitutive glutamine synthetase/glutamate synthase enzyme system. Glutamine synthetase had a Km for NH+4 of 0.38 mM whilst the nicotinamide adenine dinucleotide linked glutamate synthase had a Km for glutamine of 0.55 mM. R. acidophila utilized only a limited range of amino acids as sole nitrogen sources: L-alanine, glutamine and asparagine. The bacterium did not grow on glutamate as sole nitrogen source and lacked glutamate dehydrogenase. When R. acidophila was grown on L-alanine as the sole nitrogen source in the absence of N2 low levels of a nicotinamide adenine dinucleotide linked L-alanine dehydrogenase were produced. It is concluded, therefore, that this reaction was not a significant route of ammonia assimilation in this bacterium except when glutamine synthetase was inhibited by methionine sulphoximine. In L-alanine grown cells the presence of an active alanine-glyoxylate aminotransferase and, on occasions, low levels of an alanine-oxaloacetate aminotransferase were detected. Alanine-2-oxo-glutarate aminotransferase could not be demonstrated in this bacterium.

[The influence of umbilical cord structure on the course of pregnancy and parturition (author's transl)].

A series of 549 not selected placentas and umbilical cords were examined to show the influence of the different development of the umbilical cord structure on the umbilical cord perfusion and the course of parturition. For this purpose the quantity of Wharton's jelly, the spiraling of the umbilical vessels as well as the length of the cord are defined. At the same time anatomical pecularities and complications like umbilical cord torsions and true knots are considered. To judge the decisive condition of the newborn and with that the course of parturition, the pH-value of umbilical artery blood is determined. The analysis results that umbilical cords with much Wharton's jelly and with spiraling vessels are converted into an association with extremely better value than those with little jelly and less spiraling vessels. Short and rich jellied umbilical cords show a significantly lower incidence of loops around the fetal body. A similar trend can be demonstrated for umbilical cords with strong spiraling arteries. Rare phenomena like velamentous insertion, haematomas, edemas and true knots of the cords etc. proved to have no influence on the course of parturition in our relatively small examination series.

Absorption of magnesium by the young steer.

1. Steers with rumen and simple duodenal cannulas were allowed to graze pasture or were given diets of dried grass or flaked maize with or without hay. For an experiment a solution or suspension of magnesium chloride, polyethylene glycol (molecular weight 4000) and either 144Ce (as cerous chloride) or chromic oxide was added to the rumen with a morning feed. Conditions in the rumen were sometimes modified by adding sodium chloride or hydrochloric acid. 2. Changes in magnesium:marker in samples of strained rumen contents with time interval after adding the dose were due partly to changes in Mg distribution between different phases. Results indicated, but not unequivocably, that negligible amounts of Mg were absorbed in the first few hours. 3. Relative recoveries of Mg and markers at the duodenum indicated that proportions of Mg intake absorbed (net) varied from approximately zero for pasture to 0.2--0.5 for flaked maize. Significant correlations between absorption efficiency and sodium:potassium in rumen contents (positive) and rumen pH (negative) were observed. 4. Steers with simple duodenal and re-entrant ileal cannulas were given a diet of flaked maize and hay supplemented with different amounts of magnesium oxide. Little net change in Mg relative to an unabsorbed marker was found between these sites even for a diet containing an Mg supplement of 8 g/kg dry matter.

Dimeric nature and amino acid compositions of homogeneous canine prostatic human liver and rat liver acid phosphatase isoenzymes. Specificity and pH-dependence of the canine enzyme.

Two isoenzymes of rat liver acid phosphatase (orthophosphoric-monoester phosphohydrolase (acid optimum) EC 3.1.3.2) have been purified to homogeneity, at least one of these for the first time. Both of the rat liver isoenzymes have identical specific activities towards p-nitrophenyl phosphate. Molecular weights of the native enzymes are 92 000 for rat liver isoenzyme I and 93 000 for isoenzyme II, while the subunit molecular weights are 51 000 and 52 000 respectively. Data on substrate specificity and pH dependence are presented for the homogeneous canine prostatic enzyme, which is also isolated as a dimeric enzyme of (native) molecular weight 89 000. Carbohydrate analysis data are presented for canine prostatic acid phosphatase and it is further noted that both isoenzymes of rat liver acid phosphatase are also glycoproteins. The amino acid compositions of the two rat liver isoenzymes are presented together with those of the similar dimeric acid phosphatase of human liver and of canine prostate. Comparison of these results with published data for the amino acid composition of human prostatic acid phosphatase shows substantial similarities. However, significant differences are seen in the amino acid composition of rat liver acid phosphatase isoenzyme I as compared to a previous literature report. Most notably, 17 histidine residues are found per mol of isoenzyme I and 18 for isoenzyme II.

The pH-dependence of the non-specific esterase activity of carboxypeptidase A.

The hydrolysis of the following 6 esters by bovine pancreatic carboxypeptidase A (peptidyl-L-amino-acid hydrolase, EC 3.4.12.2) has been investigated over the range pH 5--10 at 25 degrees C, ionic strength 0.2: CH3CO2CHRCO2H (R = C6H5 (ester 3), C6H5CH2 (ester 4), 4-NO2C6H4CO2CHRCO2H (R = C6H5 (ester 5), C6H5CH2 (ester 6), CH3(CH2)2 (ester 7)), CH3CH2CO2CH(CH2C6H5)-CO2H (ester 9). For each ester the pH dependence of kcat/Km indicates that substrate binding is controlled by an acid of pKEH = 9.2 +/- 0.2 in the free enzyme, and although kcat/Km decreases in acidic solutions no simple dependence on an enzymic ionization is apparent. For esters 3, 5 and 7 the dependence of kcat on pH is 'bell-shaped' and is controlled by pKEH2S = 6.73, 6.72, 6.23, respectively and pKEHS = 9.3 +/- 0.2 for each ester. For esters 4 and 6 the 'bell-shaped' kcat (pKEH2S = 7.38, 6.28, respectively) is modified by a significant increase in kcat in the vicinity of pH 10. This latter phenomenon is also shown by ester 9, although data are only accessible over the range pH 7--10 for this latter ester due to pronounced product inhibition in acidic solutions. The common pH-dependences observed for the enzymic hydrolyses of these nonspecific ester substrates are compared with literature data for specific ester and peptide substrates, and possible assignments for the various enzymic pKa values are discussed.

Blockade by N-methylhydroxylamine of activation of guanylate cyclase and elevations of guanosine 3',5'-monophosphate levels in nervous tissues.

Hydroxylamine and N-methylhydroxylamine prevented the activation of soluble guanylate cyclase by the endogenous activator as well as by nitroso compounds such as N-methyl-N'-nitro-N-nitrosoguanidine or nitroprusside, while other derivaties of hydroxylamine were ineffective. Hydroxylamine and N-methylhydroxylamine did not alter the basal guanylate cyclase activity of purified enzyme preparations. Kinetics analysis indicated that N-methylhydroxylamine competes with N-methyl-N'-nitro-N-nitrosoguanidine for guanylate cyclase. The activation of guanylate cyclase by N-methyl-N'-nitro-N-nitrosoguanidine and its inhibition by N-methylhydroxylamine were reversible reactions. These effects of N-methyl-N'-nitro-N-nitrosoguanidine and N-methylhydroxylamine were observed with guanylate cyclase from other tissues. N-Methylhydroxylamine prevented the increase of guanosine 3',5'-monophosphate (cyclic GMP) levels in cerebellar slices of guinea pig by N-methyl-N'-nitro-N-nitrosoguanidine, veratridine and adenosine, while the elevations of adenosine 3',5'-monophosphate by these agents were not effected. N-Methylhydroxylamine also blocked the increases of cyclic GMP levels by carbachol, prostaglandin E1 and N-methyl-N'-nitro-N-nitrosoguanidine in neuroblastoma N1E 115 cells. Thus N-methylhydroxylamine prevents the activation of guanylate cyclase and the increased synthesis of cyclic GMP in response to transmitters without blocking the synthesis of cyclic GMP via basal enzyme activity.

Treatment of aplastic anaemia by antilymphocyte globulin with or without marrow infusion.

Forty-one patients, suffering from severe aplastic anaemia were treated either with ALG alone (27 patients) or ALG followed by infusion of allogeneic bone marrow (14 patients). Eighteen patients (67 per cent) are presently alive after ALG alone at over 100 to over 550 days. Fourteen (52 per cent) showed sustained improvement of haematopoiesis, two are alive without change, one recovered autologous haematopoiesis after cyclophosphamide conditioning and transfusion of HLA identical marrow and one is lost to follow-up. Eight patients (57 per cent) are currently alive after ALG and transfusion of haplotype identical marrow with self-sustaining autologous haematopoiesis at over 200 days to over four and a half years. No lethal complications occurred and none of the bone marrow infusions led to permanent engraftment or graft-versus-host disease. The mechanism of action is not known, but our results support the hypothesis that unspecified autoimmune reactions block the normal outgrowth of haematopoietic precursor cells in a substantial number of patients with aplastic anaemia. This therapeutic approach seems to offer good chances of survival, especially for those patients who do not have an HLA identical sibling. Its value should be further investigated.

Sphingolipid composition and catabolism in human fetal tissues.

Human fetal tissues derived from prostaglandin-induced abortuses (9--18 wk fertilization age) have been utilized to evaluate sphingolipid composition and catabolism. Sphingolipid composition (lipid-hexose, sulfatide, and lipid-bound NANA) was assessed in fetal brain. Sphingolipid catabolism was evaluated in fetal lung and brain through the measurement of relevant acid hydrolases (arylsulfatase A, beta-galactosidase, and hexosaminidase). During the fetal period studied, the parameters of sphingolipid composition revealed variability but no consistent pattern of change. Each acid hydrolase was readily detected. Enzyme specific activities revealed no variation during the 9 fetal wk studied. Cellulose acetate electrophoresis yielded the anticipated isoenzyme patterns for each acid hydrolase with little variation during the period of study. The compositional values support current concepts of cerebral development during this period of fetal life. Together with the catabolic analyses, these studies provide normative data relative to the assessment of metabolic abnormalities during this period of fetal development.

Enzyme reactions of ATP studied by positional isotope exchange.

Reversible gamma-PO3 transfer in ATP reactions can be recognized by exchange of 18O from the beta,gamma-bridge position to the beta-P-nonbridge positions: (see article). Such intramolecular exchange is less demanding for the detection of the bond cleavage than the usual ATP:ADP isotope exchange because it does not require dissociation of bound ADP from the intermediate complex. Acyl phosphate intermediates are indicated for the glutamine synthetase and carbamyl-P synthetase reactions by their extreme requirements for glutamate and bicarbonate, respectively, for positional oxygen exchange. No support is given for E-P or concerted mechanisms. No support is found for an active CO2 in the latter reaction, although this is not ruled out by the data. Positional isomerization in ATP occurs with lamellae from spinach chloroplast only in the light. When the ATP molecule interacts, it also undergoes complete exchange of the gamma-PO3 oxygen with water before it rejoins the pool of free ATP. The difference in rates of the two exchanges suggests that the torsional motion of ADP-beta-PO3 is greatly hindered on the enzyme. This may explain, by the argument of substrate activation, the rapid reversibility of the ATPase reaction on the enzyme.

Isolation and some properties of two deoxyribonucleases from a snail, Achatina fulica.

1. Two main DNases were found in the dried liver extract of a snail, Achatina fulica. They were purified by the phosphocellulose batch method and by phosphocellulose column chromatography. The enzyme eluted earlier from the phosphocellulose column was designated as Achatina DNase-1 and the other as Achatina DNase-2. DNase-1 was purified further by QAE-Sephadex A-25 column chromatography (twice) just before use because of the instability of the purified enzyme. By these procedures, DNase-1 and 2 were purified 200- and 130-fold, respectively. 2. Divalent or monovalent cations had no marked effect on either enzyme. The showed pH optima of 4.8 (DNase-1) and 5.2 (DNase-2). Ionic strength was found to be critical for the maximal activity. The isoelectric points of DNase-1 and 2 were both 6.9. On heating at 70--75 degrees C for 5 min, each enzymic activity fell to half of the initial value. 3. The enzyme preparations degraded native DNA 1.5--2.5 times faster than heat-denatured DNA. They both degraded heat-denatured DNA endonucleolytically, to give oligonucleotides with 3'-phosphates. 4. The 3'-phosphoryl and 5'-hydroxy termini of the resulting oligonucleotides were analyzed. DNase-1 possessed marked specificity for dThd at 3'-termini and dAdo at 5'-termini in the early stages of degradation, but only for dAdo at 5'-termini in the later stages. DNase-2 showed some preference for purine nucleotides at both 3'- and 5'-termini in the later stages of degradation.

Purification of soluble guanylate cyclase from rat lung.

The soluble form of guanylate cyclase from rat lung has been purified approximately 23,000-fold to homogeneity by isoelectric precipitation, GTP-Sepharose chromatography, and preparative gel electrophoresis. A single protein-staining band is observed after analytical gel electrophoresis on either 4 or 7.5% polyacrylamide gels. The final purified enzyme has a specific activity of about 700 nmol of cyclic GMP formed/min/mg of protein at 37 degrees C in the presence of 4.8 mM MnCl2 and 100 micrometer GTP. Bovine serum albumin appears to slightly increase guanylate cyclase activity, but mainly stabilizes the purified enzyme; in its presence, specific activities in excess of 1 mumol of cyclic GMP formed/min/mg of enzyme protein can be obtained. When Mg2+ or Ca2+ are substituted for Mn2+, specific activities decrease to approximately 21 and 40 nmol of cyclic GMP formed/min/mg of protein, respectively. The apparent Michaelis constant for MnGTP in the presence of 4.8 mM MnCl2 is 10.2 micrometer. Kinetic patterns on double reciprocal plots as a function of free Mn2+ are concave downward. The native enzyme has a molecular weight of approximately 151,000 as determined on Sephacryl S-200; sodium dodecyl sulfate-polyacrylamide gel electrophoresis results in two protein-staining bands with approximate molecular weights of 79,400 and 74,000. Thus, it appears that the soluble form of guanylate cyclase from rat lung exists as a dimer.

Pyridoxal phosphatase: cytochemical localization in GERL and other organelles of rat neurons.

