Pneumococcal isolations from patients with pneumonia and control subjects in a prepaid medical care group.

The incidence of pneumococcal infections and pneumonia among the 150,000 members of a prepaid medical care group in Seattle was assessed by an intensive study of outpatients for one year from June 1971 through May 1972. Respiratory specimens (sputum, pharyngeal, and nasal) from patients with pneumonia or febrile respiratory disease and from age- and sex-matched control subjects were cultured for pneumococci on media and in mice. Total rate of pneumonia was 11 per 1,000. Twenty-four per cent of patients with pneumonia and 12 per cent of control subjects carried pneumococci. The patients with pneumonia from whom the pneumococcus was isolated did not differ clinically on average from those without such isolates. Lobar and bacteremic pneumonia were extremely rare. The rate of pneumococcal infections and carriers was highest in the spring. Although young children had the highest rate of pneumonia from all causes there was no difference in the isolation rate between ill children and control children. The highest rate attributable to pneumococci was in those larger than 40 years of age. Pneumococcal carrier rates were much lower than those reported in the 1930s. Thirty-nine per cent of patients with pneumonia had evidence of viral or mycoplasmal infection, as determined primarily by serologic methods. Pneumococcal pneumonia was not a serious problem in this average, employed population in Seattle.


Effects of dimethyl sulfoxide on subunit proteins.

The effects of DMSO are thought to result from the formation of hydrogen bonds with proton-donor groups on biopolymers, which are stronger than those formed with water. Since DMSO contains methyl groups, however, effects on hydrophobic bonding in proteins could be expected at higher DMSO levels. Our studies of the effects of DMSO on model subunit proteins can be interpreted in the above terms. At a concentration of 20% or less, DMSO changed glutamate dehydrogenase into the inactive monomer and the effects were fully reversible with the activator (ADP). Higher DMSO levels resulted in irreversible inactivation. The predominant effect noted on beta-glucuronidase was irreversible inactivation by 20% or more DMSO at 37 degrees C. Purified beta-glucuronidase exhibited an activation in 20% DMSO at high substrate levels; this resulted from an apparent substrate inhibition in the absence of DMSO. DMSO inhibited the clotting of fibrinogen by purified thrombin, but the major effect appeared to be due to competition between thrombin and DMSO for binding sites on fibrinogen. These effects appear to be largely due to interactions between DMSO and hydrophobic bonding in fibrinogen, although DMSO also appears to interfere with the aggregation of fibrin monomers through its effects on hydrophilic groups. These results suggest that reversible alterations in protein structure are the major effect of exposure of subunit proteins to low DMSO levels at low temperatues, while irreversible denaturation of subunit proteins may be an appreciable effect a higher temperatures and higher DMSO concentrations.


Induction of T-cells differentiation in vitro by thymus epithelial cells.

Thymus-reticular epithelial cells (TE-cells) were grown in a cell culture devoid of any lymphocytic elements. These cells were able to induce T-cell differentiation in spleen cells from T-dificient mice as expressed by con-A responsiveness and GvH reactivity. It was also shown that xenogeneic rat TE cells were as effective in the induction of T-cell differentiation in vitro as syngeneic TE cells. This system is therefore ideal for the study of T-cell development.


Methylmalonic acidaemia and nonketotic hyperglycinaemia. Clinical and biochemical aspects.

The clinical and metabolic data of 2 cases of methylmalonic acidaemia with propionic acidaemia are reported together with those of 3 other patients with nonketotic hyperglycinaemia. Liver enzymatic studies showed decreased activity in vitro of the glycine cleavage enzyme in one patient with methylmalonic acidaemia as well as in 2 unrelated patients with nonketotic hyperglycinaemia, while the activity of the serine hydroxymethylase enzyme was normal. Hyperammonaemia was substantiated in one patient with methylmalonic acidaemia and also in one child with nonketotic hyperglycinaemia. The activity of the enzymes of the urea cycle, determined in the liver of this nonketotic child, was normal except for a decrease of the carbamyl phosphate synthetase enzyme to 15% of normal.


Leaching and degradation of 4-aminopyridine-14C in several soil systems.

Leaching and degradation of 4-aminopyridine (a frightening agent for protecting grain crops from blackbirds) was studied in seven soils. Carbon 14-labeled 4-aminopyridine was strongly adsorbed onto soil colloids, with the degree of adsorption related to pH. Application of seven in. of simulated rainfall over 20 days to surface-treated alkaline soils leached to 0.02 percent to 0.18 percent of the -14C; radioactivity was detected in the runoff from only one of the four acidic soils. Degradation of 4-aminopyridine-14C to -14CO(2) was negligible in soils incubated up to two months under anaerobic conditions. Under aerobic incubation, there was a one-week lag before extensive breakdown began. Degradation rates increased with increasing temperature and soil moisture during incubation, but soil composition had a greater influence. After three months at 30 degrees C and 50 percent moisture, evolution of -14CO(2) ranged from 0.4 percent for a highly acidic loam (pH 4.1) to more than 50 percent for a lighter-textured, alkaline, loamy sand (pH 7.8); the half-life of 4-aminopyridine in soils under these test conditions ranged from 3 to more than 22 months. A theoretical scheme is presented for the degradation of 4-aminopyridine in soils.


Delta-pH method for measuring blood cholinesterase. A study on the effect of temperature.

Efforts to evaluate precision of the Michel delta pH method for blood cholinesterase, performed in a beaker immersed in a 25 C water bath, resulted in unexpected variability in results of daily and day-to-day replicate analyses. Most disturbing was the considerable variation between each four-hour period of an eight-hour day (AM and PM measurements). Precision of quadruplicate sets, expressed as coefficient of variation, ranged from  plus or minus 0.5% to 8.5%. By rigidly controlling temperature with a water-jacketed reaction vessel the variation was reduced to plus or minus 1.7%. A study of effect of temperature on the method showed that a one-degree change in temperature resulted in a 5.5% change in plasma activity and a 3.0% change in red blood cell (RBC) activity. A graphing of the influence of temperature on reaction rate produced a linear relationship. Identical thermodynamic data were obtained with plasma and RBC samples that were inhibited 50% with an irreversible anticholinesterase agent.


Evaluation of the Nissen fundoplication for treatment of hiatal hernia: use of parietal cell vagotomy without drainage as an adjunctive procedure.

Twenty-nine patients who underwent Nissen fundoplication for the treatment of symptomatic, sliding, esophageal hiatal hernia are reported. Fourteen of these patients also underwent parietal cell vagotomy (PCV) without a drainage procedure. Simulatenous cineradiography and manometric studies, esophagoscopy and gastric analysis were performed pre- and postoperatively. Esophageal acid clearing and pH reflux studies were performed postoperatively. All but 3 patients had reflux and/or esophagitis preoperatively. Cineradiography and the pH reflux test were the most reliable tests for diagnosis of reflux. There was no operative mortality. The mean followup period was 20 months. Dysphagia occurred in 5 patients. Correction of dysphagia in one patient required operation. The dysphagia in the remaining patients was temporary and mild, responding to dilatation. Two patients had mild diarrhea. One patient who had had a previous gastric resection developed severe diarrhea after bilateral truncal vagotomy. No patient developed the "bloat syndrome". A close correlation did not exist between reflux and preoperative sphincter pressure. The mean LES pressure increased 10 mmH2O postoperatively and the two patients with persistent reflux postoperatively had normal LES pressure. Correction of reflux after Nissen fundoplication is probably due to some mechanism other than increased LES pressure. Recurrent or persistent hiatal hernia was diagnosed in 4 patients by cineradiography. Two of these patients had reflux but only the patient who had undergone PCV was without symptoms or esophagitis. The technical performance of the Nissen hiatal hernia repair was greatly facilitat  ed by PCV. This procedure also provided adequate treatment for patients with concomitant duodenal ulcer disease. PCV, unaccompanied by a drainage procedure, was not associated with increased morbidity, mortality or the adverse effects usually attributed to vagotomy. In the event of recurrent hernia and reflux, PCV may prevent the development of esophagitis.


Septic lung and shock lung in man.

Two series of patients were studied by serial measurements of blood gas exchange and pulmonarmonary dysfunction and to evaluate the dangers of respiratory failure in post traumatic patients. There were 27 patients who had sustained profound hemorrhagic shock and massive blood replacement averaging 9.7 liters and 38 patients who suffered general peritonitis or other forms of fulminating nonthoracic sepsis. All were supported by endotrachael intubation and volume controlled ventilators. The overall mortality for the post shock patients without sepsis was 12% while in the septic patients it was 35%. The maximal pulmonary arteriovenous shunt encountered in the post hemorrhagic shock patients at 36 hours averaged 20 plus or minus 8% and was accompanied by high cardiac indices (average 5.1 plus or minus 1.3 L/M-2/min) but no significant rise of pulmonary arterial pressure or peak inspiratory pressure (PIP). Severe pulmonary dysfunction subsequently occurred only in those patients who later became septic. The studies on the septic patients were divided according to the magnitude of the cardiac indices (the high indices averaged 4.8 plus or minus 1.6L/M-2/min) and thelow indices averaged 1.9 plus or minus 1.0 L/M-2/min. In the former, the average maximal shunt of 30 plus or minus 6% was sustained for 4 or more days, accompanied by an elevation of PIP to 36 plus or minus 6 cm H2O and by Pa pressure of 28 plus or minus 5 mm Hg. The patients in low output septic shock usually had an associated bronchopneumonia and had an average venous admixture of 34 plus or minus 8% and PIP values of 41 plus or minus 8 cm H2O. The mean Pa pressure in this group was 29 plus or minus 6 mm Hg.


Evaluation of biliary and pancreatic function in pancreolithiasis. Value of the determination of gamma-glutamyl transpeptidase.

Ten patients with pancreolithiasis and ten controls underwent a 110-minute pancreozymin-secretion test in which post-pancreozymin collection periods were prolonged to 30 minutes. Gamma-Glutamyl transpeptidase concentrations and outputs of duodenal aspirate in response to pancreozymin and to secretin were greatly increased in patients with pancreolithiasis. No correlation was noted between gamma-glutamyl transpeptidase and bile pigment concentrations. The mean concentrations and outputs of amylase in disease were much less than those in control subjects throughout the test. Two categories of pancreolithiasis were distinguished with respect to the distribution and size of the calculi and amylase secretion. We suggest that, in pancreolithiasis, there is an increase in ductal or centroacinar cell mass.


Acetazolamide treatment of hypokalemic periodic paralysis. Probable mechanism of action.

Following administration of glucose and insulin to three patients with hypokalemic periodic paralysis, serum K+ fell 1.9 mM. After administration of acetazolamide, 250 mg four times daily, serum K+ fell 0.9 mM, a substantial difference. In normal persons glucose and insulin lowered serum K+ 0.5 mM, and this was not changed substantially by acetazolamide. The metabolic acidosis induced by the drug appears to be responsible for the change in decrement of serum K+ and for the amelioration of symptoms in the patients. The findings agree with earlier reports that metabolic acidosis lowers the rate of entry of K+ into muscle, thus opposing the heightened or pathological entry of K+ into muscle cells during attacks of the disease.


[Experimental changes of the pH in the middle ear and its effect on the internal ear (author's transl)].

Irrespective of their highly differing pH-value many pharmaca are nowadays applied to the middle ear. Animal experiments are to answer the question, weather changes in the pH-value of the middle ear may cause internal ear damages, With local application of different buffers (2.3-9.1 pH) into the tympanic cavity, the membrane of the round window was examined by means of the surface preparation technique, the pH-value of the perilymph was measured and the hair cells of the organ of corti were counted and histologically evaluated. With the reduction of the pH-value in the middle ear from 4.0 to 2.3 an increasing damage of the round window membrane could be detected. The pH-value of the perilymph, however, showed only a slight statistical variation, which cannot be guaranteed. After multiple applications of extremely acid buffers into the tympanic cavity directly before the round window membrane, a small but statistically significant reduction in the pH-value of the perilymph could be noticed. Hair cell damages at the organ of corti could in no case be observed.


Effect of hyperbaric air on long-term memory organization and recall.

Three experiments are reported which investigated the effects of hyperbaric air on long-term memory. In the first, word lists were learned at 1 and 8.6 ATA using a variable input-free recall paradigm. It was found that learning was affected but not clustered memory organization, and it was concluded that disorganization of memory is not a factor contributing to the learning deficit found with hyperbaric air. In the second and third experiments it was found that the recall of words, which had been learned when non-narcotic, was disrupted at 10 ATA by hyperbaric air and that this disruption was not overcome by providing memory-cues at the time of recall. Two possible explanations for these results are discussed. A hypothesis is put forward to reconcile the results of various hyperbaric memory experiments by pointing out that a relationship between stress and learning found with nitrous oxide could be applicable to these studies also.


Epidural analgesia, fetal monitoring and the condition of the baby at birth with breech presentation.

Between December 1970 and March 1973, 138 patients with a singleton fetus presenting by the breech after 36 weeks of pregnancy were deemed suitable for vaginal delivery under epidural analgesia; 130 were delivered vaginally, 10 of them by breech extraction. There was one stillbirth and no neonatal deaths. Epidural analgesia for vaginal breech delivery seemed beneficial. In 65 cases it was possible to compare the umbilical vein pH with the Apgar score at one minute. In 35 patients a continuous recording of the fetal heart rate was used to predict the Apgar score at one minute and the results are discussed.


NAD- and NADP-dependent 7alpha-hydroxysteroid dehydrogenases from bacteroides fragilis.

Twenty strains of Bacteroides fragilis were screened for hydroxysteroid oxidoreductase activity in cell-free preparations. Eighteen strains were shown  to contain NAD-dependent 7alpha-hydroxysteroid dehydrogenase. Sixteen of the strains containing the NAD-dependent enzyme also contained NADP-depedent 7alpha-hydroxysteroid dehydrogenase, but invariably in lesser amounts. A strain particulary rich in both 7alpha-hydroxysteroid dehydrogenase activities was selected for further study. Measurement of activity as a function of pH revealed a fairly sharp optimal activity range of 9.5--10.0 for the NAD-dependent enzyme and a broad flat optimal range of 7.0--9.0 for the NADP-dependent enzyme. Michaelis constants for trihydroxy-bile acids for the NAD-dependent enzyme were in the range of 0.32--0.34 mM, whereas dihydroxy-bile acids gave a Km of 0.1 mM. Thin-layer chromatography studies on the oxidation product of 3alpha, 7alpha-dihydroxy-5beta-cholanoic acid (chenodeoxycholic acid) by the dehydrogenase revealed a band corresponding to that of synthetic 3alpha-hydroxy, 7-keto-5beta-cholanoic acid. Similarly the oxidation product of chenodeoxycholic acid by both 7alpha-hydroxysteroid dehydrogenase and commercially available 3alpha-hy-droxysteroid dehydrogenase revealed a band corresponding to that of synthetic 3,7-diketo-5beta-cholanoic acid. Neither of these two oxidation products could be distinguished from those by the Escherichia coli dehydrogenase oxidation previously reported. Disc-gel electrophoresis of a cell-free lyophilized preparation indicated one active band for NAD-dependent activity of mobility similar to that for the NADP-dependent E. coli enzyme. The NADP-dependent dehydrogenase was unstable and rapidly lost activity after polyacylamide disc-gel electrophoresis, ultracentrifugation, freezing on refrigeration at 4 degrees C. No 3 alpha- or 12alpha-oriented oxidoreductase activity was demonstrated in any of the strains examined.


Current concepts of the mechanism of acute inflammation in gouty arthritis.

The acute gouty attack develops after free crystalline monosodium urate crystals appear in the joint cavity (1). Recent developments in the investigation of urate crystal-induced inflammation have led to a better understanding of the pathogenesis of acute gouty arthritis. The purpose of this review is to summarize present concepts concerning the mechanism of the acute gouty attack.


Purification and properties of a lipid acyl-hydrolase from potato tubers.

1. A pure lipid acyl-hydrolase was prepared from potato tubers by acetone precipitation, Sephadex G-100 and DEAE-Sephadex A-50 column chromatography, and by electrofocusing. 2. The purified enzyme was an acidic protein of pI 5.0 and molecular weight of about 70 000. Km values were 0.38 mM for monogalactosyldiacylglycerol and 1.7 mM for phosphatidylcholine. 3. The hydrolytic activity of the enzyme on different substrates was determined. The relative rates were acylsterylglucoside  greater than monogalactosyldiacylglycerol greater than monogalactosylmonoacylglycerol greater than digalactosyldiacylglycerol greater than diagalactosylmonoacylglycerol, while the rates for phospholipids were lysophosphatidylcholine greater than phosphatidylcholine greater than lysophosphatidylethanolamine greater than phosphatidylethanolamine. 4. Analyses of enzymatic hydrolysis products suggested that a single enzyme had both galactolipase and phospholipase activities, and for the phospholipids it showed activities similar to phospholipase B and glycerylphosphorylcholine diesterase. 5. A competitive relation was found between monogalactosyldiacylglycerol and phosphatidylcholine as substrates of the enzyme, indicating that the active sites for both substrates may be the same. 6. It was suggested that histidine and probably serine residues were important to the enzymic activity, and that a tyrosine residue might be involved in the activity as an accessory component.


Elevation of arterial phosphorfuctokinase activity associated with susceptibility to atherosclerosis in pigeons.

The activity of phosphofructokinase (PFK, 2.7.1.11) was measured in arteries of very young (5-8 week old) pigeons known to differ in susceptibility to atherosclerosis. The activity of the arterial enzyme was significantly higher in the atherosclerosis-susceptible White Carneau (WC) pigeons than in the resistant Show Racers (SR). The difference was significant whether enzyme activity was calculated on the basis of extract protein, DNA content or fat-free dry weight. In the White Carneau arteries the activity of the enzyme was higher in the female than the male pigeons. PFK is a key regulatory enzyme of glycolysis and is subject to fine control adjustments. A low ATP/ADP ratio and a fall in citrate concentration, as for example, induced by hypoxia, are meditors of a feedback mechanism leading to a rise in PFK activity and enhancement of glycolysis for energy production. This mechanism appears to be the cause of the higher PFK activity in the WC arteries, because related studies indicate impaired Krebs cycle activity in these vessels. It is suggested that the increased dependence of the WC arteries on glycolysis facilitates the development of atherosclerosis in this pigeon strain and that the mechanism is similar to the mechanism by which tissue hypoxia causes lipid accumulation and connective tissue alterations in the arterial wall.


Cholesterol ester hydrolase in human red blood cells.

1. Cholesterol ester hydrolytic activity (sterol-ester hydrolase EC 3.1.1.13) was detected in human red blood cells. Enzyme activity appeared confined to the cell membrane and was most marked in washed preparations of red cell ghosts. 2. Hydrolytic activity was stimulated by the anti-oxidants D-alpha-tocopherol and butylated hydroxytoluene. Marked inhibition was produced by erythrocyte hemolysate, sodium taurocholate, and Triton X-100. 3. Optimal pH for the reaction was 5.4--5.7. 4. Because red cell cholesterol is all unesterified, it is speculated that the hydrolase serves to maintain the erythrocyte membrane free of esterified cholesterol.


Nicotinamide-adenine dinucleotide inhibition of pig kidney alkaline phosphatase.