A phosphatase, hydrolyzing pyridoxal-5-phosphate (P5P), a physiologically active component of the vitamin B6 complex and an essential co-enzyme in the synthesis of neurotransmitters, has been localized cytochemically in the perikarya of neurons in the peripheral, autonomic and central nervous systems of the rat. Neurons in dorsal root ganglia, sympathetic ganglia and ventral horn of spinal cord were studied by light and electron microscopy, while Purkinje cells, neurons in the dentate nucleus of the cerebellum, thalamus, and hypothalamus were studied by light microscopy only. Optimal conditions for demonstrating this activity in aldehyde-fixed tissue were determined with dorsal root ganglia. At the optimal pH of 5.0, neurons in these ganglia and in all other neurons studied show pyridoxal-5-phosphatase (P5Pase) activity in GERL. Small neurons in dorsal root ganglia also display enzyme activity in the endoplasmic reticulum (ER); activities in GERL and ER are also appreciably high at neutral pH. Small and large neurons in these ganglia, and neurons of sympathetic ganglia, show variable P5Pase activity in the Golgi apparatus. These localizations differ from the usual sites of both acid phosphatase and alkaline phosphatase activities. The P5Pase activity, demonstrated cytochemically, is a new acid hydrolase activity in GERL.

The mouse gut T lymphocyte, a novel type of T cell. Nature, origin, and traffic in mice in normal and graft-versus-host conditions.

Lymphocytes of the mouse intestinal mucosa, identified in tissue sections or purified suspensions of intraepithelial lymphocytes as T cells (gut T lymphocytes [GTL]), were studied in normal mice or in beige mice (the equivalent of the Chediak-Higashi syndrome in man, characterized by giant granules in various cell types, including mast cells). Mice were studied in normal or in germ-free conditions, or during a graft versus host (GVH) reaction resulting from the injection of parental thymocytes into lethally irradiated F1 mice, a condition leading to massive accumulation of T lymphocytes of donor origin in the host gut mucosa. In normal as well as in GVH conditions, a high percentage of the gut IE lymphocytes contain granules (up to 80% in the beige mouse). These granules have ultrastructural, hostochemical and other features resembling those of mast cell granules; in beige mice, up to 50% of them can be shown to contain histamine. Granulated T cells are also found in the lamina propria. It appears that the GTL may progressively lose their surface T antigens when the granules become more developed. Kinetics of [3H]TdR labeling of the GTL, transfer experiments with T cells of various origins, selective [3H]TdR labeling and selective irradiation of the Peyer's patches (PP), and effect of thoraic duct (TD) drainage led to the conclusion that GTL are the progeny of T cells stimulated to divide in the PP microenvironment, which endows them with a gut-homing tendency. From the PP, these cells follow a cycle, migrating to the TD and to the blood to colonize the whole intestinal mucosa, the majority of them as dividing cells undergoing a single round of traffic, with some probably able to recirculate and becoming a more long-lived variety. Antigenic stimulation within the PP is necessary for the emergence of GTL progenitors, but their gut-homing property is unrelated to the antigen as shown with fetal gut grafts, notably in GVH where grafts syngeneic to the host or donor become similarly infiltrated by GTL. On the basis of their properties and of further evidence to be reported elsewhere, it is proposed that GTL belong to a special class of T lymphocytes, related to the immune defenses of the mucosal systems in general, and capable of acting as progenitors of mucosal mast cells.

Autoxidation of polysorbates.

Aqueous solutions of polysorbate 20 undergo autoxidation on storage, with the peroxide number increasing and subsequently decreasing again, the acidity increasing continuously, the pH and surface tension falling and tending to level off, and the cloud point dropping sharply until turbidity begins at room temperature. The changes are accelerated by light, elevation of temperature, and a copper sulfate catalyst. At the same time, hydrolysis occurs, liberating lauric acid. Analysis of the alterations in these properties leads to the conclusion that hydrolysis has the major influence near room temperature and that oxyethylene undergoes chain shortening at temperatures above 40 degrees. However, evidence of degradation is detectable even in previously unopened commercial samples of polysorbates 20, 40, and 60, warranting attention to the stability of and standards for these surfactants as compared with the solid alkyl ether type of nonionic surfactant.

Evidence for anionic cation transport of lithium, sodium and potassium across the human erythrocyte membrane induced by divalent anions.

1. The passive net transport of Li+ and Na+ across the human red cell membrane was accelerated by the divalent anions carbonate, sulphite, oxalate, phosphite and malonate. Phthalate, maleate, sulphate and succinate were found additionally to stimulate downhill transport of K+. Marked differences in anion efficacy and selectivity were observed. 2. The effects of these 'carbonate type' anions were reversible and fully blocked by SITS, dipyridamole and other inhibitors of anion transfer. 3. Cation transport acceleration induced by the monovalent anions salicylate, benzoate, thiocyanate and 2,4-dinitrophenol were inhibited by dipyridamole, but not affected by SITS. A great number of mono- and polyvalent anions were without detectable influence on Li+ transport. 4. Li+ net uptake induced by oxalate exhibited a pH dependence similar to that reported for halide self exchange. 5. Transport acceleration by carbonate type anions displayed a linear, 1:1 dependence on the concentrations of both the anion and the cation and was symmetric with respect to the two sides of the membrane. 6. It is concluded that the divalent carbonate type anions form singly charged, negative 1:1 ion pairs with the respective alkali metal cations, the ion pairs traversing the red cell membrane via the anion exchange pathway. This concept of anionic formation of some of the ion pairs considered. The relative efficacies and cation selectivities of polyvalent anions can largely be explained on the basis of electrostatic interactions governing ion pair formation. However, the chelating properties, structural flexibility, polarizability of the anions and the accessibility of the ion pairs to the anion exchange pathway need also be considered. 7. An exchange of NaCO-3 ion pairs for internal HCO-3 or Cl- is discussed as a possible mode of cellular pH regulation.

Beta-adrenergic receptors: regulatory role of agonists.

Direct radioligand binding studies have been used to probe the molecular mechanisms whereby agonist catecholamines regulate the function of beta-adrenergic receptors in a model system, the frog erythrocyte. The unique characteristics of agonist as opposed to antagonist action are first, the ability to stimulate the adenylate cyclase through the receptor and second, the ability to desensitize the system by alterations induced in beta-adrenergic receptors. These properties of agonist are not shared by antagonist despite the high affinity and specificity of antagonist binding to the beta-adrenergic receptors. Agonist and antagonist receptor complexes may be distinguished in a variety of ways including differences in their sensitivity to regulatory guanine nucleotides and also by gel chromatography on AcA 34 Ultragel. The agonist receptor complex appears to elute from the columns with an apparently increased size. A "dynamic receptor affinity model" of beta-adrenergic receptor action is proposed which features several distinct conformational states of the receptor. Agonists have much higher affinity for the physiologically active or coupled state of the receptor, whereas antagonists have equal affinity for both. In addition, a third "desensitized" state of the receptor is also postulated to exist.

Pneumococcal polysaccharide immunization in infants and children.

By using indirect hemagglutination, the antibody responses of normal infants and children to an octavalent pneumococcal vaccine that contained pneumococcal polysaccharide types 1, 3, 6, 7, 14, 18, 19, and 23 were evaluated. By 2 years of age, there was a significant rise in hemagglutination titers to all the polysaccharide types, except type 19. By 6 to 8 months of age, five of the eight types of pneumococcal polysaccharides tested resulted in up to 60% responders and, by 2 years, a significant number responded to all pneumococcal polysaccharide types in the vaccine. Pneumococcal polysaccharide type 3 resulted in a significant antibody response as early as 3 months of age, whereas type 19 never resulted in a significant antibody response. Except for type 3, it seemed that when the other pneumococcal polysaccharides tested produced an antibody response, the degree of resonse did not subsequently change significantly with increasing age. The relationship of antibody response to age for pneumococcal polysaccharides is similar to that found for other polysaccharide vaccines. Based on the results of our study, we would recommend immunization with pneumococcal vaccine at 6 months of age with repeat immunization at 2 years of age, especially in high-risk children.

Phosphorus-31 NMR studies of E. coli ribosomes.

Phosphorus-31 nuclear magnetic resonance spectra, relaxation times and nuclear Overhauser (NOE) enhancement have been measured for E. coli ribosomes, subunits and rRNA. NOE and T1 experiments reveal that the phosphorus relaxation in this organelle is largely dipolar in origin. Moreover these results imply the presence of internal motion within the RNA chain with a correlation time of about 3-5 x 10(-9) sec. In all cases the predominant resonance is centered at about -1.5 ppm (relative to 85% H3PO4) as expected for a phosphodiester linkage where there is a large degree of double helix. The linewidth narrows by about a factor of four when the ribosomal proteins are removed indicating a substantial immobilization of the RNA when it is assembled into the ribosome. In addition to the phosphodiester resonance, ribosomes also reveal one or two narrower resonances shifted to low field by 1-4 ppm. Based on the observation that these resonances show a pH dependent chemical shift, we assign them to phosphate monoesters i.e. terminal 3' or 5' phosphate groups. These terminal phosphates are due to short oligomers of RNA derived from the terminus of the chain.

Polarographic estimation of antazoline hydrochloride.

The dc-polarographic investigation of antazoline hydrochloride in aqueous acidic media is described. Using potassium chloride-hydrochloric acid mixture as the supporting electrolyte (pH = 3.25), antazoline hydrochloride was electrochemically reduced at the dropping mercury electrode, with the production of two waves with E1/2 values of --1.35 and --1.65 V respectively. As revealed from the study of the effect of mercury column height, pH of the medium and concentration of the depolarizer, the polarographic reduction of the antazolinium cation is preceded by a catalytic H-wave. The diffusion-controlled nature of the electrode process permitted the quantitative determination of antazoline hydrochloride in concentrations down to 1.0 . 10(-4)M. Application of the presented procedure to the analysis of different dosage forms of the compound studied proved successful and compared favourably with official estimations of anatazoline salts. In view of its simplicity, accuracy and sensitivity, the presented polarographic method can be recommended for routine analysis of antazoline formulations.

Food-stimulated acid secretion measured by intragastric titration with bicarbonate in patients with duodenal and gastric ulcer disease and in controls.

Gastric acid secretion stimulated by a normally eaten beefsteak meal was measured for 4 h in 16 patients with duodenal ulcer disease (DU), in 9 patients with gastric ulcer disease (GU), and in 14 controls by intragastric titration with bicarbonate to a constant pH 5.5. Reproducibility of the method investigated in 6 DU and in 5 controls gave similar acid secretory values (var. coeff. = 7.5%). DU produced acid on a higher level and with longer duration after food than controls and GU (p less than 0.001). Apart from the second half of the first hour after food, when the acid secretion was higher in controls than in GU (p less than 0.025), there was no significant difference in acid output after food between GU and controls. Maximum gastrin values and 'total gastrin output' after food were significantly higher in GU than in controls, but these differences were not significant between GU and DU and between DU and controls. Fasting gastrin and gastrin levels after food were not correlated to basal acid output or acid output after pentagastrin or food in any of the groups. The maximal acid output after food was higher than the peak acid output after pentagastrin in controls, DU and GU. The relation between food- and pentagastrin-stimulated acid output was not statistically significantly different between the three groups. Instead, acid secretion after food was well correlated to acid secretion after pentagastrin in controls, DU and GU (r = 0.85).

HLA-Bw52 in Takayasu disease.

Takayasu disease is characterized by a pulseless condition which most often occurs in young females from Asian or South American areas. The cause of this disease remains obscure. Recently we encountered monozygotic, Japanese identical twin sisters, both of whom were diagnosed as having Takayasu disease. A genetically related factor was considered and HLA analysis was carried out. A population study on HLA typing analyses of 65 patients with Takayasu disease revealed a high frequency of HLA-B5 as compared with 128 healthy Japanese (chi2 :17.0, P less than 10(-4)). Subgroups of B5, Bw51 and Bw52 were successively studied in 82 patients with this disease. Bw51 antigen was found in 12.2% of patients with Takayasu disease and in 19.5% of 128 healthy Japanese. Contrarily, Bw52 antigen was confirmed in 43.9% of patients, a statistically significant frequency with the level of 26.5 in the chi2 test (cP less than 3 x 10(-4)) when compared with 12.5% in normal Japanese. Thus a genetically related factor in the pathogenesis of Takayasu disease has to be considered.

Purification and properties of rennin-like enzyme from Aspergillus ochraceus.

An active milk-clotting enzyme was purified some 40-fold from culture supernatant of Aspergillus ochraceus. The purification steps included ammonium sulfate precipitation, G-100 Sephadex gel filtration, and ion exchange chromatography, using DEAE Cellulose column. The enzyme exhibited milk-clotting activity and proteolytic behaviour, an optimum at pH 6.0 and in the range of 7--8.5, respectively. The purified enzyme was actively proteolytic against casein, haemoglobin, and bovine serum albumin at pH 8. The milk-clotting activity was greatly enhanced by manganous ions and by increasing concentrations of calcium chloride. Copper, zinc, and ammonium ions were potent inhibitors of the milk-curdling activity of the purified enzyme. Significant inhibition was also noted with sodium chloride at concentrations of 3% or more. Under the specified reaction condition, maximum rate of proteolysis against casein was obtained at 0.4% substrate concentration, whereas the milk-clotting time was linear proportional to dry skim milk concentration in the range of 8 to 24%. The results are discussed in comparison with other microbial milk-clotting enzymes, and limitations of applicability are also presented.

Pressure passive cerebral blood flow and breakdown of the blood-brain barrier in experimental fetal asphyxia.

Cerebral blood flow (CBF) was studied in non-exteriorized near-term sheep fetuses using the radioactive microsphere technique. By partially occluding the umbilical vessels for a period of 1--1 1/2 hours a progressive and severe asphyxia with a final arterial pH of 6.90 was achieved. Varying the mean arterial blood pressure in the fetuses by blood withdrawal or infusion in this state, CBF was measured at different perfusion pressures (mean arterial blood pressure (MABP) minus central venous pressure (CVP)). A passive flow/pressure relationship--loss of autoregulation--was found, with hyperemia reaching CBF values up to 6 times normal at normal MABP of about 60 to 70 mmHg, and severe ischemia reaching CBF values close to zero in large cortical areas at MABP of 30 mmHg. CVP remained essentially unchanged at 10--15 mmHg. The severe and prolonged asphyxia rendered the blood-brain barrier leaky to the albumin tracer Evans blue. In four other fetuses umbilical cord clamping was omitted. However, only in one of these cases was acidosis completely avoided, and CBF autoregulation maintained. The three other fetuses were acidotic at the end of the surgical procedure and had impaired autoregulation.

The in vitro interaction of Trypanosoma cruzi bloodstream forms and mouse peritoneal macrophages.