1. The interaction of NAD+, NADH and various nucleotide analogues with pig kidney alkaline phosphatase (orthophosphoric-monoester phosphohydrolase (alkaline optimum) EC 3.1.3.1) has been investigated by kinetic means. Some inhibitors act uncompetitively whereas others markedly increase the slopes of double reciprocal plots suggesting they have some affinity for the free enzyme. 2. The compounds seem to bind to alkaline phosphatase through interactions of their bases with a relatively non-specific region of the enzyme, although it is likely that for those nucleotides having some affinity for the free enzyme there is some attraction between the pyrophosphate backbone and the active site. 3. From studies of the effect of NAD+ and NADH on ATPase activity it was concluded that the substrate inhibition that is characteristic of the ATPase activity of alkaline phosphatase originates from binding of ATP to the site assumed to exist for NAD+ and NADH. The potentiation of NAD+-inhibition of ATPase activity by Mg-2+ is probably a result of the depletion of [ATP-4-] the true substrate. The depletion allows NAD+ to complete more effectively for the active site. 4. Binding of NADH is favoured by protonation of an enzymic group with a pK of approx. 9.0 belonging possibly to a tyrosine residue or a zinc hydrate. 5. A large entropy decrease was found to accompany the binding of NAD+ and NADH to alkaline phosphatase. This may be further evidence of an "induced-fit" mechanism previously suspected because of the synergistic inhibitory effects of adenosine and nicotinamide.


Purification of a derepressible arylsulfatase from Chlamydomonas reinhardti. Properties of the enzyme in intact cells and in purified state.

Arylsulfatase (aryl-sulfate sulfohdydrolase, EC 3.1.6.1) has been purified from SO4-2-minus-starved cells of Chlamydomonas reinhardti. The enzyme was isolated from acetone-powder extract by (NH4)2SO4 precipitation, Sephadex G-200 filtration and ion-exchange chromatography. Only one fraction of aryl-sulfatase was found. The final preparation was homogenous by the criteria of sedimentation, diffusion and polyacrylamide gel electrophoresis. The purified enzyme had a molecular weight of about 150 000, estimated by ultracentrifugation and gel filtration, and an isoelectric point of 9.0. The properties of the enzyme as investigated in intact cells and in the purified state were found to be very similar except for the temperature optimum. Imidazole strongly increased the enzyme by increasing the V, but reduced the affinity for the substrate. The enzyme activity was competitively inhibited by borate with a greater affinity for borate than for the substrate. The Chlamydomonas enzyme is a Type I arylsulfatase since it was inhibited by CN-minus, but not SO4-2-minus and phosphate.


Proteolytic activities in yeast.

Studies on the mechanism and time course of the activation of proteinases A (EC 3.4.23.8), B (EC 3.4.22.9) and C (EC 3.4.12.--) in crude yeast extracts at pH 5.1 and 25 degrees C showed that the increase in proteinase B activity is paralleled with the disappearance of proteinase B inhibitor. Addition of purified proteinase A to fresh crude extracts accelerates the inactivation of the proteinase B inhibitor and the appearance of maximal activities of proteinases B and C. The decrease of proteinase B inhibitor activity and the increase of proteinase B activity are markedly retarded by the addition of pepstatin. Because 10-minus 7 M pepstatin completely inhibits proteinase A without affecting proteinase B activity, this is another indication for the role of proteinase A during the activation of proteinase B. Whereas extracts of yeast grown on minimal medium reached maximal activation of proteinases B and C after 20 h of incubation at pH 5.1 and 25 degrees C, extracts of yeast grown on complete medium had to be incubated for about 100 h. In the latter case, the addition of proteinas A results in maximal activation of proteinases B and C and disappearance of proteinase B inhibitor activity only after 10--20 h of incubation. With the optimal conditions, the maximal activities of proteinases A, B and C, as well as of the proteinase B inhibitor, were determined in crude extracts of yeast that had been grown batchwise for different lengths of time either on minimal or on complete medium. Upon incubation, all three proteinases were activated by several times their initial activity. This reflects the existence of proteolytically degradable inhibitors of the three proteinases and together with the above mentioned observations it demonstrates that the "activation" of yeast proteinases A, B and C upon incubation results from the proteolytic digestion of inhibitors rather than from activation of inactive zymogens by limited proteolysis.


Properties and specificity of the major metal-chelator-sensitive proteinase in the keratinolytic larvae of the webbing clothes moth.

The metal chelator-sensitive proteinase activity from the larvae of the webbing clothes moth, Tineola bisselliella, was fractionated into two components by chromatography on DEAE-cellulose and the properties of the major fraction investigated. The approximate molecular weight obtained by gel filtration was 24 000. The pH optimum of 9.4 and the high stability between pH 9.0 and 11.5 are consistent with the alkaline conditions known to be present in the larval mid-gut. The enzyme also showed a second region of high stability around pH 2.3. The cleavage specificity against S-carboxy-methyl A and B chains of insulin was quite different to that of the metal chelator-sensitive proteinases from snake venoms and microorganisms. 10 bonds in the A-chain and 8 bonds in the B-chain were cleaved and the tentative rules governing the specificity limitations of this metal-chelator-sensitive proteinase are discussed.


Isolation of an extracellular neutral proteinase from Pseudomonas fragi.

A single proteolytic enzyme (EC 3.4.4.-) was isolated from culture supernatants of Pseudomonas fragi with 20% yielded and 60-fold purification by means of stepwise DEAE-Sephadex batch adsorption, ammonium sulfate precipitation, gel filtration and DEAE-cellulose chromatography. The enzyme was Zn-2+ activated and Ca-2+ stabilized, had optimum activity at pH 6.5--8.0 and 40 degrees C. The molecular weight range was 40 000--50 000 as determined by dodecylsulfate  gel electrophoresis, gel filtration and Zn assay. This proteinase has properties similar to other extracellular bacterial neutral proteinases.


Kinetic studies of a beta-lactamase by a computerized microacidimetric method.

On-line computerized treatment of enzyme kinetic data allows the precise measurement of Michaelis--Menten constants (Km and V) from a single progress curve. This method has been used to determine the kinetic constants of a beta-lactamase extracted from an Escherichia coli strain. In the profile of enzymatic activity there obtained, Km and V are a function of the pH. From these results some information is derived about the mechanism of the enzyme--substrate binding.


Functionally distinct classes of K+ sites on the (Na+ + K+)-dependent ATPase.

K+ interactions with a rat brain (Na+ + K+)-dependent ATPase and the associated K+-dependent nitrophenyl phosphatase activity were examined. Classes of sites for K+ were distinguished, initially, on the basis of affinity estimated by kinetic analysis in terms of KO.5 (the concentration for half-maximal activation), and by K+-accelerated enzyme inactivation by F-minus, which permits evaluation of a dissociation constant for K+, KD. Moderate-affinity sites ("alpha sites"), with a KD near 1 mM, were demonstrable for the phosphatase activity and for the "free" enzyme. High-affinity sites ("beta sites"), with a KD near 0.1 mM, were seen for the overall ATPase activity and under conditions in which enzyme phosphorylation by substrate also occurs. Further differentiation between alpha and beta sites was made in terms of (i) the characteristic changes in affinity with pH, and (ii) the efficacy of Li+ relative to K+, Rb+, Cs+, and Tl+ at these two classes of sites. Low-affinity sites ("gamma sites") through which K+ inhibits enzymatic activity were also detectable, with a KD around 140 mM. These data are incorporated into a model for the reaction sequence to accommodate both transport processes and certain K+/ATP antagonisms.


Rat liver cysteine sulfinate decarboxylase: purification, new appraisal of the molecular weight and determination of catalytic properties.

Rat liver cystein sulfinate decarboxylase (L-cystein sulfinate carboxylase) was purified approximately 500-fold. By cellulose acetate and polyacrylamide gel electrophoresis or by analytical ultracentrifugation, the purified enzyme appears to be nearly homogeneous. The Stokes radius (3.4 nm) and sedimentation coefficient (6.5 S) were determined. The molecular weight, calculated and experimentally estimated is around 100 000 and the enzyme is constituted of two identical subunits whose molecular weights are 55 000. The role of pyridoxal phosphate as coenzyme was demonstrated and the requirement for free sulhydryl groups for activity was studied. The ability of native pure cysteine sulfinate decarboxylase to also decarboxylate cysteate was stressed: therefore, we concluded that in rat liver a single protein catalyzed both reactions, although only the decarboxylation of cysteine sulfinate is of physiological interest.


19-F NMR studies of the binding of a  fluorine-labeled phosphonate ion to E. coli alkaline phosphatase.

The interaction of a fluorinated phosphonate with Zn-2+-and Mn-2+-alkaline phosphatase as studied by 19-F NMR revealed a stoichiometry of 1:1 for the binding of the phosphonate anion to the enzyme. In the presence of two metal ions, one fluorinated phosphonate ion was found to interact strongly with the enzyme, while a different interaction was observed when the number of metal ions per enzyme exceeded two. Phosphate replaced enzyme bound phosphonate, as is shown by the 19-F NMR spectra. No direct interaction between the fluorinated phosphonate and the metal ion responsible for enzyme activity was indicated by the 19-F NMR data. This observation supports the idea of a considerable distance between metal ion and substrate binding site in Escherichia coli alkaline phosphatase.


Electron donors and inhibitors of nitrate reductase from Cyanidium caldarium.

Studies on nitrate reductase (NAD(P)H:nitrate oxidoreductases EC 1.6.6.2) of Cyanidium caldarium revealed that the enzyme is inhibited by excess of electron donor, NADPH, reduced benzylviologen and FMN. Also dithionite, used to reduce benzylviologen and FMN, inactivates nitrate reductase: however, FMN at an optimal concentration and nitrate, added before the dithionite, protect the enzyme against this inactivation. Cyanide, cyanate and carbamyl phosphate inhibit the enzyme competitively with respect to nitrate, and Ki values are reported. Organic mercurials, 0.1 mM, act preferentially on NADPH activity, whereas Ag+ and Hg-2+ at the same concentration inactivate 80--90% of the benzylviologen and FMN activities. ADP is very poor inhibitor. Urea 4 M in 2 h destroys 90% of the NADPH activity and only 30% of the benzylviologen and FMN activities. The apparent Km values for NADPH, benzylviologen, FMN and nitrate have been determined.


Purification, properties and induction of a specific benzoate-4-hydroxylase from Aspergillus niger (UBC 814).

An inducible benzoate-4-hydroxylase has been partially purified from crude extracts of the mycelial felts of Aspergillus niger. This enzyme catalyzes the transformation of benzoate to p-hydroxybenzoate with equimolar consumption of NADPH and O2. It requires tetrahydropteridine as a prosthetic group. The optimum activity was found at pH 6.2 with a Km value at 30 degrees C of 1.6-10-minus 4 for NADPH and 1.3-10-minus 4 M for benzoate. Fe-2+ (iron) is required for the enzyme activity. The enzyme is stabilized by the inclusion of benzoate, EDTA and glutathione in the extracting buffer. The enzyme is specific for benzoate as substrate. Sulfhydryl groups(s) are essential for enzyme activity as indicated by p-chloromercuri-benzoate and N-ethylmaleimide inactivation. Benzoate-4-hydroxylase activity is decreased in the mycelial felts of Aspergillus niger grown in the presence of higher concentrations of benzoate. Maximum activity of the enzyme was observed at 36 h after inoculation.


Effects of sulfhydryl agents on the activation of tryptophan-5-monooxygenase from bovine pineal glands.

Partially purified tryptophan-5-monooxygenase (L-tryptophan, tetrahydropteridine: oxygen oxidoreductase (5-hydroxylating) EC 1.14.16.4)from bovine pineal gland was activated by preincubation with sulfhydryl agents such as dithiothreitol, L-cysteine, cysteamine, L-cysteine ethylester, N-acetyl-L-cysteine, 2-mercaptoethanol and reduced glutathione, at alkaline pH (optimum pH equals 8.5). Dithiothreitol was the most effective of these, leading to approximately 50-fold activation of the enzyme after preincubation. Fe-2+ or other reducing agents such as borohydride, dithionite and ascorbate facilitated the velocity of the activation in the presence of sulfhydryl agents. In the absence of sulfhydryl agents, no activation was observed even in the presence of Fe-2+ or other reducing agents, suggesting an obligatory role or sulhydryl agents during the activation. The relative velocity and full extent of the activation were dependent on the concentrations of both the sulfhydryl agent and the enzyme in the activation mixture. The kinetic analysis of the activation indicated that the sulfhydryl agent reacts with more than 2 sites in the enzyme; one type of site is reduced by sulfhydryl agents, Fe-2+ or other reducing agents and the other specifically modified by a sulfhydryl agent. The activated enzyme did not require any exogenous Fe-2+ for its catalytic activity, but some roles of iron maybe exist in its catalytic reaction. The optimum pH for catalytic reaction of the activated enzyme was approximately 6.5. The apparent Km for L-tryptophan and pteridine cofactor, tetrahydro-pteridine (2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropterin), of the activated enzyme were 30 and 35 muM respectively.


Purification and properties of rat liver adenine phosphoribosyltransferase.

The adenine phosphoribosyltransferase (AMP: pyrophosphate phosphoribosyltransferase, EC 2.4.2.7) of rat liver was purified to a specific activity of 1.1 mumol of AMP formed per min per mg. The enzyme activity is associated with an apparently homogenous protein as shown by isoelectrofocusing, acrylamide gel electrophoresis, and N-terminal amino acids analysis (phenylalanine). The molecular weight of the enzyme was estimated to be approx. 20 000 by acrylamide gel electrophoresis in the presence of sodium dodecylsulfate and by sucrose density gradient zone sedimentation. The rat liver enzyme exhibited initial burst synthesis of AMP when 1-pyrophosphorylribose 5-phosphate was added. The 1-pyrophosphorylribose 5-phosphate initial-burst activity copurifies with the adenine phosphoribosyltransferase activity. A PH optimum of 10.0 was demonstrable for the adenine phosphoribosyltransferase. The initial-burst and steady-state phases of AMP synthesis catalyzed by highly purified rat liver adenine phosphoribosyltransferase have been partially characterized by the use of ligands which bind to sulfhydryl groups. Studies utilizing p-chloromercuribenzoate and HgCl2 as inhibitors of AMP sulfhydryl during the initial-burst and steady-state phases have revealed that sulfhydryl groups with different rates of ligand binding are present in the enzyme. The initial-burst phase was thereby delineated from the steady-state phase by use of these mercurial ligands. This delineation was also accomplished by titration with the Mg-2+ chelator, EDTA. The inhibitory effects of mercurials and EDTA were reversed by beta-mercaptoethanol and excess Mg-2+, respectively. Quantitative binding studies with 5,5'-dithiobis(2-nitrobenzoic acid) and p-chloromercuribenzoate yielded values of 3.65 and 3.6 mol of sulfhydryl per mol of enzyme, respectively. 3.3 mol of cysteic acid per mol of performic acid-oxidized enzyme were found by amino acid analysis.


Cholesteryl ester hydrolytic acitivity of rat liver plasma membrane.

Cholesteryl ester hydrolyzing activity of rat liver plasma membranes was studied using acetone-dispersed [4-14-C] cholesteryl oleate as substrate. In contrast to whole liver homogenates which displayed ample activity at both acid (4.5) and neutral (6.2-7.4) pH, purified plasma membrane fractions contained little activity at neutral pH as compared to acid pH. Moreover, rate-zonal sucrose density-gradient centrifugation patterns of plasma membrane rich fractions suggested a specific association with plasma membrane only in the case of the acid activity. These findings suggest that in vivo hepatic cell surface membranes contain little or no cholesteryl ester hydrolytic activity at extracellular pH. They support the possibility that plasma lipoprotein cholesteryl esters enter hepatic parenchymal cells prior to hydrolysis.


Isoelectric focusing of human serum high density lipoprotein in the presence of radioactive ampholines.

Preparative isoelectric focusing of a serum high density lipoprotein fraction (rho 1.063-1.125 g/ml) in the presence of radioactive Ampholines showed that there were no significant 14-C-activity peaks in the lipoprotein region of the pH gradient. This suggests that the discrete lipoprotein subspecies obtained by electrofocusing are not artefacts caused by the binding of Ampholines to serum high density lipoprotein.


Effects of cyanide on stearyl-coenzyme A desaturase activities in microsomes from various mammalian tissues.

The effect of potassium cyanide on the desaturase activity for stearyl-CoA in microsomes of various mammalian tissues has been investigated. Potassium cyanide inhibited the desaturase activities in microsomes from livers of rat, hen, guinea pig and rat lung, but not the activities in microsomes from rabbit liver, pig thyroid and bovine adrenocortex, It is concluded that the so-called "cyanide-sensitive factor" does not seem to be common to all the desaturase activities of stearyl-CoA in the microsomes of mammalian tissues.


Studies on lipase from Mucor javanicus. I. Purification and properties.

1. Lipase produced by a mold, Mucor javanicus, was purified about 180-fold from the ethanol precipitate of the culture filtrate. Purification was achieved by acid precipitation followed by gel filtrations on Sephadex G-200 (at low ionic strength) and Sephadex G-75 (at a high ionic strength). The purified enzyme preparation showed unusual behavior on polyacrylamide gel electrophoresis. The molecular weight was estimated to be 21 000. The enzyme had a positional specificity towards the position 1 and 3 of triacylglycerols. 2. Lipase in the crude preparation takes an aggregated form. aggregated form was achieved by raising the ionic strength of the medium. 3. The purified lipase preparation from Mucor javanicus exhibits phospholipase A1 activity, hydrolyzing the carboxyl ester at the 1-position of phosphatidylcholine. This activity seems to be due to the action of the lipase itself and not due to any other specific phospholipases.


Induction of tryptophan oxygenase by dexamethasone in isolated hepatocytes. Dependence on composition of medium and pH.

Hepatocytes were isolated from perfused rat livers. 4 x 10-6 cells/ml were incubated at at 37 degrees C in different media in the absence and presence of a steroid hormone, dexamethasone phosphate (2 x 10-5 M). 1. Hormonal enzyme induction occurred in cells suspended in a simple salt medium, devoid of amino acids and macromolecules. This induction was completely blocked by addition of either actinomycin D (2 mu-g/ml) or cycloheximide (50 mu-g/ml). 2. Incubation of cells in media containing defatted albumin did not enhance hormonal enzyme induction, although disintegration of cells during incubation was reduced. Addition of a crude albumin fraction reduced tryptophan oxygenase induction and dextran completely blocked enzyme induction by dexamethasone. 3. An increase of dexamethasone concentration in the presence of albumin to 9 x 10-5 M was unable to raise enzyme induction further, and a still higher concentration of hormone, 3 x 10-4 M, resulted in reduced enzyme induction. 4. The hormonal induction of tryptophan oxygenase was most pronounced when the pH of the medium was between 7.0 and 7.6, with an optium at 7.3. No induction was found when the pH of the medium was either 6.6 or 7.8. The basal tryptophan oxygenase activity was much less influenced by similar pH variations. It is concluded that hepatocytes in suspension are able to carry out hormone-stimulated enzyme synthesis and that factors influencing this process may be studied under controlled conditions in such systems.


Analysis of polypeptide disposition in human erythrocyte membranes employing membrane inversion.

High resolution segregation of erythrocyte membrane polypeptides achieved by isoelectric focusing in 8 M urea was employed in conjunction with surface-restricted radioiodination to analyze the disposition of polypeptides within the human erythrocyte membrane. Several membrane polypeptides showed significant uptake of radioiodine, with the principal labeled component migrating between pH values of 3.0 and 3.5. Two approaches were taken in examining membrane polypeptide disposition on both faces of the erythrocyte membrane. Saturation labeling of the outer face of the membrane with one iodine isotope followed by cell lysis and reiodination with a second iodine isotope did not prove feasible and another procedure based on surface iodination with 125-I, formation of sealed inside-out vesicles and re-iodination with 131-I was adopted. Studies of sialic acid release from the membrane surface and trypsin cleavage of radioiodinated peptides indicated that selectively labeled, sealed inside-out vesicles had been formed. The ratio of 125-I to 131-I in membrane polypeptides separated by isoelectric focusing confirmed the existence of externally disposed, internally disposed and spanning proteins.