The uptake and further development of bloodstream forms from T. cruzi Y and CL strains in mouse peritoneal macrophages have been investigated. Parasites from the Y strain (which present predominance of slender forms) are 20 to 30-fold more infective to macrophages than those from CL strain in which stout forms highly predominate. A complete amastigote-trypomastigote cycle is observed in normal or thioglycollate-induced macrophages infected with parasites from both strains.--Opsonization significantly increases the uptake by normal macrophages of parasites from both strains. The fate of the opsonizated parasites is, however, different: the Y trypomastigotes present a normal cycle which culminates with the release of newly formed trypomastigotes whereas CL parasites are extensively destroyed by normal macrophages.--The differences in the uptake and fate displayed by both T. cruzi populations are not well understood. They are apparently related to parasite membrane components or macrophage receptors differences, which are probably influencing endocytosis and the further intracellular development of the parasites.

Influence of pH on elastic deformability of the human erythrocyte membrane.

Fresh human blood was diluted 1:5000 in buffered saline-sucrose solution and titrated to a pH varying from 4.5 to 10.5 with 0.1 N HCl or 0.1 N NaOH. Circular regions of the membrane of individual cells were then deformed at 25 degrees C by aspiration into a micropipette having an internal tip diameter of 0.9-1.4 micron. A membrane surface elasticity modulus, mu (dyn/cm), was computed from the relationship between length of the aspirated membrane and the deforming pressure according to a two-dimensional membrane model. Surface elasticity increases with decreasing pH and with time after the cell suspension is acidified, rising several orders of magnitude with a t1/2 of 1--5 h as pH is lowered from 7.2 to 4.6. This increase in mu is only partially reversible. pH greater than 7.2 had little effect on mu. Membrane surface elasticity is not affected by variations in external [Ca2+] over the range of 0--50 mM, tonicity of the suspension medium from 275--400 mosM, or age of 0--50 h. Addition of 50 mM NaHCO3 to the medium increases the rate of change of mu at a given pH. These results suggest that the elastic properties of the red cell membrane are largely determined by interactions among structural proteins located on the cytoplasmic surface of the membrane and that these interactions are initiated by changes in intracellular pH.

Importance of chloride for acute inhibition of renin by sodium chloride.

To evaluate the contribution of chloride to acute renin inhibition by sodium chloride, plasma renin activity (PRA) was measured before and after peripheral venous infusion of NaCl, NaHCO3, NaBr, NaNO3, lysine monohydrochloride, or lysine glutamate in NaCl-deprived rats. In contrast to controls and animals infused with other sodium salts, PRA decreased (P less than 0.01) after infusion with NaCl [from 28.3 +/- 2.8 to 13.3 +/- 1.8 ng/ml per h (SE)] and NaBr (from 40.6 +/- 6.2 to 21.8 +/- 3.9 ng/ml per h), and renal tubular halide reabsorption increased (P less than 0.05). Arterial pressure, plasma volume, inulin clearance, net sodium balance, serum Na+ and K+, and pH were not different among sodium-loaded groups. PRA was also suppressed (P less than 0.01) by infusion with lysine monohydrochloride (from 51.6 +/- 5.4 to 32.4 +/- 5.1 ng/ml per h) but not with lysine glutamate. These results suggest that inhibition of renin by sodium is dependent on an intrarenal effect of chloride. During infusion with sodium salts which suppressed renin, negative free water clearance (TcH2O) increased, whereas infusion with sodium salts that did not inhibit renin resulted in either no change or decreased TcH2O. The association of renin inhibition and increased TcH2O indirectly supports the hypothesis that renin suppression by chloride is related to the magnitude of absorptive chloride transport in the thick ascending limb of the loop of Henle.

[New results on ageing of connective tissue (author's transl)].

Some new results on ageing of connective tissue are demonstrated, regarding not only mesenchymal, but also parenchymal organs of human and rat (both sexes). These results show that ageing of connective tissue is more a dynamic process (with measurable metabolic parameters of the several connective tissue cells and their products) than a passive or so-called degenerative connective tissue process. The bradytrophy concept of connective tissue cannot be accepted any longer. Then connective tissue cells can produce metabolic rates of the same level like parenchymal cells. The different organs possess partly common basic processes partly differences in connective tissue ageing, dependent on the composition and patterns of proteoglycans resp. of GAG and collagen types, furthermore dependent on localisation, structure and function. The results on ageing of connective tissue are useful for better understanding of ageing processes of parenchymal organs. Then their ageing is influenced essentially by the ageing of the own connective tissues. The turnover, more than the number of mesenchymal and parenchymal cells, decreases with ageing. More old than young cells seem to exist in old tissues and organs. The performance of ageing connective tissue cells can be constant or increase or decrease. Many mesenchymal and parenchymal organs develop a so-called "ageing-fibrosis".

Behavior therapy, supportive psychotherapy, imipramine, and phobias.

In a controlled outcome study of phobias, 111 adult patients (69% women, 31% men) received a course of 26 weekly treatment sessions consisting of (1) behavior therapy and imipramine hydrochloride (2) behavior therapy and placebo, or (3) supportive psychotherapy and imipramine. Patients were classified as agoraphobic, mixed phobic, or simple phobic. The great majority of patients in all groups showed moderate to marked global improvement (70% to 86%, depending on rater). In agoraphobics and mixed phobics (both groups experiencing spontaneous panic attacks), imipramine was significantly superior to placebo. There was no difference between behavior therapy and supportive therapy, both resulting in high improvement rates (76% to 100%, depending on rater). In simple phobic patients, there was a high rate of improvement with all treatment regimens (72% to 93%, depending on rater), with no significant difference between imipramine and placebo or between behavior therapy and supportive therapy. Of 88 moderately to markedly improved patients followed up for one year after completing treatment, 83% maintained their gains and 17% relapsed. No patients showed symptom substitution. Eighteen percent of the patients receiving imipramine hydrochloride showed marked stimulant side effects on from 5 to 75 mg/day.

Effect of the mouse mutants testicular feminization and sex reversal on hormone-mediated induction and repression of enzymes.

The mouse mutants testicular feminization and sex reversal have been used to investigate hormone-mediated induction and repression of enzymes. Tfm/Y animals were already known to be androgen insensitive, rendering the androgen-inducible enzymes ADH and beta-glucuronidase noninducible because of an inherited deficiency of a cytosol androgen-receptor complex. The animals display female secondary sexual characteristics. Sxr/+,XX animals display male primary and secondary sexual characteristics with small testes. We demonstrate (1) that the Tfm mutation is pleiotropic, preventing repression of an androgen-repressible enzyme (ornithine aminotransferase) as well as induction of androgen-inducible enzymes, (2) that an estrogen-inducible enzyme (histidine decarboxylase) is not affected by the Tfm mutation, and (3) that Sxr/+,XX animals produce enough androgen for malelike activities of androgen-sensitive enzymes. It was also discovered that histidine decarboxylase repressed by androgen in normal animals, rather than being unaffected by it in Tfm/Y animals, is in fact induced. This unexpected phenomenon is discussed and an explanation is suggested for it.

Cerebral oxidized and reduced nicotinamide-adenine dinucleotide phosphate and glucose 6-phosphate dehydrogenase in mice during exposure to high oxygen pressure.

NADP+, NADPH and glucose 6-phosphate dehydrogenase were determined in the cerebral cortex of mice exposed to high O2 pressure for 0, 8 and 16 min. These time intervals corresponded to 0, 50 and 100% of the CT50 (the time taken for 50% of the mice to convulse). Cerebral NADP+, NADPH and glucose 6-phosphate dehydrogenase also were determined in O2-exposed mice exhibiting hyperactivity, convulsions, and in mice killed 10s after convulsions. Similar increases in cortical NADP+ and decreases in NADPH were found in mice exposed to 610kPa (6 atm.) of 100% O2 for 0, 50 and 100% of the CT50, during hyperactivity, onset of seizure and 10s after convulsions. The NADP+/NADPH ratio increased approx. 25% at 0% of the CT50, and remained at this increased value at all O2-exposure periods including the hyperactive state, onset of seizure and 10s after convulsions. Identical changes in cerebral NADP+ , NADPH and the NADP+/NADPH ratio were found in mice exposed for 16min to 100% O2 at 100, 350 or 610kPa. No change in cerebral glucose 6-phosphate dehydrogenase was found in mice exposed to 610kPa of 100% O2 during the various stages of O2 toxicity. Only in the 10s post-convulsive group was a statistically significant decrease in glucose 6-phosphate dehydrogenase observed. Disulfiram [bis(diethylthiocarbamoyl) disulphide], an effective O2-protective agent, did not prevent the O2-induced increase in cerebral NADP+ and the NADP+/NADPH ratio, or decrease in NADPH.

Zymogen activation in serine proteinases. Proton magnetic resonance pH titration studies of the two histidines of bovine chymotrypsinogen A and chymotrypsin Aalpha.

Reversible unfolding of bovine chymotrypsinogen A in 2H2O either by heating at low pH or by exposure to 6 M guanidinium chloride results in the exchange of virtually all the nitrogen-bound hydrogens that give rise to low-field 1H NMR peaks, without significant exchange of the histidyl ring Cepsilon1 hydrogens. These preexchange procedures have enabled the resolution of two peaks, using 250-MHz correlation 1H NMR spectroscopy, that are attributed to the two histidyl residues of chymotrypsinogen A. Assignments of the Cepsilon1 hydrogen peaks to histidine-40 and -57 were based on comparison of the NMR titration curves of the native zymogen with those of the diisopropylphosphoryl derivative. Two histidyl Cepsilon1 H peaks were also resolved with solutions of preexchanged chymotrypsin Aalpha. The histidyl peaks of chymotrypsin Aalpha were assigned by comparison of NMR titration curves of the free enzyme with those of its complex with bovine pancreatic trypsin inhibitor (Kunitz). The NMR titration curves of histidine-57 in the zymogen and enzyme and histidine-40 in the zymogen exhibit two inflections; the additional inflections were assigned to interactions with neighboring carboxyl groups: aspartate-102 in the case of histidine-57 and aspartate-194 in the case of histidine-40 of the zymogen. In bovine chymotrypsinogen A in 2H2O at 31 degrees C, histidine-57 has a pK' of 7.3 and aspartate-102 a pK' of 1.4, and the histidine-40-aspartate-194 system exhibits inflections at pH 4.6 and 2.3. In bovine chymotrypsin Aalpha under the same conditions, the histidine-57-aspartate-102 system has pK' values of 6.1 and 2.8, and histidine-40 has a pK' of 7.2. The results suggest that the pK' of histidine-57 is higher than the pK' of aspartate-102 in both zymogen and enzyme. A significant difference exists in the structure and properties of the catalytic center between the zymogen and activated enzyme. In addition to the difference in pK' values, the chemical shift of histidine-57, which is highly abnormal in the zymogen (deshielded by 0.6 ppm), becomes normalized upon activation. These changes may explain part of the increase in the catalytic activity upon activation. The 1H NMR chemical shift of the Cepsilon1 H of histidine-57 in the chymotrypsin Aalpha-pancreatic trypsin inhibitor (Kunitz) complex is constant between pH 3 and 9 at a value similar to that of histidine-57 in the porcine trypsin-pancreatic trypsin inhibitor complex [Markley, J.L., and Porubcan, M. A. (1976), J. Mol. Biol. 102, 487--509], suggesting that the mechanisms of interaction are similar in the two complexes.

Sequence of reactions which follows enzymatic oxidation of allylglycine.

The pathway following flavoprotein-catalyzed oxidation of allylglycine (2-amino-4-pentenoate) has been studied and found to be dependent on the incubation conditions. In N-2-hydroxyethyl-N'-2-ethanesulfonic acid (Hepes) buffer, the oxidation product 2-iminium-4-pentenoate predominantly reacts to form 2-amino-2,4-pentadienoate, a strong noncovalent inhibitor of D-amino-acid oxidase. However, in pyrophosphate buffer, the more rapid reaction is hydrolysis to form 2-keto-4-pentenoate, which has been found to be a substrate for L-lactic dehydrogenase. 2-Keto-4-pentenoate is in rapid equilibrium with 2-hydroxy-2,4-pentadienoate, which is also a strong noncovalent inhibitor of D-amino-acid oxidase. In both systems, these metastable intermediates react in subsequent slower steps to yield trans-2-keto-3-pentenoate, which accumulates in the incubation. Syntheses of trans-2-amino- and trans-2-keto-3-pentenoate are described. Comparisons between the reactivities of acetylenic and olefinic species have been made based on the differences between this pathway and that following oxidation of propargylglycine [Marcotte, P., and Walsh, C. (1978), Biochemistry 17 (preceding paper in this issue)].

The role of Zn(II) in calf intestinal alkaline phosphatase studied by the influence of chelating agents and chemical modification of histidine residues.

Alkaline phosphatase from calf intestine (orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) is reversibly inhibited at pH 8.0 by incubation with chelating agents. Complete reactivation may be achieved by stoichiometric addition of Zn2+. Atomic absorption spectrometry was used to demonstrate the linear correlation between Zn2+ content and degree of reactivation. The reversibly inhibited enzyme contained 1 Zn2+ per subunit whereas 2 Zn2+ were found in both the reactivated and the native enzyme. At more alkaline pH-values, inactivation by chelating agents becomes irreversible; under such conditions the inactivated alkaline phosphatase still contains 1 Zn2+ per subunit. The conformational changes resulting from the loss of Zn2+ and leading to irreversible inactivation were investigated by optical rotatory dispersion, immunological techniques, and ultraviolet and fluorescence spectroscopy. Azocoupling of the alkaline phosphatase with diazonium-1-H-tetrazole and Zn2+ content measurement of azocoupled enzyme probes indicated that 2 histidine residues per subunit are involved in binding of the catalytically important Zn2+.

Laser Raman studies on cobrotoxin.

Laser Raman spectra of cobrotoxin under various conditions have been obtained. Comparison of the spectra of native cobrotoxin in lyophilized form and in aqueous solution indicates that the secondary structures of cobrotoxin are not significantly affected by the removal of the aqueous solvent. On going from the native to the partially reduced and the completely reduced, carboxy-methylated forms, characteristic peaks of the C-S-S-C and tyrosine ring in the region of 500--900 cm-1 showed definite changes in structure. The partially reduced form gave two peaks at 502 and 524 cm-1, suggesting difference in the conformation of the remaining disulfide bonds. As indicated by the present work, the conformation of the main chain of cobrotoxin in the native unperturbed state, in the partially reduced and in the completely reduced forms are the coexistence of beta-pleated sheet with random-coil structure, predominantly random coil, and predominantly random coil with the existence of an alpha-helix type structure, respectively. The effect of pH on the conformation of cobrotoxin in solution appeared to give rise to the change of the local structure of two aromatic residues common to all snake neurotoxins.

Collagen-induced platelet aggregation and release. I Effects of side-chain modifications and role of arginyl residues.