The purification and characterization of plasma membranes and the subcellular distribution of adenylate cyclase in mouse parotid gland.

1. Plasma membranes have been purified 17-fold from mouse parotid gland homogenates prepared in hypertonic sucrose media using differential centrifugation. The method is fast and simple. The membranes were characterised by electron microscopy, enzyme composition and chemical composition. Further purification was achieved by isopycnic centrifugation in discontinuous sucrose gradients. 2. The purified membranes contain an adenylate cyclase activity which is stimulated by isoproterenol and fluoride. Only 50% of the total adenylate cyclase activity sedimented in the plasma membrane fraction. The rest of the activity resided in the crude nuclear and mitochondrial pellets. However, this adenylate cyclase activity was not associated with these organelles but with membrane fragments in the pellets. Purified nuclei did not contain adenylate cyclase activity. 3. Adenylate cyclase activity was also localised by electron microscopic cytochemistry. Besides being found at the plasma membrane, large amounts of adenylate cyclase were found in a small proportion of the vesicles within the acinar cells, which appeared to be secondary lysosomes. 4. Adenylate cyclase activities, under standard assay conditions, are proportional to the time of incubation and the concentration of enzyme. The enzyme requires both Mg-2+ and CA-2+ for activity. Isoproterenol increased activity 2-fold and this increase is abolished by beta-adrenergic blocking agents.


Distribution of NAD(P)H-dependent cytochrome P-450 mixed function oxidase system in the brush border membrane of rabbit kidney cortex.

Optical and magnetic studies were made on subfractions of rabbit kidney cortex. Cytochrome P-450 and cytochrome b5-dependent mixed function oxidase systems were localized mainly in the brush border membranes and microsomes. Cytochrome P-450-dependent mixed function oxidases in the membranes comprised both an NADPH-dependent system and an NADH-dependent system.


On the effects of propionate and other short-chain fatty acids on sodium transport by the toad bladder.

1. Propionate and other unbranched short-chain fatty acids, butyrate, pentanoate, hexanoate and octanoate were found to both stimulate and inhibit active sodium transport by the toad bladder, as measured by the short-circuit current (s.c.c.). 2. Stimulation alone followed addition of low concentrations of fatty acids (0.1-1.0 mM) to either the serosal or mucosal bathing medium; stimulation was also seen after an initial period of inhibition in response to higher concentrations (approx. 5 mM) of some compounds. 3. Inhibition alone followed addition of high concentrations (5-20 mM) of these compounds. The duration and magnitude of the inhibition varied with increasing concentration and chain length of the fatty acid, and was greater following mucosal addition than serosal addition. 4. The inhibitory effect of mucosal propionate increased with decreasing pH of the mucosal bathing medium. 5. Inhibition by the fatty acids was completely reversed upon removing the compound from the bathing medium, and stimulation characteristically followed. 6. In studies designed to evaluate the role of metabolism of the fatty acids in their mucosal inhibitory effects it was found that 14-c-labelled propionate, when added to the mucosal surface of the bladder, was converted to 14-CO2, and mucosal succinate and alpha-oxoglutaric acid at 20 mM inhibited the s.c.c. slightly. However, malonate did not interfere with inhibition by mucosal propionate and two non-metabolizable acids, dimethylpropionate and benzoate, induced inhibition (and no stimulation) of the s.c.c. 7. In the presence of an inhibitory concentration of fatty acid, the ability of the bladder to respond to added pyruvate was reduced in proportion to the reduction in the level of the s.c.c., whereas the natriferic response to vasopressin was largely intact. 8. We conclude that stimulation of sodium transport by propionate and other short-chain fatty acids is due to metabolism of the compounds and provision of energy to the sodium transport mechanism. The basis of the inhibition appears complex. It may in part depend on metabolism of the fatty acids and/or uncoupling of oxidative phosphorylation, with resultant reduction in net ATP production for the sodium transport mechanism. However, the inhibition may also be caused in part by a direct effect on the mucosal entry of sodium into the transporting epithelial cells.


Osmotic behaviour of Acholeplasma laidlawii B cells with membrane lipids in liquid-crystalline and gel state.

The osmotic behaviour of Acholeplasma laidlawii B cells was investigated with combined spectrophotometric and enzymatic measurements. The conclusion could be drawn that this osmotic behaviour depends largely on the physical state of the membrane lipids. When part of the membrane lipids is in the liquid-crystalline phase the cell is able to swell and behaves as a good osmometer. However, when the membrane lipid is in the gel phase, the cell is unable to swell and the change in absorbance of the cell suspension is then completely due to lysis.


Effect of morphine on adrenergic transmission in the mouse vas deferens. Assessment of agonist and antogonist potencies of narcotic analgesics.

1. Morphine inhibits the electrically evoked (0.1-0.15 Hz, 1 ms) contractions of the longitudinal muscle of the mouse vas deferens but not of the rabbit, guinea-pig, rat, cat, hamster or gerbil. This effect is stereospecific and is antagonized by naloxone or naltrexone. 2. Normorphine is equiactive with morphine but its effects are more rapid in onset and decline. 3. In the mouse vas deferens, the resting outflow of tritium-labelled catecholamines is unaffected by morphine. The electrically evoked outflow is depressed by morphine or normorphine in a dose-dependent manner. The ID50 for inhibition of contraction and for depression of outflow is 0.5 muM. 4. The relative agonist potencies of compounds without antagonist component (codeine, pethidine, morphine, normorphine, heroin, levorphanol, Ba-20227, etorphine) show good correlation with the relative agonist potencies determined in the guinea-pig ileum and for analgesia in man. 5. For compounds with dual agonist and antagonist properties, the dose-response curves for agonist activity are shallow. When the lowest concentrations giving a depression of the contraction of the mouse vas deferens are used, a good correlation is obtained with the guinea-pig ileum. 6. The relative antagonist potencies of naloxone, nalorphine, levallorphan, and cyclazocine agree well with those obtained in the guinea-pig ileum; these, in turn, correlate well with the values obtained in the morphine-dependent monkey. 7. The fact that the agonist effects of drugs with dual agonist and antagonist action show little or no dependence on concentration, makes the mouse vas deferens particularly suitable for the assay of assay of antagonist activity. 8. As an assay preparation, the mouse vas deferens is less robust and consistent in its responses than the guinea-pig ileum.


Action of beta-adrenoceptor antagonists on the response to isoprenaline in the oestrogen dominated rabbit uterus.

1. Log dose-response curves to isoprenaline from spontaneously contracting muscle strips from rabbit uterus have been obtained. The effects of the alpha-adrenoceptor antagonists phentolamine and phenoxybenzamine, the beta- adrenoceptor antagonists propranolol and practolol, and (+)-propranolol on the log dose-response curves were studied. 2. Phentolamine 5.3 times 10- minus 7 M had a stimulating effect on the muscle strips, moving the log dose-response curve of isoprenaline to the right. Phenoxybenzamine 2.9 times 10- minus 5 M had no effect on the curves. 3. Propranolol 3.4 times 10- minus 6 M had no effect on the curves from circular muscle strips, with either phentolamine 5.4 times 10- minus 7 M or with phenoxybenzamine 2.9 times 10- minus 5 M as alpha-blocker. The curves from the longitudinal muscle strips were shifted somewhat to the right, the same shift, however, being obtained with (+)-propranolol 3.4 times 10- minus 6 M. 4. Practolol 3.8 times 10- minus 6, 3.8 times 10- minus 5 and 3.8 times 10- minus 4 M was without effect on the curves, either in the circular or in the longitudinal strips. 5. It is concluded that neither propranolol at 3.4 times 10- minus 6 M nor practolol act as beta-adrenoceptor antagonists in oestrogen dominated rabbit uterus. In all other tissues investigated, propranolol or practolol block the effect of isoprenaline. Whether the effect of isoprenaline in this tissue may be termed a beta-effect, is then a question of definition. In addition there is no evidence for any extraneuronal uptake mechanisms for isoprenaline. 6. Variations in sensitivity to beta-stimulation with the time of the year were observed, the sensitivity being greatest in the winter and lowest in the summer.


Determination of the Optimum temperature for regional renal hypothermia during temporary renal ischaemia.

To determine the optimum temperature at which the in situ kidney should be maintained while it is ischaemic, 47 mongrel dogs were studied. 35 of these underwent 90 minutes of left renal ischaemia with the kidney temperature maintained at 37 degree, 30 degree, 22 degree, 15 degree and 0 degree C respectively. The effect on renal function was determined by measurements of G.F.R. before and at regular 15-minute intervals after the inschaemic period. Computer statistical analysis exposed the optimum temperature to be 15 degree C. Renal artery blood flow, renal histology, 15-Cr labelled platelets and renal arteriography were used to determine the mechanism of ischaemic injury. Quantitation of renal cell injury confirmed that no additional protection to ischaemia could be gained by colling below 15 degree C. 15 degree C is recommended as the optimum temperature for use in clinical renal hypothermia.


Immunological effects of orchidectomy.

Orchidectomy caused a non-specific increase of immunological reactivity in mice evidenced by accelerated rejection of skin allografts, increased response to oxazolone and sheep erythrocytes. These changes were associated with an increase of thymic size. The response to pneumococcal polysaccharide, which depends upon B-cells, was not increased. Orchidectomised mice showed protection against induced methylcholanthrene sarcomas and transplantable tumours; tumours which are not obviously hormone dependent.


Relief of duodenal ulcer sysmptons by oral metiamide.