To investigate the mechanisms governing collagen interaction with blood platelets, the effects of side-chain modifications on collagen-induced platelet aggregation and release of serotonin were studied. Since many chemical modifications alter the ability of collagen to form fibers that, according to current theory, may complicate interpretation of data, we eliminated this possibility by using collagen stabilized in a native-type fibrillar structure by treatment with either glutaraldehyde or ultraviolet irradiation. Acetylation, methylation, succinylation, treatment with 2,4-dinitrofluorobenzene, 2,4,6-trinitrobenzene sulfonic acid or 1,2-cyclohexanedione, and deguanidination with hypobromite were used to modify collagen side-chain reactive groups: amino, carboxyl, hydroxyl and guanidino. Both unmodified monomeric dispersed and fibrillar collagen preparations initiated platelet aggregation and release, although the kinetics and magnitude of the response were different. Monomeric collagen which had been modified by deguanidination, methylation or succinylation, failed to polymerize in physiological conditions and did not induce platelet aggregation and release. However, none of the chemical modifications of stabilized native-type collagen fibers, except treatment with hypobromite or cyclohexanedione, had an effect on collagen-induced platelet aggregation and release. Both hypobromite and cyclohexanedione modified guanidino groups of arginyl residues. Results showed that the ability of a collagen sample to induce platelet aggregation and release of serotonin is dependent on the arginine content of fibrillar collagen. These data demonstrate that manipulation of amino, carboxyl and hydroxyl groups is unimportant as long as the native-type fibrillar structure is maintained, and that arginyl residues are directly involved in collagen-platelet interaction. Moreover, the data suggest that only the arginyl residues in the Y position of the tripeptide unit Gly-X-Y of collagen are responsible.

Conditions limiting the use of ionophore A23187 as a probe of divalent cation involvement in biological reactions. Evidence from the slow fluorescence quenching of type A spinach chloroplasts.

The conditions under which ionophore A23187 can be used as a probe of Mg2+ involvement in the reactions of intact (Type A) spinach chloroplasts have been investigated by monitoring ionophore-induced reversal of slow fluorescence quenching. The following observations were made: (1) A23187-dependent reversal of quenching is a strong function of pH. This is consistent with competition between protons and divalent cations for the carboxylic acid moiety of the ionophore. (2) In the presence of exogenous Mg2+, quenching reversal by A23187 is significantly slowed. It is suggested that formation of the dimeric A23187 . Mg2+ complex delays action of the ionophore at the thylakoid membrane by slowing equilibration of the ionophore among chloroplast membrane phases. (3) In the absence of Mg2+, significant interaction of A23187 with certain monovalent cations--Li+ and Na+, but not K+--is observed. Evaluations of the interaction of ionophore A23187 with specific biological systems and inferences of divalent cation involvement, or lack thereof, must take these limitations into account.

Effect of salts on D-glycerate dehydrogenase kinetic behavior.

Bovine liver D-glycerate dehydrogenase (D-glycerate:NAD (NADP) oxidoreductase, EC 1.1.1.29) adapts its kinetic behaviour to a sequential mechanism. The presence of NaCl causes an appreciable variation in the Km and V values. relative to the both substrates in the hydroxypyruvate/D-glycerate dehydrogenase/NADH system, which does not happen in the D-glycerate/D-glycerate dehydrogenase/NAD system. The former system is inhibited by high concentrations of NaCl and activated by low salt concentrations. The hydroxypyruvate concentration causing substrate inhibition increases as the concentration of NaCl increases; excess NADH inhibition is independent of the salt concentration. The variation of the initial rates of both systems, in the presence of chlorides having monovalent and divalent cations, or sodium halides, Na2SO4 and NaNO3 (at constant ionic strength) suggests that the anions have a specific action on the enzyme. An increase in the NaCl concentration causes a displacement of the optimum D-glycerate dehydrogenase pH (with hydroxypyruvate and NADH as substrates) towards the acid area. The enzyme stability, at varying pH, varies with the salt concentration.

Characteristics of the contingent negative variation in patients suffering from specific phobias.

The essential clinical feature of phobic neurosis is the anticipatory fear of certain objects and situations. Since an important factor in the generation of the contingent negative variation (CNV) is the anticipation of the imperative stimulus, records of CNVs were used as an indicator of electrocortical activity in a group of 14 patients suffering from specific phobias. After clinical evaluation, a CNV to a picture of a nondisturbing object was obtained. The amplitude of the CNV and duration of the PINV (postimperative negative variation) were taken as reference values against which response to the phobic objects were evaluated. Reaction time was automatically measured between the period of S2 and the button press. Significant differences, viz., larger CNV amplitude, longer PINV duration, and shorter reaction time, were found with phobogenic than with nondisturbing stimuli. After behavioral recovery with behavior therapy, no differences were noted in the CNVs obtained with the presentation of non-disturbing and the ones that were phobogenic stimuli. The significance of the results for an understanding of phobic neurosis and behavior therapy are discussed.

Brain alpha-adrenergic receptors: comparison of [3H]WB 4101 binding with norepinephrine-stimulated cyclic AMP accumulation in rat cerebral cortex.

The ability of a series of adrenergic agents to displace the binding to brain membranes of [3H]WB 4101, a potent alpha-adrenergic antagonist (WB 4101 = 2-[2-(2,6-dimethoxyphenoxy)ethylaminomethyl]-1,4-benzodioxane hydrochloride), has been compared with the potency of these agents in stimulating or inhibiting the alpha-adrenergic component of cyclic AMP accumulation in rat cerebral cortex slices. [3H]WB 4101 rapidly bound to a high affinity site (KD = 2.7 nM) in membranes from cerebral cortex. Binding came to equilibrium by 2 min at 37 degrees C and was rapidly reversed in the presence of phentolamine. The potencies of adrenergic agents (WB 4101 greater than phentolamine greater than naphazoline) in displacing binding of [3H]WB 4101 were comparable to the potencies of these agents as inhibitors of the alpha-adrenergic component of norepinephrine-stimulated cyclic AMP accumulations. Phenoxybenzamine, clonidine, chlorpromazine and haloperidol were about 10--30 times more potent in inhibiting cyclic AMP accumulation than in displacing [3H]WB 4101 binding. The potency of classical alpha-adrenergic agonists in displacing WB 4101 (epinephrine greater than norepinephrine greater than methoxamine) correlated with the ability of these agonists to increase cyclic AMP levels. Overall a significant correlation (r = 0.87, P less than 0.005) was found between WB 4101 binding and alpha-adrenergically mediated cyclic AMP accumulation in brain. Several ligands bind to specific sites in brain membranes with alpha-adrenergic receptor properties. The identification of these binding sites as receptors depends on a correlation of binding with a known alpha-adrenergic receptor-mediated response in brain. These data demonstrating that WB 4101 correlates with norepinephrine-stimulated cyclic AMP accumulation suggest that WB 4101 may bind to the membrane receptor sites mediating the alpha-adrenergic accumulation of cyclic AMP in rat cerebral cortex.

Cellulase and beta-glucosidase production by a basidiomycete species.

The optimisation of cellulase and beta-glucosidase production by a basidiomycete species was studied and cellulase and cellobiase production by this and Trichoderma viride (and its mutants) in shake flasks were compared. The former produced an active cellulase comparable to that of T. viride when tested on filter paper, carboxymethylcellulose, and cotton; however, it produced 20 to 26 times larger amounts of cellobiase. Both cellulase and beta-glucosidase were obtained in good yield only when cellulose was the carbon source. The production of these enzymes was not repressed by readily assimilated carbon sources in the presence of cellulose. Only traces of cellulase and beta-glucosidase were formed on glucose, fructose, maltose, and cellobiose although good growth was obtained on these substrates. These enzymes were not induced on sophorose, lactose, mannitol, or glycerol and growth was poor on these substrates. Cellobiose octaacetate was a less effective inducer of cellulase and beta-glucosidase than was cellulose.

The American Cleft Palate Association: its first 36 years.

History involves memory, and sometimes memory is faulty. History requires documentation, and sometimes documents cannot be found. History demands winnowing, and sometimes the wheat and the chaff cannot be completely separated. History is an assembly of details, and details may become burdensome. History is a challenge, to which the historian tries to respond. Surely, earlier Historians of the American Cleft Palate Association must have encountered this challenge. The present Historian acknowledges the work of her predecessors: George H. Foster, W. J. Robinson, and William Harkins from dentistry and Gretchen M. Phair and Asa J. Berlin from speech pathology. In preparing this review of events that took place from 1943 to 1978, in the United States and in other countries, during times of war and times of relative peace, through difficulties and accomplishments, I have attempted to be as accurate as possible. If I have distorted facts and if I have omitted people and happenings that should have been included, I have not done so intentionally. As Historian of the American Cleft Palate Association, I have appreciated the opportunity to learn about the organization and the people, past and present, who have built and maintained it. Thank you for giving me this assignment.

Sympathetic and parasympathetic components of reflex cardiostimulation during vasodilator treatment of hypertension.

1. Haemodynamic responses to diazoxide (300 mg intravenously) were studied in 15 hypertensive patients before and after chronic beta-adrenoreceptor blockade by 320 mg of propranolol daily. After diazoxide alone, mean arterial pressure and total peripheral resistance were lowered by 24 +/- 3 and 35 +/- 5% (mean +/- SEM) respectively. Cardiac output and heart rate rose by 25 +/- 9 and 21 +/- 3%. During beta-adrenoreceptor blockade, the percentage changes of mean arterial pressure, heart rate, cardiac output and total peripheral resistance after vasodilatation were not significantly different from those after diazoxide alone. 2. Atropine, 0.04 mg/kg body weight, was given to 12 hypertensive patients chronically treated with beta-adrenoreceptor blockade, before acute vasodilatation by diazoxide. Diazoxide caused no increase in heart rate after combined beta-adrenoreceptor and parasympathetic blockade. However, cardiac output rose by 14 +/- 5%. 3. We conclude that withdrawal of parasympathetic tone is an important determinant of circulatory homeostasis after acute vasodilatation during beta-adrenoreceptor blockade.

Localization of putative transmitters in the hippocampal formation: with a note on the connections to septum and hypothalamus.

Biochemical assays on microdissected samples, denervation studies, subcellular fractionation, and light and electron microscopic autoradiography of high affinity uptake have been performed to study the cellular localization of transmitter candidates in the rat hippocampal formation. High affinity uptake of glutamate and aspartate is localized in the terminals of several excitatory systems, such as the entorhino-dentate fibres (perforant path), mossy fibres (from granular cells) and pyramidal cell axons. Thus, in stratum radiatum and oriens of CA1, 85% of glutamate and asparate uptake and 40% of glutamate and aspartate content are lost after lesions of ipsilateral plus commissural fibres from CA3/CA4. Hippocampal efferents also take up aspartate and glutamate, since these activities are heavily reduced in the lateral septum and mamillary bodies after transection of fimbria and the dorsal fornix. The synthesis (by glutamic acid decarboxylase), content and high affinity uptake of gamma-aminobutyrate (GABA) are not reduced after lesions of these or other projection fibre systems. A localization in intrinsic neurons is confirmed by a selective loss of glutamic acid decarboxylase after local injections of kainic acid. Peak concentrations of the enzyme occur near the pyramidal and granular cell bodies, corresponding to the site of the inhibitory basket cell terminals, and in the outer parts of the molecular layers. Some 85% of glutamic acid decarboxylase is situated in 'nerve ending particles'. Acetylcholine synthesis (by choline acetyltransferase) disappears after lesions of septo-hippocampal fibres. Since 80% of the hippocampal choline acetyltransferase is in 'nerve ending particles', the characteristic topographical distribution of this enzyme should reflect the distribution of cholinergic septo-hippocampal afferents. Serotonin, noradrenaline, dopamine and histamine are located/synthesized in afferent fibre systems. Some monoamine-containing afferents to the hippocampal formation pass via the septal area, others via the amygdala. The hippocampal formation also contains nerve elements reacting with antibodies against neuroactive peptides, such as enkephalin, substance P, somatostatin and gastrin/cholecystokinin.

Isolation and characterization of the acidic phosphoproteins of 60-S ribosomes from Artemia salina and rat liver.

Eucaryotic L7/L12-type proteins are present in ethanol/salt extracts (P1 protein) of ribosomes from Artemia salina and rat liver. These proteins are partially phosphorylated and occur in two forms of closely related structure: a major form eL12 having methionine at the N-terminal position and a minor form of eL12 (eL12') which seems slightly elongated and contains a blocked N terminus. Purification of the four different forms of this protein, eL12, eL12-P, eL12' and eL12'-P, was performed by ion-exchange chromatography on carboxymethyl-cellulose and DEAE-cellulose. Using a radioimmuno assay, 1.8 copies of eL12 and 0.9 of eL12' were found on the 80-S A. salina ribosome. In ribosomes of both rat liver and A. salina, eL12 is present in a larger quantity than eL12'. 40-S and 60-S ribosomal subunits extracted with ethanol/salt were essentially free of eL12 proteins. A large pool of eL12 was found in the cytosol after removal of the ribosomes by centrifugation or molecular sieving. The proteins of rat liver and A. salina are similar with regard to their isoelectric points and molecular weights. Sedimentation equilibrium studies indicated that the isolated protein eL12 occurs as a dimer.

Comparison of disposition and effect of timolol and propranolol on exercise tachycardia.

The kinetic disposition and beta-adrenergic blocking action in relation to plasma level of a single oral dose of either timolol or propranolol has been compared in healthy male volunteers. The disposition profiles clearly disclosed different properties of the two drugs, although their half-lives were similar. The available fraction of timolol in the systemic circulation was estimated to be approximately 60% of the dose, and 17.4% was exereted unchanged in urine. The logarithm of plasma concentration showed a significant correlation with the beta-blocking activity assessed by an exercise test. The mean potency ratios of timolol to propranolol as an antagonist of chronotropic effects on exercise tachycardia were 11 to 17 and 3.6 to 5.5 in dose- and concentration-effect relationships, respectively. The absolute reduction of exercise heart rate gave the best coefficient of all measures of beta-blockade. When drug action was measured as beta-blockade assessed by a given response to exercise tachycardia, the effect declined linearly with time, even though plasma levels fell exponentially. This results suggest that the pharmacokinetic t1/2 is much shorter than the pharmacological t1/2.

A difference in effects of physiological Ca2+ concentrations on activity of guanylate cyclase preparations obtained from the taenia caecum of guinea pig and from the longitudinal muscle of rat duodenum.