Thirty patients with symptoms of duodenal ulceration were treated for five to eight weeks in a double-blind trial with either metiamide 1 g daily by mouth or a placebo. In the 15 patients receiving metiamide there were significant reductions in nocturnal pain and antacid consumption. Daytime pain was diminished. The results suggest that histamine H2-receptor antagonists are likely to be useful in the medical management of the symptoms of duodenal ulceration.


Long-term study of indomethacin and alclofenac in treatment of rheumatoid arthritis.

Indomethacin and alclofenac were compared for 13 months under double-blind conditions in 109 patients with active, classical, or definite rheumatoid arthritis at a relatively early stage of the disease. Both indomethacin and alclofenac were clearly effective: most patients either improved or remained as well controlled as on entry. Alclofenac proved the more effective drug, however, producing a significantly greater reduction in morning stiffness, articular index, and erythrocyte sedimentation rate, and only in the alclofenac-treated group did functional capacity improve and latex-agglutination titres diminish. Comprehensive laborabory tests showed no significant deviation from normal which could have been attributed to either drug.


Comparison of intravenous AH 5158 (ibidomide) and propranolol in asthma.

The cardiac and bronchial effects of AH 5158 and propranolol were compared in a double-blind, placebocontrolled intravenous study on 10 asthmatics. AH 5158, like propranolol, is a non-cardioselective beta-adrenoceptor-blocking drug, but unlike propranolol it also has an alpha-adrenoceptor-blocking action. Both drugs produced equivalent cardiac(beta1) blockade, but propranolol produced bronchoconstriction, whereas AH 5158 did not. Hence the alpha-blocking action of AH 5158 seems to prevent the bronchoconstrictor effects of propranolol in these patients.


Response of lymphocyte guanyl cyclase to propranolol, noradrenaline, thymoxamine, and acetylcholine in extrinsic bronchial asthma.

The lymphocyte guanyl cyclase response to alpha-agonists was studied in 10 normal people and 12 patients with bronchial asthma. In the normal subjects alpha-adrenergic stimulation with noradrenaline plus propranolol and cholinergic stimulation with acetylcholine evoked significant increases in cyclic guanosine monophosphate formation. In addition the alpha-receptor blocking drug thymoxamine produced a significant stimulation of this enzyme system, and the effects of thymoxamine and acetylcholine were additive. This suggests that receptors for cholinergic and alpha-adrenergic agents are independent. In contrast, lymphocyte guanyl cyclase activity did not show a significant response to these agents in patients with acute asthma. In asthmatic patients in remission the responses were partially restored. The significance of these results for control of bronchomotor tone and the relation of guanyl cyclase activity to cyclic adenosine monophosphate in normal subjects and patients with asthma is discussed.


Controlled study of atenolol in treatment of hypertension.

The antihypertensive effect of atenolol, a new beta-1-receptor blocking agent, was studied in a double-blind trial in which 45 patients with essential hypertension were randomly assigned to placebo or atenolol treatment. Atenolol caused a statistically significant and clinically relevant reduction of blood pressure. The optimum daily dose for moderately severe hypertension was considered to be 200 mg. Several irrelevant side effects were collected by the use of a check list, but there was no difference in the number of complaints during placebo and active treatment. Atenolol has a useful antihypertensive effect and, at least theoretically, has advantages over other beta-adrenergic blocking agents.


Neurons from fetal rat brain in a new cell culture system: a multidisciplinary analysis.

A new culture system for cells from the mammalian brain was developed by a modification of a previously established technique. This modification involved the use of fluorodeoxyuridine and adult horse serum. The cultures contained large, easily visualized neurons both isolated from other neurons and in networks of varying complexity. These cells were large enough to permit reliable intracellular electrophysiologic recording and were often sufficiently dispersed to allow examination of membrane responses to iontophoretically applied neurotransmitter candidates. Many responses characteristic of central neurons in situ were seen, including evoked and spontaneous action potentials, complex patterns of inhibitory and excitatory post-synaptic potentials, and neurotransmitter-induced membrane responses. These preparations were examined by phase contrast microscopy, by light microscopy after silver impregnation and by Nomarski interference optics. Total choline acetyltransferase (CAT) activity was little changed and specific activity was increased in the new culture system as compared with the earilier system. Conditions which gave the highest specific activity of CAT also provided the best cultures from the standpoint of electrophysiologic and morphologic analysis. This new approach will allow, in culture, detailed multidisciplinary analyses of individual neurons and small networks of neurons from the mammalian brain.


The inotropic and chronotropic responses of the guinea pig and dog myocardium to isoprenaline and salbutamol.

The relative effects of isoprenaline and salbutamol on the inotropic and chronotropic responses of the denervated myocardium of the chloralose anesthetized dog and of the isolated guinea pig atrium, and the inotropic response of the isolated dog papillary muscle were studied. Both the in vivo dog heart and the in vitro guinea pig atrium displayed a similar relative response pattern to isoprenaline and salbutamol with regard to their inotropic and chronotropic responses. However, a comparison of the relative inotropic responses of the dog heart in vivo and in vitro showed that in vitro, salbutamol has a much lower affinity and efficacy for the adrenergic receptors than isoprenaline.


Magnesium binding in the digesta of the chicken.

Two experiments involving laying hens and mature roosters were carried out to determine the effect of pH on the ultrafilterable fraction of Mg((Mg)u) present in the digesta along the gastrointestinal tract. Concurrently, the approximate molecular weight of the (Mg)u was determined. The average pH's for laying hens were: crop 4.88, proventriculus 5.27, ventriculus 4.77, duodenum 5.57, jejunum 6.15, ileum 7.82, and colon 6.65. Similar pH values were obtained for mature roosters. The (Mg)u fraction decreased significantly in the ileum where the pH was greater than 7.00. However, treating the ileal digesta with an acid pH buffer released about 70% of the nonfilterable Mg. The molecular weight of the (Mg)u was found to be less than 700, suggesting that the Mg was present in digesta as an inorganic salt or as an organic complex of low molecular weight.


Long-term therapy of chronic bacterial prostatitis with trimethoprim-sulfamethoxazole.

Trimethoprim (TMP) meets all of the theoretical requirements of diffusion into, and actual concentration in, human prostatic fluid. When TMP is combined with the sulfonamide sulfamethoxazole (SMX), potentiation of antibacterial activity is achieved and the development of resistant bacterial strains is less likely to occur. In our initial use of TMP-SMX in the treatment of 13 men with chronic bacterial prostatitis due to gram-negative organisms, patients were given two tablets of TMP-SMX twice daily for only 14 days. The results were that two patients (15%) were cured, nine patients (70%) were improved (sterile prostatic fluid during therapy) but eventually relapsed, and two patients (15%) were unchanged by therapy. In our present study 19 patients (31.6%) were totally cured and 9 of 23 (39.1%) gram-negative organisms were permanently cleared from prostatic fluid; 8 of the 9 patients (42.1%) were improved but eventually relapsed with the same organism; 5 of the 19 patients (26.3%) were considered unchanged by therapy.


Encephalitis after administration of live measles vaccine.

In a previously well child with no evidence of pre-existing immunologic defect a fatal encephalitis developed 10 days after administration of measles vaccine. There was pathologic evidence of an early viral encephalitis characterized by perivascular mononulcear infiltrates. Although the virus was not recovered, the diagnosis of a measles virus infection and encephalitis is supported by the postmortem findings of Warthin-Finkeldey cells in lymphoid tissues, an intranuclear inclusion in the brain and histologic changes of encephalitis.


Characterization and subunit analysis of ferritin isolated from normal and malignant human liver.

Ferritin was purified from normal, fetal, and malignant liver tissue. Ferritin purified from hepatoma tissue migrated slightly faster than normal human liver ferritin in polyacrylamide gel electrophoresis. Hepatoma and fetal liver ferritin contained an acidic components in gel and liquid isoelectric focusing not found in normal liver ferritin. We have called it a carcinofetal isoferritin. The subunit compositions of ferritins purified from human liver cell carcinoma and normal liver were then compared. Both ferritin consisted of a subunit species with an identical molecular weight of approximately 18,500. A single subunit of similar molecular weight was also demonstrable after dissociation of 8 M urea and by gel filtration in urea. Two subunits were demonstrable in normal liver ferritin by means of acrylamide electrophoresis in 8 M urea in acid pH. The same two subunits were also demonstrable in ferritin isolated from human liver cell carcinoma. However, a third subunit, intermediate in charge between the two normal liver subunits, was demonstrable in different amounts in ferritins from two hepatomas. Ferritins from normal and malignant livers were immunologically indistinguishable. The tumor-specific acidic isoferritin was isolated and antisera were prepared. The isolated acidic isoferritin was found to be immunologically identical to normal liver isoferritins. It is concluded that the multiple isoferritins of the human liver ferritin consist of two subunits, which are identical in molecular weight but which differ in net charge. Ferritin, isolated from two human liver carcinoma tissues, was composed of the same two subunits and a third unique subunit. Different amounts of these subunits may account for the several normal isoferritins and a unique tumor-specific acid isoferritin found in hepatoma.


Stability and dissociation of P3H4-1 Burkitt's lymphoma cell soluble complement-fixing antigen identified with human serum.

The soluble complement-fixing antigen of the P3HR-1 Burkitt's lymphoma cell line, identified with human serum containing antibody against Epstein-Barr virus viral capsid antigen, loses activity under a variety of conditions. The major characteristic reported here is retention of activity on exposure to specific physical or chemical conditions followed by loss of activity after subsequent neutral dialysis. Antigen of untreated cell lysates, which retains activity after dialysis against large volumes of neutral buffers or 0.14 M NaCl, will lose activity if the lysate is first heated to 56 degrees or if the lysate is exposed to acid perchlorate, and the resulting precipitate and supernatant are redialyzed separately to neutral pH. The P3HR-1 soluble complement-fixing antigen appears to be an aggregate including a labile, dissociable component.


Changes in fine structure accompanying estrogen-induced tumorigenesis of Leydig cells in the mouse testis.