A cholinergic stimulant, butyltrimethylammonium bromide and serotonin increased the tissue levels of cyclic GMP in the taenia caecum of guinea pig but not those in the longitudinal muscle of rat duodenum. On the other hand, physiological Ca2+ concentrations enhanced the activity of a guanylate cyclase preparation obtained from the taenia caecum of guinea pig, while guanylate cyclase in the longitudinal muscle of rat duodenum was not influenced by Ca2+. The difference in the effects of the smooth muscle stimulants on the tissue levels of cyclic GMP in two different smooth muscles in attributed to differences in the properties of guanylate cyclase of smooth muscles.

A comparison of socio-demographic and fertility characteristics of women sterilized in hospitals and camps.

Socio-demographic characteristics of 5846 women undergoing sterilization in hospitals and 1752 women undergoing sterilization in camps in India are analyzed. The average age of women accepting sterilization was 29.8 years; the mean number of living children was 4.2. Women sterilized in hospitals were of significantly higher age and parity than those sterilized in camps. The mean parity for all women was 4.6. A steep increase in median parity was observed with increasing age in both hospitals and camps. The study indicates that, while the level of education may affect acceptance of sterilization, there is no minimum level of education necessary for its acceptance. Most of the women in the study were not gainfully employed. As expected most of the women were Hindus. Muslims were underrepresented in the hospital series; in camps, however, Muslims showed a much higher rate of acceptance of sterilization. The rate of pregnancy wastage was significantly higher in the hospital and the child loss rate was significantly higher for the camp cases. While the rate of previous abortions was higher for low parity women, the child loss rate was higher for high parity women. The child loss parity ratio was significantly higher for the camps than for the hospital cases. The vast majority of women in both hospitals and camps reported no previous contraceptive practice. Sterilization appears to have been the method of choice for most of these women.

[Presynaptic receptor systems in central noradrenergic neurons].

Slices of rat occipital cortex, hypothalamus or cerebellar cortex were stimulated either electrically or by high potassium (in few experiments by tyramine). 1. Electrical or potassium stimulation elicit physiologically noradrenaline release in contrast to tyramine. 2. Stimulation-induced overflow of tritium, reflecting noradrenaline release, was diminished by a) alpha-adrenoceptor agonists, b) morphine and enkephaline, c) prostaglandin E1. The effect of agonists was abolished only by specific antagonists. Metabolism of 3H-noradrenaline was unchanged. 3. It is concluded that transmitter release from the noradrenergic neurones of several brain areas is modulated by drugs acting on alpha-adrenoceptors, opiate receptors and prostaglandin receptors. In contrast to other tissues there was no evidence obtained for presynaptic dopamine, beta-, muscarine, nicotine and angiotensin receptors. It seems likely that the receptors, involved in the modulation of noradrenaline release, are located at the noradrenergic nerve ending themselves, i.e. that they are presynaptic receptors.

Extractability of cell wall polysaccharide from lactobacilli and streptococci by autoclaving and by dilue acid.

Autoclaving cell wall of Streptococcus mutans Ingbritt for 15 min under the Rantz and Randall conditions released one-tenth of the total cell wall carbohydrate, whereas two-thirds was extracted after autoclaving for 180 min. The extract contained the serotype c-specific antigen but lacked the lipoteichoic acid component extracted when whole cells were autoclaved. Autoclaving cell wall preparations from other strains of S. mutans and also Streptococcus salivarius and Streptococcus mitis in 0.85% NaCl for 180 min released the major proportion of the wall polysaccharide fraction. Approximately 50 to 90% of wall carbohydrate of Lactobacillus fermentum and Lactobacillus casei was released when cell wall preparations were autoclaved in 0.85% NaCl for 180 min. For wall preparations from several strains of S. mutans, autoclaving for 60 min at pH 3.75 released only 39 to 62% of wall carbohydrate, whereas almost total release could be achieved with the lactobacilli. Heating S. mutans Ingbritt cell wall for 24 h at 60 degrees C in 0.1 N H(2)SO(4) released only two-thirds of the wall carbohydrate; by comparison nearly all of the wall carbohydrate was released in 3 h from L. casei and L. fermentum. Autoclaving L. casei cell wall and purified soluble wall fractions hydrolyzed the phosphodiester bond between the polysaccharide and peptidoglycan. This was shown by the release of reactive N-acetylhexosamine in both cases and the presence of a phosphomonoester in the autoclaved soluble wall fractions. The results indicate that autoclaving can hydrolyze covalent linkages, and this must be considered when the Rantz and Randall procedure is used to obtain antigen preparations.

In vitro cultivation of leprosy bacilli in hyaluronic acid-based medium. 2. Progress and developing concept of the role of hyaluronic acid suggested by culture and armadillo infection studies.

Progress is summarized relating to the verification, identification of M. leprae and understanding of the process of adaptation the pathogen passes through before in vitro growth takes place. It is recognized that hyaluronic acid apparently does not serve as a source of energy but the possibility is presented that it plays a role in the reconstruction of M. leprae cell walls made "leaky" by constant intracellular life. This apparently occurs, in culture, initially by the development of coccoid forms which after a period of weeks finally give rise to acid-fast bacilli. If these understandings are correct and the bacillary cell walls are vitated by enzyme and other action occasioned by intra-macrophage existence, then cell wall antigenicity may also be vitiated or altered by intracellular parasitism and restored by in vitro cultivation. The possible importance of this hypothesis in the understanding of immunologic responses in leprosy, and in the possibilities for therapeutic use and vaccine development are discussed.

Mixing technique for study of oxygen-hemoglobin equilibrium: a critical evaluation.

In the mixing technique for study of oxygen-hemoglobin equilibrium, the O2 saturation (SO2) of a blood mixture is calculated from the volume ratio at which an oxygenated sample is mixed with a deoxygenated sample, and the PO2 in the mixture is measured polarographically. Any predetermined level of SO2 may be obtained by proper choice of the volume ratio. It is shown that the volume and oxygen saturation of the mixed samples are by far the most critical parameters in calculating SO2, and a method is suggested by which the volume ratio is accurately measured by weighing the blood samples before mixing. Other parameters that influence determination of SO2, e.g., the O2 capacity of the blood, are much less important. The method has been applied to establish the O2 dissociation curve in human blood, and good reproducibility and agreement with standard curves were obtained. Measurements in rabbit blood yielded similarly satisfactory results. The technique is particularly applicable to problems that require exact adjustment of SO2 to a predetermined value, such as determination of the half-saturation pressure or of the Bohr effect at various levels of O2 saturation.

Direct fluorometric determination of a dissociation constant as low as 10(-10) M for the subtilisin BPN'--protein proteinase inhibitor (Streptomyces subtilisin inhibitor) complex by a single photon counting technique.

It was found that an increase in fluorescence intensity at 340 nm is observed on the binding of Streptomyces subtilisin inhibitor (SSI) with subtilisin BPN' in the pH range 6--10. The dissociation constant, Ki, of the enzyme-inhibitor complex was determined as a function of pH and temperature by direct fluorometric titration utilizing the single photon counting technique in the protein concentration range of 10(-9) M. Ki values as low as 10(-10) M could be obtained with reasonable accuracy by this high-sensitivity detection method. From the temperature dependence of Ki, it was found that the binding is endothermic, and is entirely "entropy-driven" in nature. The effect of pH on Ki suggested the participation of an ionizable group with pKapp = 8.5 in the binding.

Purification and properties of an NAD(P)+-linked formaldehyde dehydrogenase from Methylococcus capsulatus (Bath).

Crude soluble extracts of Methylococcus capsulatus strain Bath, grown on methane, were found to contain NAD(P)+-linked formaldehyde dehydrogenase activity. Activity in the extract was lost on dialysis against phosphate buffer, but could be restored by supplementing with inactive, heat-treated extract (70 degrees C for 12 min). The non-dialysable, heat-sensitive component was isolated and purified, and has a molecular weight of about 115000. Sodium dodecyl sulphate gel electrophoresis of the protein suggested there were two equal subunits with molecular weights of 57000. The heat-stable fraction, which was necessary for activity of the heat-sensitive protein, was trypsin-sensitive and presumed to be a low molecular weight protein or peptide. A number of thiol compounds and other common cofactors could not replace the component present in the heat-treated soluble extract. The purified formaldehyde dehydrogenase oxidized three other aldehydes with the following Km values: 0.68 mM (formaldehyde); 0.075 mM (glyoxal); 7.0 mM (glycolaldehyde); and 2.0 mM (DL-glyceraldehyde). NAD+ or NADP+ was required for activity, with Km values of 0.063 and 0.155 mM respectively, and could not be replaced by any of the artificial electron acceptors tested. The enzyme was heat-stable at 45 degrees C for at least 10 min and had temperature and pH optima of 45 degrees C and pH 7.2 respectively. A number of metal-binding agents and substrate analogues were not inhibitory. Thiol reagents gave varying degrees of inhibition, the most potent being p-hydroxymercuribenzoate which at 1 mM gave 100% inhibition. The importance of possessing an NAD(P)+-linked formaldehyde dehydrogenase, with respect to M. capsulatus, is discussed.

Content uniformity of potent drugs in tablets.

The type of distribution of low dosage drugs that occurs in batches of commercially available tablets has been examined. The uniformity of content of ethinyloestradiol tablets 10 micrograms B.P. from different sources, assessed by single tablet assay showed that three batches exhibited some positive skewness and one marked positive skewness. At the high level of dilution required, the mixing theory indicates that particles of drug must be very fine if acceptable content uniformity is to be obtained. Cohesion of particles impairs the efficiency of the mixing process. It is shown theoretically that unless the drug is dispersed into its component particles the shape of the distribution curve for the content of drug per tablet, in a batch of tablets, will be positively skewed. The relation between the tensile strength of the powdered drug and the degree of skewness of the drug content is also discussed. A positively skewed distribution for tablets containing a small amount of potent drug is unacceptable because this can lead to the presence of relatively large doses of drug in a single tablet. Where one unexpectedly high result occurs in the quality control of the content uniformity of tablets containing potent drugs it is suggested that sufficient single tablet assays be performed to allow the shape of the distribution curve to be assessed. A test for skewness should be included in compendial standards.

A comparison of the cardiovascular effects of centrally administered clonidine and adrenaline in the anaesthetized rat.

Intracerebroventricular (i.c.v.) injections of clonidine and adrenaline-induced hypotension and bradycardia in urethane anaesthetized spontaneous hypertensive rats. The hypotension induced by clonidine (3 microgram i.c.v.) was antagonized by pretreatment with the alpha-antagonists piperoxan, which also antagonized clonidine-induced bradycardia, and yohimbine. The hypotension and bradycardia induced by adrenaline (10 microgram i.c.v.) were unaffected by alpha-antagonist pretreatment, while beta-antagonist pretreatment with (--)-propranolol or metoprolol was effective against adrenaline but not clonidine-induced hypotension and bradycardia. Pretreatments with the histamine H2-receptor antagonists metiamide and cimetidine antagonized clonidine but not adrenaline-induced hypotension. These data indicate that different central mechanisms are involved in mediating the hypotension and bradycardia induced by centrally administered clonidine and adrenaline and do not, therefore, support the hypothesis that the hypotensive effects of clonidine (i.c.v.) are mediated by central adrenaline receptor activation in urethane-anaesthetized spontaneous hypertensive rats.

Characterization of Rauscher murine leukemia virus envelope glycoprotein receptor in membranes from murine fibroblasts.

Plasma membrane preparations from KA31 (mouse) cells contained receptors for the binding of Rauscher murine leukemia virus (R-MuLV) envelope glycoprotein, gp70. This binding was demonstrated by gel filtration of a mixture of the microsomal fraction of the cells and 125I-labeled gp70. A rapid and convenient assay was developed to measure the complex formation between the membrane receptors and gp70 involving specific precipitation of the complex by 3 to 4% polyethylene glycol. The complex formation was responsive to the concentrations of both the receptor and gp70 and also to changes in temperature and pH. The gp70 binding was a noncooperative, saturable process, and an association constant of 3.5 X 10(8) M-1 was estimated from the binding data. The complex formation was reversible and a near-total exchange of 125I-labeled gp70 in the complex was achieved by incubation with excess of unlabeled gp70. The complex formation was inhibited by protein denaturing agents, guanidine-hydrochloride and urea. Pretreatment of the membrane fractions with either chymotrypsin or phospholipase C led to a loss of the membrane-associated receptor activity, indicating that a lipoprotein structure was important for the receptor function, consistent with the observation that nonionic detergents strongly inhibited the complex formation.

Relationship between gastric secretion and serum gastrin levels in dogs anesthetized with morphine and urethane.

The serum levels of immunoreactive gastrin (IRG) and secretion of gastric juice were simultaneously determined in dogs anesthetized with morphine and urethane. There was a significant positive linear correlation between secretion and serum IRG level in these dogs. Serum IRG level and gastric secretion were reduced by bilateral vagotomy at the neck. The amount of gastric juice was reduced dose-dependently by an intravenous injection of atropine (0.001--0.016 mg/kg), hexamethonium (0.064--1 mg/kg) and secretin (2--8 U/kg). The reduction of gastric secretion paralleled that of the serum IRG level. However, the reduction of gastric secretion did not parallel that of serum IRG level under the influence of prostaglandin E1 (0.002--0.008 mg/kg i.v.) and duodenal acidification. Prostaglandin E1 and duodenal acidification reduced gastric secretion without the reducing serum IRG level. These findings were discussed in relation to the mechanism of gastric juice stimulation by morphine, and it is suggested that endogenous gastrin release through the vagal and non-vagal pathways participates in morphine-induced gastric secretion. The difference in inhibitory effect between duodenal acidification and secretin suggests the possibility that substances other than secretin may participate in the regulation of gastric secretion in dogs.

Phosphorylation and dephosphorylation of spectrin.

The phosphorylation of spectrin polypeptide 2 is thought to be involved in the metabolically dependent regulation of red cell shape and deformability. Spectrin phosphorylation is not affected by cAMP. The reaction in isolated membranes resembles the cAMP-independent, salt-stimulated phosphorylation of an exogenous substrate, casein, by enzyme(s) present both in isolated membranes and cytoplasmic extracts. Spectrin kinase is selectively eluted from membranes by 0.5 M NaCl and co-fractionates with eluted casein kinase. Phosphorylation of band 3 in the membrane is inhibited by salt, but the band 3 kinase is otherwise indistinguishable operationally from spectrin kinase. The membrane-bound casein (spectrin) kinase is not eluted efficiently with spectrin at low ionic strength; about 80% of the activity is apparently bound at sites (perhaps on or near band 3) other than spectrin. Partitioning of casein kinase between cytoplasm and membrane is metabolically dependent; the proportion of casein kinase on the membrane can range from 25% to 75%, but for fresh cells is normally about 40%. Dephosphorylation of phosphorylated spectrin has not been studied intensively. Slow release of 32Pi from [32P] spectrin on the membrane can be demonstrated, but phosphatase activity measured against solubilized [32P] spectrin is concentrated in the cytoplasm. The crude cytoplasmic phosphospectrin phosphatase is inhibited by various anions--notably, ATP and 2,3-DPG at physiological concentrations. Regulation of spectrin phosphorylation in intact cells has not been studied. We speculate that spectrin phosphorylation state may be regulated 1) by metabolic intermediates and other internal chemical signals that modulate kinase and phosphatase activities per se or determine their intracellular localization and 2) by membrane deformation that alters enzyme-spectrin interaction locally. Progress in the isolation and characterization of spectrin kinase and phosphospectrin phosphatase should lead to the resolution of major questions raised by previous work: the relationships between membrane-bound and cytoplasmic forms of the enzymes, the nature of their physical interactions with the membrane, and the regulation of their activities in defined cell-free systems.