The development of estrogen-induced Leydig cell tumors in cryptorchid BALB/c mice was studied with the electron microscope. Changes in Leydig cell fine structure are apparent by 10 days after the s.c. implantation of a pellet of diethylstibestrol (DES). The smooth endoplasmic reticulum is diminished, and there is an increase in lipid droplets and free polysomes as compared with untreated cryptochid controls. These alterations persist as the Leydig cells proliferate to form focal areas of hyperplasia in the interstitial tissue. During this period of proliferation, activated macrophages containing large residual bodies appear among the Leydig cells. If DES treatment is continued for several months, malignant Leydig cell tumors, result. They are characterized by a nuclear and cytoplasmic pleomorphism of the Leydig cells and a decreased macrophage population. Virus-like particles are rarely seen within the cell during the period of tumorigenesis. Along with the reduction in smooth endoplasmic reticulum in the Leydig cells after DES treatment, evidence from the literature suggests that there is also a decrease in testosterone biosynthesis. However, it is not clear whether these two effect are correlated, since the level of the microsomal enzymes of steroid biosynthesis may vary independently of either the amount of smooth endoplasmic reticulum or the level of androgen secretion. The increase in lipid droplets seen in Leydig cells after DES treatment suggest the accumulation of precursors from the steroid biosynthetic pathway. The macrophages are though to represent scavenger cells, rather than a primary tumor cell population. The paucity of virus-like particles within altered Leydig cells implies that formed virus is not a prerequisite for tumorigenesis.


Nuclear protein changes in rat hepatomas correlating with growth rate.

The nature of nuclear proteins that are soluble in 8 M urea-50 mM phosphate, pH 7.6, was compared in rat liver and Morris hepatomas, Isoelectric focusing, using carrier ampholytes for a pH gradient of 3.5 to 10, indicated that with increasing growth rate of the hepatomas there was a progressive tendency for a decrease in nonhistone nuclear proteins with isoelectric points in the range 7.5 to 8.9 and an increase in the range 5.1 to 6.7. Studies on the influence of time on the pH gradient revealed that a nonuniform drift provided a better resolution of the pH range 7.5 to 8.9 at 7 hr than at 24 hr, while the latter time for electrofocusing gave an improved resolution of the pH range 5.1 to 6.7 Polyarcylamide gel electrophoresis in a urea-acetic acid system showed that 8 M urea-50 mM phosphate; pH 7.6 extracted a small part of the histones from nuclei of both liver and hepatomas. There was less extraction of histones from the hepatoma nuclei, especially in two rapidly growing hepatomas with the most notable difference being seen in the lysine-rich H1 histone. The results suggested that in addition to qualitative or quantitative changes in nonhistone nuclear proteins in liver cancer there are alterations in the binding of histones to chromatin.


Altered distribution and excretion of N-1-methylnicotinamide in rats with Walker 256 carcinosarcoma.

Levels of nicotinamide and N-1-methylnicotinamide in serum, liver, and kidney as well as renal clearances and 24-hr urine levels of N-1-methylnicotinamide were compared in normal rats and rats bearing Walker 256 tumors. There was no significant difference between normal and tumor-bearing rats with regard to nicotinamide levels. With regard to N-1-methylnicotinamide, tumor-bearing rats had significantly lower serum and liver levels and significantly higher 24-hr urine levels and renal clearances. Walker 256 tumor tissue and liver and kidney from a normal and a tumor-bearing rat were separately examined for S-adenosylmethionine:nicotinamide methyltransferase activity. The specific activity in tumor tissue extract was greater than that in each liver extract, which, in turn, was much greater than the specific activity in each tissue (liver and kidney) from the tumor-bearing rat was equal to the specific activity in the corresponding tissue of the normal rat. S-adenosylmethionine:nicotinamide methyltransferase was obtained with 18-fold purification from a tissue extract of Walker 256 tumor. The enzyme activity required activation by thiols, and maximal activity was observed at pH 8.6. The Km's for the substrates, S-adenosylmethionine and nicotinamide, were 7.0 x 10--3 mM and 0.50 mM respectively. The Ki's for the products, S-adenosylhomocysteine and N-1-methylnicotinamide, were respectively, 25 x 10--3 mM and greater than 5 mM.


Differential alkali-extraction of hemicellulose and hydroxyproline from non-delignified cell walls of lupin hypocotyls.

The extraction by alkali of hemicellulose polysaccharides and polymer hydroxyproline from non-delignified, primary cell-walls of lupin hypocotyls has been studied, using sequential extractions at 0 degrees and 18-22 degrees. 10% Aqueous potassium hydroxide at 0 degrees rapidly removed about two-thirds of the hemicellulose normally extracted in 10% KOH at 18-22 degrees and including nearly all of the hemicellulose-A. Little hydroxyproline was released at 0 degrees. When the temperature was subsequently raised to 18-22 degrees, the remaining 10% KOH-soluble hemicellulose, along with most of the hydroxyproline, was released. The monosaccharide composition of these fractions changed markedly with the time of extraction. Arabinose increased from 35% of the polysaccharide extracted during the first hour at 18-22 degrees to similar to 65% of the polysaccharide extracted between 16 and 20 h at 18-22 degrees. Hydroxyproline changed similarly as a proportion of polymer. The implications of these and other results are discussed in relation to polysaccharide and polymer extraction without prior delignification and to models of the primary cell-wall.


Purification and properties of coffee-bean alpha-D-galactosidase.

A purification method for alpha-D-galactosidase from Coffea canephora is described. Two enzymes, alpha-D-galactosidases I and II, having molecular weights of 28,000 and 36,500, respectively, were found and extensively purified. The reaction mechanism of alpha-D-galactosidase II was studied. The enzyme hydrolyzed aryl and alkyl alpha-D-galactopyranosides and was severely inhibited by excess of these substrates. No inhibition occurred with raffinose. The influence of para substituents on the reaction rate of phenyl alpha-D-galactopyranosides, the effect of added alcohols, and the non-competitive inhibition by methyl alpha-D-galactopyranoside were investigated. A two-step mechanism with the formation of an enzyme-galactosyl complex is proposed. With aryl galactopyranosides, the reaction of the enzyme-galactosyl complex with water is rate-limiting. Influences of the substituents on the inhibition constant were investigated by linear free-energy relationships, and significant correlations between this constant and electronic parameters could be calculated. The influence of pH on the reaction is complex.


The structure of Klebsiella serotype II capsular polysaccharide.

Using periodate oxidation, methylation analysis, the characterization of oligosaccharides obtained by partial acid hydrolysis, p.m.r. spectroscopy, and analytical ultracentrifugation, the structure of the (mildly alkali-treated) Klebsiella serotype 11 capusular polysaccharide has been elucidated. The tetrasaccharide repeating-unit comprises the sequence yields 3)-beta-D-Glcp-(1 yields 3)-beta-D-GlcUAp-(1 yields 3)-alpha-D-Galp-(1 yields with a 4,6-O-(1-carboxyethylidene)-alpha-D-galactosyl residue linked to O-4 of the glucuronic acid residue. The structural basis for some serological cross-reactions of the Klebseilla K11 antigen is discussed, and it is shown that rabbit antisera against the Klebsiella K11 test-strain predominantly contain K agglutinins specific for branch-terminal 4,6-O-(1-carboxyethylidene)-D-galactose.


Bacteriophage-borne enzymes in carbohydrate chemistry. Part I. On the glycanase activity associated with particles of Klebsiella bacteriophage No. 11.

The preparation and use of particles of Klebsiella bacteriophage No. 11 are described. A glycanase activity associated with the viruses catalyses the depolymerization of (alkali-treated) Klebsiella serotype 11 capsular polysaccharide, ultimately to a mixture of oligosaccharides consisting of one or two repeating units. Mainly glucosidic bonds are hydrolysed. The substrate specificity of the viral enzyme has been characterized by using derivatives of serotype-11 polysaccharide, as well as 81 heterologous, bacterial, capsular glycans. It is concluded that the glycanase will (at least) also depolymerize all polysaccharides containing the unsubstituted chain-trisaccharide repeating-unit of its natural substrate.


Studies on the structure and mechanism of an exo-(1 yields 3)-beta-D-glucanase from Basidiomycete QM806.

A method for the large-scale production of a (1 yields 3)-beta-D-glucan glucohydrolase (EC 3.2.1.58) from the culture filtrate of Basidiomycete QM806 is described. The final preparation is homogeneous by disc electrophoresis under non-dissociating and denaturing conditions, by ultracentrifugation, and by isoelectric focusing. Various physical and chemical characteristics of the enzyme have been determined, including terminal amino acid residues, extinction coefficient, and stability to pH extremes. The N-terminal amino acids are leucine and serine (Sanger's method) and the C-terminal amino acids are alanine, serine, and glycine (hydrazinolysis). pH profile studies show that no group titrating in the region 2.5-8 is directly involved with substrate binding and that a single group having a pKa of 6.5 is involved in the catalysis. Photooxidation of the enzyme caused rapid inactivation. The pH-dependence of this photooxidation, and amino acid analysis of the photooxidized enzyme, indicate that decomposition of histidine is probably responsible for the loss of activity. Other chemical modifications performed were: treatment with hydrogen peroxide under acidic conditions, esterification with diphenyldiazomethane, and oxidation with N-bromosuccinimide. Oxidation with N-bromosuccinimide indicated that a tryptophan side-chain is involved in, but not necessary for, the catalytic activity.


Ultrastructural study of crystalloids in sertoli cells of the normal, intersex and experimental cryptorchid swine.