[Drug interactions].

Every alteration of the effect of a drug within the human body due to substances introduced from outside are considered drug interactions. Several types of these mutual actions are differentiated. They can be influenced with varying success according to the site of the interaction. An unusual variant of the interactions sometimes appears in genetically induced disturbances in the metabolism of drugs.

Localization of the site of adenylylation of glutamine synthetase by electron microscopy of an enzyme-antibody complex.

Antibodies to the nucleosidel,N(6)-ethenoadenosine have been used to localize the site of adenylylation of the glutamine synthetase [L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2] of Escherichia coli. Antibodies were induced in rabbits by injection of a bovine albumin-ethenoadenosine conjugate. The resulting antisera strongly bound ethenoadenosine, its 5'-nucleotide, or protein conjugates of the nucleoside; little or no crossreaction was seen to adenosine, AMP, or the protein carrier. Ethenoadenylylated glutamine synthetase was prepared by modification of the enzyme by the E. coli adenylyltransferase, using etheno-ATP as a substrate. The ethenoadenylylated glutamine synthetase was precipitated by antibodies to ethenoadenosine in conjunction with goat anti-rabbit gamma globulin. Electron micrographs of reaction mixtures of ethenoadenylylated glutamine synthetase and anti-ethenoadenosine showed individual enzyme molecules complexed with one or more antibodies and pairs of enzyme molecules crosslinked by a single antibody. The approximate site of adenylylation was located from the apparent area of contact between enzyme and antibody. We conclude that the adenylylation sites are on the periphery of the bilayered hexagonal disc, offset by 15 +/- 10 degrees from the 2-fold axis of symmetry through a vertex of the hexagon and 20 +/- 10 A from the plane between the layers of the disc.

Transport of model compounds across the peritoneal membrane in the rat.

The absorption into the systemic circulation of compounds administered intraperitoneally in large volumes was investigated in the rat. The influence on absorption of molecular weight, lipid-water partition coefficient (K), and dissociation constant (pKa) was studied. Nine neutral compounds ranging in molecular weight from 18 to 2 million demonstrated absorptions that decreased with increasing molecular weight. Five compounds were tested with variable lipid partition (K) values (0.001--3.3) and the absorptions increased from 57 to 96% as the K values increased. A series of nine acids and bases covering a wide range of pKa values )0.9--9.9) were investigated. For the acids, absorption increased with increasing pKa value, while for the bases absorption decreased with increasing pKa. For both groups of compounds absorption was directly related to the extent of ionization at physiologic pH. As has been documented for other biological membranes, the peritoneal membrane in the rat was found to behave in a lipoid manner. Unionized or lipid-soluble compounds are absorbed to a greater extent than ionized or lipid-insoluble compounds, and neutral compounds are absorbed in relation to their molecular weights.

The peripheral platelet count in response to adrenergic alpha-and beta-1-receptor stimulation.

The aim of the present work was to investigate the effect of adrenergic alpha- and beta-1-receptor stimulation on the peripheral platelet count. The experiments were carried out on 8 healthy male volunteers using radioisotopically labelled platelets. 3 subjects received i.v. infusions of adrenaline (0.09 microgram X kg-1 X min-1) before and after the ingestion of 40 mg propranolol. In response to the first infusion there was an instant increase in the venous platelet-bound radioactivity (PBR) which amounted to 12% over basal value. This effect of adrenaline seemed to be potentiated by propranolol pretreatment. 5 subjects received i.v. infusions of the highly selective beta-1-receptor agonist H 133/22 (prenalterol, Hässle, Sweden). In response to a cumulative dose of 4.75 mg prenalterol a slight but significant (P less than 0.05) decrease in PBR occurred. It is concluded that alpha-receptor stimulation causes a depletion of platelets from the exchangeable splenic platelet pool resulting in a concomitant increase in the peripheral platelet count. Beta-receptor stimulation has an opposite effect on the spleen. The trapping of platelets by the spleen is mediated both via beta-1- and beta-2-receptors, but the effect of beta-2-receptor stimulation seems to predominate.

Difference in the effect of bucolome on the hepatic transport maximum of sulfobromophthalein and indocyanine green.

The effects of bucolome (BC, 1-cyclohexyl-5-n-butyl-2,4,6-trioxoperhydropyrimidine, a non-steroid antiinflammatory agent), a potent choleretic, on the maximal biliary excretion rates (Tm) of sulfobromophthalein (BSP) and indocyanine green (ICG) were investigated in rats. With continuous infusion of BSP or ICG, Tm and bile flow rate in control rats and rats given BC (40--100 min after 20 mg/100 g body weight i.p. injection) were compared. The BSP Tm was not significantly different in control and BC administered rats (BC rats), while the bile flow rate and endogenous bile salts excretion rate were significantly higher in BC rats. On the other hand, ICG Tm was significantly higher in BC rats, although ICG concentration in bile was significantly lower in BC rats. The rates of the rise in plasma concentration of the dyes were significantly lower in BC rats. However, the plasma concentration of ICG was significantly higher in BC rats at corresponding time intervals than in control rats throughout the experimental period. The difference in the effect of BC on the transport of these two dyes indicates variability in the regulatory mechanisms for hepatic anion transport.

Metabolism of arachidonic acid by platelets: utilization of arachidonic acid by human platelets in presence of linoleic and dihomo-gamma-linolenic acids.

In vitro human platelet prostaglandin synthesis has been studied from added radioactive arachidonic acid (i) as function of substrate concentration, (ii) as function of platelet concentration and (iii) as function of pH. Platelets, as in platelet rich plasma when labelled with arachidonic acid, washed and treated with thrombin, released radioactivity mainly from phosphatidylcholine and phosphatidylinositol. The released radioactivity was mostly accounted for by the formation of the previously identified oxygenation products of arachidonic acid. Platelet utilization or arachidonic acid was also studied in presence of linoleic and dihomo-gamma-linolenic acids, the two essential fatty acids known for antithrombotic effect. At its high concentrations linoleic acid decreased platelet cyclo-oxygenase activity as seen by a decreased formation of endoperoxides from arachidonic acid. Dihomo-gamma-linolenic acid was found to be a mutually competitive substrate with arachidonic acid for the platelet prostaglandin synthetase thus causing reduced utilization of arachidonic acid as shown by measuring the various oxygenation products of arachidonic acid. These two acids were utilized differently by platelet prostaglandin synthetase.

[Histamine and its role in peptic gastric diseases: the discovery of histamine-H2-receptor antagonists].

For the definition of histamine receptors the following prerequisites must be fulfilled: (1) Course of dose-response curves according to the mass-action law; (2) parallel displacement of these curves to the right in the presence of antagonists; (3) inhibition only by specific histamine antagonists; (4) slope of a Schild-plot not significantly different from unity. For H2-receptors these prerequisites could ideally be fulfilled, especially by the development of highly specific H2-receptor antagonists. However, this new class of compounds acts not only by mere competitive inhibition of histamine at its H2-receptor, but also by activating the metabolism of this secretagogue. A further explanation of the action of H2-receptor antagonists in the treatment of chronic duodenal ulcer may be given by studying the pathogenetic role played by histamine in the development of this disease: duodenal ulcer patients showed an increased liberation of histamine from mucosal mast cell stores as well as a decreased activity of histamine methyltransferase (i. e. longer action of histamine!). The rise in histamine content and histamine methyltransferase activity after vagotomy may be the basis for a biochemical explanation of the acid-reducing effect of this operation.

Illicit drug use among urban adolescents. A decade in retrospect.

Over the past decade (1967 to 1977), 76,000 adolescents were screened for a history and somatic signs of illicit drug use at a detention center for juveniles and at an adolescent inpatient unit of a university-affiliated hospital. Dramatic changes in the patterns of drug abuse are reported. Opiate use was prominent in the first half of the decade with a peak in 1970 to 1971 and marijuana use more prominent in the last five years. Inhalant abuse as represented by glue and halogenated cleaning fluids was documented only early in the decade, while the existence of stimulant and depressant abuse follows still other patterns over the decade. Hospital admissions for serious somatic complications of illicit drug use, namely, overdose, drug-related death, hepatic coma, detoxification, and viral hepatitis, were correlated only with trends in the use of opiates. Awareness of drug abuse patterns among adolescents is important for the health professional so that complications can be diagnosed and treated and educational efforts properly directed.

Continuous intrapartum monitoring of fetal scalp pH.

Forty patients were monitored intrapartum with a continuous fetal scalp tissue pH electrode in the mean time of 2.39 hours. The monitoring records were considered "accurate" with good correlation to the intermittent fetal scalp capillary pH values in 76.9 per cent. The correlation coefficient was 0.74. The "accuracy" improved in the latter 23 cases to 87 per cent with a correlation coefficient of 0.82. This improvement was probably due to modification of the application technique, as well as to a new calibration method at 37 degrees. Continuous fetal scalp pH monitoring was clinically useful in 65 per cent, it was partially useful in 20 per cent, but of no value in 15 per cent of the patients studied. There were no apparent maternal complications with the use of this technique and 38 of the infants had no sequelae. Two infants had complications: one developed inflammation of the electrode site. This was treated with antibiotics. One electrode broke during the application and a fragment of the electrode tip remained in the fetal scalp. All the infants were grossly normal and there was a good correlation between the continuous pH readings and the immediate neonatal outcome.

[The solubilizer of diazepam (valium)--its action on respiration (author's transl)].

Investigations carried out in volunteers showed that when compared with physiological saline the solubilizer of diazepam caused hyperventilation. Among the constituents of the solubilizer propylene glycol and ethyl alcohol have to be excluded, likely benzyl alcohol was due to the respiratory response. Benzyl alcohol has local anesthetic properties and its action on respiration has never been examined up to now. Therefore a further study was performed to compare ampoule solutions of diazepam with or without benzyl alcohol in the solubilizer used. The solutions were given intravenously, the dose of diazepam was 0.35 mg/kg, that of benzyl alcohol 1.1 mg/kg respectively. The measurements showed that solutions containing benzyl alcohol produced a statistical significant increase of respiratory rate and of minute volume. In case of intravenous administration of Valium using ampoule solutions diazepam and in addition a second active substance are applicated. The latter defined as benzyl alcohol has stimulating effects on respiration.

[Osmotic colloidal pressure: measurement and clinical importance].

Knowledge of osmotic pressure has long existed and its practical use in the treatment of patients under intensive care has been widely developed over the past few years following the introduction of simple electronic osmometers. It is related more to plasma albumin than to globulins. It varies in relation with a certain number of physiological factors and in different pathological circumstances. The value of OP and above all of the difference OP--PCP in the prevention, diagnosis, treatment and prognosis of pulmonary oedema has been clearly demonstrated in several recent studies. Its value in relation to total serum proteins is that it takes into account any possible dysproteinaemia and that it is directly expressed in units of pressure which makes possible the calculation of the OP--PCP difference which represents the difference between the only OP--PCP difference which represents the difference between the only two intravascular forces which participate in fluid exchanges at the level of the pulmonary capillary.

Adenylate cyclase in bloodstream forms of Trypanosoma (Trypanozoon) brucei sp.

1. The adenylate cyclase in Trypanosoma brucei is located in the plasma membrane. 2. A partial kinetic analysis of the properties of the enzyme revealed a Km for ATP of 1.75 mM and a Km for Mg2+ of 4mM. 3. At low concentrations, Mg2+ activated the enzyme directly in addition to its effect of lowering the concentration of inhibitory free ATP species. 4. At high concentrations, Mg2+ inhibited the enzyme. Furthermore, the enzyme was inhibited at any Mg2+ concentration if the concentration of ATP exceeded that of Mg2+. 5. The opposing effects of Mg2+ at low and high concentrations would be consistent with more than one binding site for Mg2+ on the enzyme. 6. A study of the patterns of product inhibition revealed little or no effect of 3':5'-cyclic AMP, but a profound inhibition by pyrophosphate, which was competitive with respect to ATP (Ki 0.135 mM). This result suggests that the substrate-binding domain on T. brucei adenylate cyclase interacts mainly with the triphosphate portion of the ATP molecule. 7. The enzyme activity was unaffected by the usual mammalian enzyme effectors glucagon, adrenaline, adenosine, GTP and guanyl-5'-yl imidodiphosphate. 8. The enzyme was not activated by fluoride, instead a powerful inhibition was found. The enzyme was also inhibited by relatively high concentrations of Ca2+ (1 mM).

Yellow lupin (Lupinus luteus) aminoacyl-tRNA synthetases. Isolation and some properties of enzyme-bound valyl adenylate and seryl adenylate.

As a continuation of our studies on plant (yellow lupin, Lupinus luteus) aminoacyl-tRNA synthetases we describe here formation and some properties of valyl-tRNA synthetase-bound valyl adenylate (EVal(Val-AMP)) and seryl-tRNA synthetase-bound seryl adenylate (ESer(Ser-AMP)). Valyl-tRNA synthetase-bound valyl adenylate was detected and isolated by several approaches in the pH range 6--10. In that range inorganic pyrophosphatase increases the amount of valyl adenylate by factor 1.8 regardless of pH. 50% of valine from the EVal(Val-AMP) complex isolated by Sephadex G-100 gel filtration was transferred to tRNA with a rate constant greater than 4 min-1 (pH 6.2, 10 degrees C). The ratio of valine to AMP in the enzyme-bound valyl adenylate is 1 : 1 and it is not changed by the presence of periodate-oxidized tRNA. In contrast to enzyme-bound valyl adenylate, formation of ESer(Ser-AMP) is very sensitive to pH. Inorganic pyrophosphatase increases the amount of seryl adenylate by a factor 6 at pH 8.0 and 30 at pH 6.9 60% of serine from the ESer(Ser-AMP) complex was transferred to tRNA with a rate constant greater than 4 min-1 (pH 8.0, 0 degrees C). The ratio of serine to AMP in the enzyme-bound seryl adenylate is 1 : 1. The rate of synthesis of the enzyme-bound aminoacyl adenylates was measured by ATP-PPi exchange. Michaelis constants for the substrates of valyl-tRNA and seryl-tRNA synthetases in ATP-PPi exchange were determined. Effects of pH, MgCl2 and KCl on the initial velocity of aminoacyl adenylate formation are described. For comparison, catalytic indices in the aminoacylation reactions catalyzed by both lupin enzymes are given and effects of pH, MgCl2 and KCl on tRNA aminoacylation are presented as well. Under some conditions, e.g. at low pH or high salt concentration, lupin valyl-tRNA and seryl-tRNA synthetase are active exclusively in ATP-PPi exchange reaction.

[Electrophoretic analysis of substrate specificity of wheat alcohol dehydrogenases].

Electrophoresis in polyacrylamide gel slabs has been used to study the isoform composition and substrate specificity of alcohol dehydrogenases in the embryo and young seedlings of the diploid wheat Triticum monococcum L., the tetraploid T. dicoccon (Schrank) Schuebl and the hexaploid T. spelta L. Three alcohol dehydrogenases of different substrate specificity and developmental pattern were distinguished: a) the NAD-dependent alcohol dehydrogenase, catalyzing the oxidation of different primary and secondary aliphatic and aromatic alcohols, as well as certain compounds with several hydroxyl groups (tris, triethanolamin) and revealing, after electrophoresis, one major band in the diploid wheat and three bands in both polyploid wheats; b) the NADP-dependent aromatic alcohol dehydrogenase (substrate--cinnamic alcohol), revealing, after electrophoresis, one major fast moving band in the diploid wheat and two bands in polyploid wheats; c) an aromatic alcohol dehydrogenase (2-3 bands after electrophoreis) with no specificity to the cofactors (NAD or NADP).

[Interaction of aliphatid alcohols with cytochrome P-450 from rat liver microsomes].

The interaction of alyphatic alcohols and cyclohexanol with cytochrome P-450 in microsomes has been investigated. All alchohols induced the modified 11 type spectral changes by mixing with microsomes. These changes are characterized by lambdamax = 412 and lambdamin = 380-382 nm in difference spectra. The dissociation constants of the alcohol cytochrome P-450 complexes are determined. On this dissociation constants influence pH and Triton X-100 presence. The interaction of the alcohols with cytochrome P-450 in phosphate buffer pH = 6,0 in the detergents absence is characterized by one dissociation constant for MeOH, EtOH, n-BuOH and cyclohexanol and by two dissociation constants for i-PrOH, i-BuOH and tert.-BuOH. The interaction of the alcohols with cytochrome P-450 in Tris-HCL-buffer (pH 7.5) in the Triton X-100 presence is characterized for all above alcohols by the dissociations constants, which are described by Taft equation with coefficient rho =-1.55. This fact confirms the interaction of alcohols HO-groups with heme iron of cytochrome P-450. The scheme of interaction of alcohols with cytochrome P-450 is discussed.

Prevention and reversal of isolation-induced systolic arterial hypertension in rats by treatment with beta-adrenoceptor antagonists.

1. Rats were made hypertensive by 5 days of continuous isolation in glass metabolic cages; in the text "hypertensive" means having a systolic blood pressure greater than 145 mmHg. 2. Daily intraperitoneal injections of either propranolol (5 mg/kg) or atenolol (5 mg/kg) reduced systolic blood pressure within 3 days and the systolic blood pressure remained low provided that the treatment was continued. 3. Treatment with metoprolol also lowered the systolic blood pressure of isolated rats but only when a larger dose (10 mg/kg) was given. 4. Systolic blood pressure returned to hypertensive levels following withdrawal of treatment after 15 days of isolation. However, following cessation of treatment after 27 days of isolation, the systolic blood pressure did not rise. 5. Rats given propranolol in the drinking water (intake equivalent to a daily dose of 5 mg/kg) before and during isolation did not develop hypertension. 6. The possibility that suppression of sympathetic function by the beta-adrenoceptor antagonists was responsible for these changes is discussed.

Effects of fluoride on in vitro calcification of tendon matrix.

Ca2+ and Pi uptake induced in vitro by a collagenous matrix derived from bovine tendon is inhibited by 1 X 10(-6) to 2 X 10(-5) M NaF and stimulated by 2 X 10(-5) to 2 X 10(-3) M NaF. Fluoride uptake occurs only over the latter concentration range. The uptake of Ca2+, Pi, and F-1 progresses toward a limiting extent at which the molar Ca/P and Ca/F values are 1.6 to 1.7 and 4.5 to 5.7, respectively. Although the matrix-bound mineral, previously formed in the absence of NaF, readily undergoes dissolution when exposed to a Ca2+- and P-free medium of pH less than 7.4, the bound mineral phase formed in the presence of NaF does not. We conclude that fluoroapatite is the primary matrix-bound mineral. The uptake of fluoride, Ca2+. amd Pi by both uncalcified and previously calcified matrices is inhibited by methylenediphosphonate and by phosphonoacetate as is calcification in the absence of NaF. Kinetic studies indicate that formation of a CaP complex precedes the uptake of F-1 and suggest that F-1 and OH-1 compete for interaction with that CaP complex during the calcification process. We concluded that fluoroapatite formation induced by the collagenous matrix occurs by a multistep pathway comparable to that proposed previously for hydroxyapatite formation.

Glucose-6-phosphate dehydrogenase deficiency in Sicily. Incidence, biochemical characteristics and clinical implications.

This report deals with the incidence, type and clinical implications of G6PD deficiency in Sicily. Of 3347 male subjects examined, 56 were deficient in G6PD. They were distributed throughout the island. The G6PD levels in RBC were almost zero; in leukocytes, platelets and saliva they were found to be 26%, 18% and 16%, respectively, of controls. The Michaelis constant for NADP and G6PD was lower than for controls. Conversely, the utilization of the analogous Ga16P and 2dG6P was higher. The thermostability of the enzyme was lower and the pH optima (6.5 and 9.5) were different from the controls. An identical electrophoretic pattern was found both in normal and deficient subjects. This pattern is superimposable on that described as Mediterranean variant. The analysis among 270 subjects admitted to our Clinic with hemolysis due to G6PD deficiency demonstrated that the most frequent disease is favism, followed by neonatal jaundice, while hemolysis due to drugs is very rare. Ingestion of fresh fava beans was the most frequent cause of favism, but cases occurred after breast feeding and inhalation of pollen.

Acute effects of piretanide in normal subjects.

Acute clearance studies were performed in normal subjects to assess the actions of the new diuretic, piretanide, on renal function. The drug increased both glomerular filtration rate and effective renal plasma flow in roughly proportionate amounts, so that filtration fraction did not change. In a dosage of 2 to 3 mg, it induced an increase in sodium excretion of almost 13% of filtered load, and there was an associated 2- to 3-fold increase in potassium excretion. The abstraction of solute-free water from the collecting duct was markedly reduced, but the drug induced no significant decline in the generation of free water. The rate of bicarbonate excretion, as well as that of titratable acid and ammonium, was increased approximately proportionately so that there was no increase in urinary pH or net hydrogen ion excretion. There was no phosphaturia, a unique finding, since all other drugs and maneuvers that cause a bicarbonate diuresis are also phosphaturic. Piretanide increased calcium excretion by approximately 19% of filtered load. The data suggest that the drug acts largely in the ascending limb of the loop of Henle and that it also affects the proximal tubule. Despite its sulfonamide structure, none of the drug's effects appear to be related to inhibition of carbonic anhydrase.

Release of nickel from cooking utensils.

The release of nickel to boiling water from new and used saucepans of different material was measured. No nickel was released from aluminium, teflon and enamel. Certain amounts of nickel were released from stainless steel, but only at acid pH.

beta-Galactosidases in mouse duodenal mucosa.

beta-Galactosidase activity, in fetal mice, first appears at 16 days of gestation and has a pH optimum of 4. In postnatal development the enzyme activity of cell homogenates tends to show bimodal pH at 4 and 5.6. There are two molecular forms of the enzyme, separable both by molecular-sieve chromatography and electrophoresis. One of the molecular forms of the enzyme is active over a wider range of pH (3.2-6.2) and has half as much activity at 5.6 as it does at 4. This isoenzyme is continuously present in both fetal and postnatal stages. The second isoenzyme first appears at birth, is active over a narrower range of pH (4.6-6.2) and inactive at PH 4. The bimodal pH optima observed in postnatal stages in the cell homogenates, appears to be due to the combined activity of the two molecular forms. In isolated brush border membranes, isoenzyme 2 is the only one present. The other organelles (mitochondria, microsomes, lysosomes, nuclei and cytoplasm) have variable proportions of both isoenzymes, as indicated by the activity ratio at pH 4/5.6.

Affinity purification and properties of cathepsin-E-like acid proteinase from rat spleen.

A unique acid proteinase different from cathepsin D was purified from rat spleen by a method involving precipitation at pH 3.5, affinity chromatography on pepstatin-Sepharose 4B and concanavalin A-Sepharose 4B, chromatography on Sephadex G-100 and DEAE-Sephacel, and isoelectric focusing. A purification of 4200-fold over the homogenate was achieved and the yield was 11%. The purified enzyme appeared to be homogeneous on electrophoresis in polyacrylamide gels. The isoelectric point of the enzyme was determined to be 4.1-4.4. The enzyme hydrolyzed hemoglobin with a pH optimum of about 3.1. The molecular weight of the enzyme was estimated to be about 90000 by gel filtration on Sephadex G-100. In sodium dodecylsulfate polyacrylamide gel electrophoresis, the purified enzyme showed a single protein band corresponding to a molecular weight of about 45000. The hydrolysis of bovine hemoglobin by the enzyme was much higher than that of serum albumin. Various synthetic and natural inhibitors of the enzyme were tested. The enzyme was inhbited by Zn2+, Fe3+, Pb2+, cyanide, p-chloromercuribenzoate, iodoacetic acid and pepstatin, whereas 2-mercaptoethanol, phenylmethyl-sulfonyl fluoride and leupeptin showed no effect.

Ovarian and peripheral venous blood prostaglandin F2alpha levels in pregnant and nonpregnant women.

Prostaglandin F2alpha (PGF2alpha) was measured by radioimmunoassay in human blood plasma collected from both ovarian veins and from a peripheral vein in eight pregnant and seven nonpregnant women. In the pregnant women, significantly higher levels of PGF2alpha were found in venous blood plasma taken from the ovary which contained the corpus luteum than were found in either the ovarian vein blood plasma from the opposite ovary or in the blood plasma from a peripheral vein. Thus, PGF2alpha levels in the venous blood plasma from the ovary without a corpus luteum were not significantly different from those found in the peripheral venous blood plasma. In seven nonpregnant women, significantly higher levels of PGF2alpha were found in the venous blood plasma taken from the ovary which contained a corpus luteum than were found in the blood plasma taken from a peripheral vein. The former levels did not differ significantly from those found in the venous blood plasma taken from the ovary without a corpus luteum in these nonpregnant women. Thus, in nonpregnant women a significant difference was found between PGF2alpha levels in the venous blood plasma from the ovary without a corpus luteum and those in peripheral venous blood plasma.

Effect of leukocyte hydrolases on bacteria. X. The role played by leukocyte factors, cationic polyelectrolytes, and by membrane-damaging agents in the lysis of Staphylococcus aureus: relation to chronic inflammatory processes.

A heat-stable factor present in extracts of human blood leukocytes is capable of lysing young Staphylococcus aureus at pH 5.0. Lysis is characterized by breakdown of cell-wall components as judged by electron microscopic and biochemical analysis. The leukocyte extracts can be replaced by a variety of agents known to injure cell membranes, e.g., leukocyte cationic protein histone, polymyxin B, colimycin, phospholipase A, and lysolecithin. The mechanisms by which all these agents bring about the degradation of the staphylococcal walls was studied. By using 14C-labeled cell walls devoid of cytoplasmic structures, it was demonstrated that none of the above-mentioned agents had a direct lytic effect on purified cell walls. On the other hand, when any of these agents first interacted with intact staphylococci, a factor (presumably an autolysin) was generated that directly lysed the cell walls. Lysis of cell walls in the presence of intact staphylococci used as a source of autolysin was strongly inhibited by a variety of anionic polyelectrolytes such as heparine and liquoid. The possible role played by bacterial autolysins in the generation of microbial cell-wall components capable of triggering chronic inflammation is discussed.

Metabolism of phenol and resorcinol in Trichosporon cutaneum.

Trichosporon cutaneum was grown with phenol or resorcinol as the carbon source. The formation of beta-ketoadipate from phenol, catechol, and resorcinol was shown by a manometric method using antipyrine and also by its isolation and crystallization. Metabolism of phenol begins with o-hydroxylation. This is followed by ortho-ring fission, lactonization to muconolactone, and delactonization to beta-ketoadipate. No meta-ring fission could be demonstrated. Metabolism of resorcinol begins with o-hydroxylation to 1,2,4-benzenetriol, which undergoes ortho-ring fission yielding maleylacetate. Isolating this product leads to its decarboxylation and isomerization to trans-acetylacrylic acid. Maleylacetate is reduced by crude extracts to beta-ketoadipate with either reduced nicotinamide adenine dinucleotide or reduced nicotinamide adenine dinucleotide phosphate as a cosubstrate. The enzyme catalyzing this reaction was separated from catechol 1,2-oxygenase, phenol hydroxylase, and muconate lactonizing enzyme on a diethyl-aminoethyl-Sephadex A50 column. As a result it was purified some 50-fold, as was the muconate-lactonizing enzyme. Methyl-, fluoro-, and chlorophenols are converted to a varying extent by crude extracts and by purified enzymes. None of these derivatives is converted to maleylacetate, beta-ketoadipate, or their derivatives. Cells grown on resorcinol contain enzymes that participate in the degradation of phenol and vice versa.

Mannosyl transfer by membranes of Aspergillus niger: mannosylation of endogenous acceptors and partial analysis of the products.

A smooth membrane fraction of Aspergillus niger catalyzed the transfer of mannose from GDP-mannose to endogenous lipid and protein acceptors. The mannolipid was acidic, as judged by diethylaminoethyl-cellulose chromatography, and had a mobility similar to ficaprenyl phosphate on thin-layer chromatograms. Mannose transfer occurred optimally at pH 6.5 to 7.5 and required Mn(2+) for use of the protein as acceptor, but either Mn(2+) or Mg(2+) with the lipid as acceptor. Glycopeptides of the mannosylated protein ([(14)C]gly) and of an alpha-glucosidase (alpha-glu) secreted by the organism were produced by Pronase digestion and separation of the products on Sephadex G-25. Because ovalbumin has a carbohydrate composition similar to that of alpha-glu and because the carbohydrate structure of ovalbumin is known, ovalbumin glycopeptides (Ov) were similarly obtained and used as standards in determining carbohydrate structures. Oligosaccharide chains of [(14)C]gly, alpha-glu, and Ov were obtained by treatment of the respective glycopeptides with endo-beta-N-acetylglucosaminidase, reduction with NaBT(4), and concanavalin A-Sepharose chromatography. The (3)H-labeled oligosaccharides obtained were subjected to the following treatments: (i) digestion with alpha- and beta-mannosidases, (ii) Smith degradation, and (iii) acetolysis. Subsequently, changes in paper chromatographic mobilities were detected. Also, alpha-glu was permethylated, and the partially methylated alditol acetates were analyzed by gas-liquid chromatography. The resultant proposed structure shows that the oligosaccharide chain of alpha-glu is almost identical to that of an Ov chain, while [(14)C]gly has a structure which is probably the same as that of alpha-glu. It is suggested that the transferase(s) involved in [(14)C]gly synthesis in vitro may be responsible for glycosylation of secreted enzymes.

Kinetic study of soybean beta-amylase. The effect of pH.

1) Beta-Amylase [EC 3.2.1.2] was prepared from defatted hawk eye soybean flour. The enzyme concentration dependence of the initial velocity for the hydrolytic reaction was investigated at pH 5.4 in the range of the enzyme concentration from 6.6 x 10(-10) M to 5.0 x 10(-6) M. It was found that the initial velocity was proportional to the enzyme concentration in this range. 2) The hydrolyses of maltodextrin (DPn = 74.4) and soluble starch catalyzed by soybean beta-amylase were investigated in the pH range from 3.0 to 9.1 at 25 degrees C, and the Michaelis constant, Km, and the maximum velocity, V, for each substrate were determined at each pH. The pH-rate profile showed a bell-shaped curve, and the pH "optimum" was at 5.85. From Dixon plots of V and V/Km, the pK values were found to be 3.5 and 8.2 for the free enzyme, and 3.5 and 8.5 for the enzyme-substrate complex. The pH-rate profile in the presence of 25% methanol (v/v) was also obtained at alkaline pH. The pKe values were the same as those in the absence of methanol. Based on these results, it was estimated that the ionizable acidic group was an amino group and the basic group was a carboxyl one.

Ca2+-induced bioluminescence in Renilla reniformis. Purification and characterization of a calcium-triggered luciferin-binding protein.

A Ca2+-triggered luciferin-binding protein (BP-LH2) from the bioluminescent marine coelenterate, Renilla reniformis, has been purified by conventional methods. One kilogram of processed animals yields approximately 2.7 mg of pure protein with an overall yield of 55%. Physicochemical studies show that BP-LH2 is a globular protein containing one single polypeptide chain with one disulfide bond. Ultracentrifugation studies, amino acid analysis, and sodium dodecyl sulfate-gel electrophoresis show that BP-LH2 has an average molecular weight of 18,500. BP-LH2 has a Stokes radius of 23 A, a sedimentation coefficient, S020,w, of 2.3 S, and an isoelectric point of 4.3. The acidic nature of the protein was confirmed by amino acid analysis, which showed that 27% of the residues are acidic. The protein contains no carbohydrate, phosphate, or tryptophan. There is one noncovalently bound molecule of coelenterate type luciferin resulting in distinct protein spectral properties with absorption maxima at 276 nm (epsilon 0.1% 276 = 1.31) and 446 nm (episoln 0.1% 446 = 0.47) and a fluorescence emission at 520 nm (uncorrected). In the presence of Ca2+, BP-LH2 will react with Renilla luciferase to give the characteristic in vitro blue bioluminescence. Ca2+ binding produces a distinct change in the spectral properties of BP-LH2 including a 4-fold enhancement of tyrosine fluorescence at 332 nm and a 5-fold fluorescence enhancement at 520 nm. In addition, the visible absorption maximum shifts from 446 nm to 420 nm. The fluorescence enhancement at 320 nm occurs over the range from 1 to 10 micrometer Ca2+. BP-LH2 has two Ca2+-binding sites with an estimated Kd of 0.02 micrometer, in 10 muM Tris at pH 7.2. BP-LH2 was compared to several well studied Ca2+-binding proteins and was found to possess similar Ca2+-binding and physicochemical properties. This study clearly demonstrates that BP-LH2 is capable of triggering a bioluminescent flash in response to an intracellular Ca2+ transient.

Synthesis and use of a new spin-labeled analogue of ADP with platelet-aggregating activity.

A new spin-labeled derivative of ADP, 2-(4-acetamido-2,2,6,6-tetramethylpiperidine-1-oxyl)thioadenosine-5'-diphosphate, has been synthesized. The compound causes both the reversible and irreversible phases of aggregation of human blood platelets at concentrations similar to those required for similar phases of aggregation by ADP itself. The spin-labeled ADP also rivals ADP as a substrate for pyruvate kinase. The interaction of intact human blood platelets and of isolated platelet membranes with the platelet-aggregating spin-labeled derivatives of ADP has been studied. The dramatic decrease in the ESR signal of the spin label is primarily due to chemical reduction of the nitroxide, rather than immobilization of the label. When platelets and spin-labeled ADP are mixed, a rapid burst of nitroxide reduction occurs, followed by a much slower reduction similar in time course to that seen for other spin labels. The rapid burst of reduction, but not the slow reduction, is inhibited by adenosine, an inhibitor of ADP-induced platelet aggregation, and by sulfhydryl-blocking agents. Experiments conducted with Ellman's reagent and platelet membranes or washed platelets revealed a 10 to 30% increase in the number of reactive membrane sulfhydryl groups when ADP was present. These results indicate that there is an increase in the number of reactive sulfhydryl groups on the platelet surface when platelets or membranes are stimulated by ADP.

Evidence for a high molecular weight factor(s) in serum which increases alkaline phosphatase specific activity in HeLa.

HeLa (substrain Ho) grown in serum free medium showed an increase in the specific activity of alkaline phosphatase when fetal calf serum (10%) was added to the medium (9.7 nmoles/sec/mg protein to 86.8). Under the same conditions, eight intracellular enzymes showed no increase in activity. Similar results were obtained using a different serum or medium, and with a second strain of HeLa (substrain ATC). For a given set of growth conditions, the effect of serum was dependent on its concentration and required one or more culture generations to develop. The type of isozyme expressed did not change. Neither zinc nor a total serum lipid extract would substitute for serum. The enzyme expressed by HeLaHo was not induced by prednisolone, while that in HeLaATC was. However, for cells grown in excess prednisolone without serum, the specific activity was 25% of that found for cells grown with prednisolone and serum. Cortexolone, an antagonist of prednisolone, was without effect for HeLaHo grown in A3 medium with or without serum. The serum factor had the following characteristics. It was not lost on dialysis, treatment with DNase and RNase, or removal of lipoproteins. It was reduced after heating by 65% and after treatment with Pronase by 82%. The data are interpreted to indicate the presence of a factor (s) in serum, probably a protein, which is involved in stimulating alkaline phosphatase specific activity.

The metabolism of floctafenin in man and rodents.

Floctafenin (FFn), 2,3-dihydroxypropyl--N--(8--trifluoromethyl--4--quinolyl) anthranilate, a new nonnarcotic analgesic drug, was studied in man, mice, and the isolated perfused rat liver. In all species the drug is rapidly hydrolyzed to floctafenic acid (FFa). In seven volunteer subjects who each received a single oral dose of 400 mg floctafenin on an empty stomach, the blood concentration of FFa usually reached a maximum between 1 and 2 hours (mean 1.57 +/- 1.28 microgram/ml at 1.5 hours) and declined over the next 6 hours. Eight hours after drug administration the mean concentration of FFa in the blood of the volunteers was 0.1 +/- .05 microgram/ml. Approximately 25 per cent of the administered dose of floctafenin was recovered as FFa and hydroxy-FFa in the urine collected from each subject for 48 hours after drug administration. In mice each having received a single intraperitoneal dose of floctafenin (2 mg), the concentration of floctafenin declined by about 50 per cent in 15 minutes, and this decline was accompanied by a rise in the concentration of FFa that remained constant for 3 hours. The analgesic effect observed after administration of floctafenin to humans is likely to be mediated by its major metabolite, FFa. In these volunteers no free floctafenin was detected in the blood.

Immunological studies of T-cell receptors. II. Limited polymorphism of idiotypic determinants on T-cell receptors specific for major histocompatibility complex alloantigens.

These studies explore the extent of genetic polymorphism in the expression of anti-MHC receptors by T cells in different strains of rats. This question was approached with the use of the model of specifically induced GVH resistance in F1 rats which has been shown to reflect a specific T-cell mediated immune response against parental strain T cell anti-MHC receptors specific for host alloantigens. When A/B F1 rats derived from MHC incompatibile matings are immunized with lymphocytes from one parental strain (A they display a specific resistance to anti-B GVH reactivity caused by T cells from that parental strain, but not anti-AGVH reactions from the other. In addition, they resist anti-B GHV reactivity by T cells from third-party donors (C, D, E,...), a finding taken to indicate that the idiotypes of anti-MHC receptors on T cells, recognized by other T cells, show little or no polymorphism. This conclusion suggests that anti-MHC receptors are shared in the species and may be encoded, at least partially, by germ-line genes.

Change in brain guanosine 3',5'-monophosphate (cGMP) content by thyrotropin-releasing hormone.

Thyrotropin-releasing hormone (TRH) causes a significant increase in rat cerebellar guanosine 3',5'-monophosphate (cGMP) content after parenteral administration. A smaller but still significant increase in cGMP was also observed in brain stem, whereas no significant changes were observed in cGMP in other gross brain regions or in adenosine 3',5'-monophosphate in any brain region. TRH also caused a similar increase in cerebellar cGMP content in hypophysectomized rats indicating that the increase is independent of an intact pituitary. The time course of cGMP elevation in the cerebellum after administration of 10 mg/kg of TRH i.v. showed a peak at 2.5 to 5.0 min followed by a less rapid decrease. The time course of TRH immunoreactivity in the same cerebellar homogenates roughly paralleled these changes. Only those TRH analogs which were previously shown to antagonize pentobarbital sleeping time in mice caused an elevation in cGMP content in the cerebellum. TRH also caused a significant increase in cerebellar cGMP in rats pretreated with phenobarbital and chlordiazepoxide. The TRH-induced increase in cerebellar cGMP was not affected by cerebellar climbing fiber lesions caused by 3-acetylpyridine nor blocked by haloperidol, suggesting that TRH acts by mechanisms different from harmaline or apomorphine in raising cerebellar cGMP.

[Metabolism of carbromal during detoxication with combined hemoperfusion and hemodialysis (author's transl)].

Combined charcoal hemoperfusion and hemodialysis was performed on three occasions in two patients with severe carbromal intoxication. The concentration of carbromal, its organic metabolites and of bromide was determined in arterial blood before and after passage of the charcoal column and behind the dialyzer cartridge. Results show a rapid metabolic degradation of carbromal including cleavage of bromide. Besides carbromal and its main metabolite 2-brome-2-ethylbutyramide (=carbromide) debromised organic metabolites appear to be responsible for the severity of the intoxication. Bromide plays no role in the pathogenesis of acute intoxication. Carbromal and its organic metabolites are eliminated through a large surface dialyzer almost as effectively as with charcoal hemoperfusion. The effectivity of detoxication is enhanced by the combination of both procedures. Free bromide is not adsorbed at charcoal but is readily dialyzable. Values of carbromal, calculated out of bromide levels, do not correspond to directly measured blood levels. Treatment of carbromal intoxication with combined hemoperfusion-hemodialysis should be performed early in all severe, risky or otherwise complicated cases until the patients awakens.

Beta-D-glucosidase in fractions from rat brain.

Beta-Glucosidase activity has been determined in homogenate and in centrifugation fractions of 7-day-old and adult rat brain; maximum activity was found at pH 4 and pH 5. Of the adult brain, more than 50% of the activity was concentrated in the 800-g sediment fraction (P1), while in the brain of 7-day-old rat about 20% was found in the corresponding fraction. The activity maximum in all fractions after a 2% Triton X-100 treatment occurs at pH 5. Addition of Triton to adult brain homogenate enhances the activity, but this stimulation is less than the sum of the activities observed at pH 4 and pH 5 in the absence of Triton. Triton addition to brain homogenate of 7-day-old rat results in a fall in activity at pH 4 and in a maximum at pH 5. In rat brain homogenate subjected to sonication, a loss of activity is observed at pH 4, scarcely at pH 5; the activity loss is completely abolished and turned into an increase under the influence of Triton. This increase equals the level obtained when Triton is added to an untreated brain homogenate. Sonication of rat brain homogenate leads to changes in the distribution pattern; about 25% of the activity of the adult brain is found in the P1 fraction compared to 50% in the corresponding fraction of the untreated brain. Fractionation of a sonicated brain homogenate from adult rat reveals that at pH 4 most activity (52%) is concentrated in the 20,000-g pellet (P2), 23% in supernatant fluid (S2); at pH 5 the opposite is observed; most activity (49%) is found in the 20,000-g supernatant (S2) and 23% in the 20,000-g pellet (P2). In the presence of Triton the activity of the sonicated brain homogenate of adult rat increases; this stimulation roughly equals the sum of the corresponding activities measured at pH 4 and pH 5 in the absence of Triton.

Studies on the mechanisms of central action of alpha-adrenergic receptor blocking agents.

The article summarizes the results of experiments, partly published, on the central action of alpha-adrenergic receptor blocking drugs given systemically in rats. Phenoxybenzamine, phentolamine and aceperone increase utilization of noradrenaline (NA in whole brain and antagonise NA--stimulated formation of cyclic AMP in brain cortical slices. They also counteracts locomotor hyperactivity and flexor reflex stimulation induced by NA receptor agonists. The investigated alpha-adrenolytics do not antagonize brain dopamine (DA) or acetycholine (ACh) receptor stimulation, although they weakly reduce the effects of central serotonin (5-HT) system activation. It is concluded, that phenoxybenzamine, phentolamine and aceperone block directly central NA receptors. They do not block DA or ACh receptors in the brain but they are weak antagonists of 5-HT system stimulation. The investigated compounds decelerate utilization of DA and increase those of ACh in the brain, these effects seem to be secondary to NA system hypofunction.