Spindle- or needle-shaped crystalloids are observed in Sertoli cells of the intersex and experimental cryptorchid swine in the light and electron microscopes. Small crystalloids are also observed in Sertoli cells of the normal swine only by electron microscopy. These crystalloids consist of fine filaments. The filaments are about 5 nm in diameter and arranged parallel to the long axis of the drystalloid. In cross sections of the crystalloid, the close backing of the filaments shows hexagonal arrays. The interfilamentous distance is about 5 nm. In all animals, bundles of short filaments, which are 5nm in diameter, are observed in the basal part of the Sertoli cells. Ultrastructural similarities among the crystalloids, the bundles of fine filaments, and the filamentous layer in the junctional specialization of the Sertoli cell are shown. These morphological similarities suggest that the crystalloids are formed by the aggregation of the bundles in the Sertoli cells of azoospermic testes.


D-valine as a selective agent for normal human and rodent epithelial cells in culture.

A nutrient medium has been developed to enable the growth of normal epithelial cells while selectively inhibiting fibroblast proliferation. In this medium, D-valine is substituted for L-valine; and only those cells containing D-amino acid oxidase can convert the D-amino acid into its essential L-enantiomer. The ability to select for cells with this enzyme has enabled us to maintain epithelial cell populations free from fibroblast overgrowth. The presence of D-amino acid oxidase has been histochemically confirmed in the epithelial cells selected from renal cell suspensions and explants. The ability to proliferate in the selective medium is transmitted to the clonal progeny of these cells. Moreover, epithelial cell proliferation of this medium indicates the presence of D-amino acid oxidase, which we have detected in tissues where it had not previously been reported-fetal human kidney, lung, and cord. Fibroblasts will not grow in the selective medium, but will proliferate normally if the product of the D-amino acid oxidase reaction is supplied.


"Superinduction" of tyrosine aminotransferase by actinomycin D: a reevaluation.

Reexamination of the effects of actinomycin D (AMD) on the intracellular level and rate of synthesis of tyrosine aminotransferase (TAT) in hepatoma tissue culture (HTC) cells reveals that much apparent controversy can be resolved with acknowledgment of the multi-faceted nature of this inhibitor's action. AMD can slow overall protein synthesis and inhibit the degradation of both TAT and its mRNA as well as block the synthesis of RNA. The extent of these secondary actions of the inhibitor depend somewhat upon the growth condition of the cells. The effects of cordycepin (3'-deoxyadenosine) on the metabolism of TAT and its mRNA are also complex, but differ in several respects from those of AMD.


Interaction of cortisol with the neural retina of the chick embryo in culture.

The uptake of cortisol and the kinetics of hormone-receptor interaction in the cytosol and cell nucleus were investigated in the intact tissue in organ culture. Cortisol is concentrated by the neural retina. The accumulation of the free steroid is temperature dependent but the effect of temperature decreases with the increase of cortisol in medium. Cortisol binding to specific receptors in the cytosol shows a sigmoidal type of kinetics which correlates well with the kinetics of glutamine-synthetase induction by cortisol. The temperature dependent translocation of the receptor-hormone complexes to the nuclei and the effect of detergents on the binding to nuclei are presented.


Vascular responses to prostaglandin F 2 alpha in isolated cat lungs.

Changes in perfusion pressure in response to graded doses of prostaglandin F 2 alpha (PGF 2 alpha) were measured during both forward and retrograde perfusion of isolated cat lungs perfused at a constant flow rate. During forward perfusion, PGF 2 alpha produced a dose-dependent increase in pulmonary artery pressure and a decrease in lung fluid volume. During retrograde perfusion, PGF 2 alpha also produced a dose-dependent increase in perfusion pressure; however, the dose required was fivefold greater than that needed to produce an identical change in pressure during forward perfusion. In addition, during retrograde perfusion, the lung fluid volume increased in response to PGF 2 alpha. These results suggest that the major site of activity of PGF 2 alpha is on the arterial side of the pulmonary vascular bed and that inactivation of PGF 2 alpha by the lung occurs primarily distal to this arterial site of vasomotion. The changes in perfusion pressure in response to PGF 2 alpha were markedly dependent on pH and oxygen tension (Po-2), being abolished by severe alkalosis and potentiated by both acidosis and hypoxia. In contrast, neither serotonin nor norepinephrine exhibited such a pH or Po-2 dependency. Since the ratio of the forward response to the retrograde response was not decreased by alterations of pH or Po-2, their influence on the responses appears to be through interaction at the site of vascular activity rather than through alteration of the rate of prostaglandin inactivation.


"Prorenin" in human plasma?

An increase in plasma renin activity was demonstrated in plasma from normal subjects and most patients with essential hypertension after prolonged storage of the plasma at -20 degrees C. No change in renin substrate concentration was observed after storage for 12 months, and the rate of angiotensin generation during incubation with a fixed amount of added human renin was not increased. It therefore seems unlikely that the observed increase in renin activity was due to changes in plasma activators or inhibitors. The data are consistent with the activation of an inactive form of renin, tentatively called "prorenin" in normal subjects averaged from 1 to 2 ng/ml/hr, and did not change during sodium deprivation. However, three of four patients studied who had low-renin essential hypertension secreted abnormally large amounts of "prorenin" in response to sodium depletion. In these patients, subsequent sodium administration was associated with a return of "prorenin" to baseline levels.


Dissociation between renin and arterial pressure responses to beta-adrenergic blockade in human essential hypertension.

Studies were carried out in 69 patients with essential hypertension to examine the relationship between changes in plasma renin activity (PRA) and arterial pressure (BP) in response to a beta-adrenergic blocking agent, propranolol. PRA had no consistent relationship with BP during treatment, either in patients receiving propranolol alone (r = 0.12) or in those receiving a combination of diuretics and propranolol (r = 0.18). Furthermore, long-term beta-adrenergic blockade failed to inhibit increases of PRA induced by diuretics or rapid sodium depletion. These results indicate that (1) beta-adrenergic blockade can reduce BP by mechanisms other than PRA suppression; and (2) the beta-adrenergic nervous system is important, but not essential, for renin release.


Dopamine induced relaxation of isolated canine renal, mesenteric, and femoral arteries contracted with prostaglandin F2-alpha.

The purpose of this investigation was to develop a system for studying the effects of dopamine on isolated blood vessels. Canine renal, mesenteric, and small femoral arteries (less than 1 mm outside diameter) were exposed to phenoxybenzamine 10-5 M for one hour and contracted with prostaglandin F2-alpha. Cumulative concentrations of dopamine ranging from 10-6 to 10-4 M caused dose-related relaxation of the arteries. Propranolol 10-6 M did not affect the relaxation in concentrations which markedly antagonized the effects of isoproterenol. Large femoral arteries (greater than 1 mm outside diameter) did not relax with similar concentrations of dopamine. N-methyldopamine (epinine) produced similar relaxation; 3-methoxytyramine was inactive. Specific antagonism could not be demonstrated by the postulated dopamine antagonists-haloperidol, chlorpromazine, apomorphine, or bulbocapnine-in concentrations up to 10-5 M. Higher concentration of these agents could not be used because they caused the arteries to relax. This study demonstrated that PGF2-alpha-contracted arteries pretreated with phenoxybenzamine are suitable for further investigations of putative dopamine agonists and antagonists.


Effects of glucose, insulin and potassium infusion on tissue metabolic changes within first hour of myocardial infarction in the baboon.

The effects of infusions of glucose, insulin and potassium (GIK) on the heart tissue metabolic changes found in adult baboons 60 min after coronary artery ligation were studied. Biopsies taken from 11 baboons without coronary artery ligation gave control values. A second group of 46 baboons had coronary artery ligation. A third group of 17 baboons received an infusion of KCl after coronary artery ligation. A fourth group of 26 baboons received infusion of GIK. Coronary artery ligation resulted in the expected fall of ATP, creatine phosphate, glycogen, tissue (K+/Na+) ratio, and tissue pH, and rise of inorganic phosphate, lactare, lactate/pyruvate ratio and alpha-glycerophosphate in the infarction zones. Compared with ligation, additional infusions of GIK approximately doubled the contents of creatine phosphate and glycogen in the infarct zones, increased the content of ATP in the central infarct zone, and decreased the content of inorganic phosphate in the peripheral infarct zone. Other GIK effects were that the tissue (K+/Na+) ratio rose in the peripheral infarct zone, and the content of both glycogen and lactate rose in the peri-infarct and non-ischemic zones; the pH of tissue homogenates did not decrease. KCl infusions had few effects compared with the ligation group. GIK infusions exerted a beneficial effect when compared with infusions of KCl in that tissue creatine phosphate rose in the peripheral infarct and nonischemic zones; the tissue K+/Na+ ratio rose in the peripheral infarct, peri-infarct, and nonischemic zones; and the lactate/pyruvate ratio fell in the infarct zone. It is proposed that GIK counteracted early tissue metabolic deterioration in the infarcting baboon heart.


Calibration of ion-selective electrodes for use in biological fluids.

We measured electromotive force at 37 degrees C with pH, sodium glass, valinomycin, and the Simon calcium electrodes in synthetic electrolyte mixtures simulating serum, in cells both with and without liquid junction. Evidently these electrodes respond in a near-Nernstian manner to changes in concentration of the ion sensed. We su-gest that such mixtures may serve as calibrating standards for ion-selective electrodes in clinical analysis. The effect of ionic strength on electrode response can be accounted for almost wholly by alterations in the activity coefficients. The lanthanum fluoride electrode activity coefficients. The lanthanum fluoride electrode shows promise as a reliable reference in cells without liquid junction.


Quantitation of free, total, and antibody-bound insulin in insulin-treated diabetics.

We describe a simplified method for measuring free, total, and antibody-bound insulin in insulin-treated patients in whom antibodies to insulin are present. The free, active insulin is extracted from the serum with a polyethylene glycol solution. Total insulin is extracted from the serum with a polyethylene glycol solution after dissociation of the antibody-antigen complex with dilute HCI. Aliquots of the extracts are used in the radioimmunoassay system. The figure for antibody-bound insulin is the difference between the total and free insulin values and reflects the concentration of insulin antibodies present. A commercially available ("Phadebas") radioimmunoassay for immunoreactive insulin was used to quantitate the insulin present in the two extracts. Recovery of added insulin averaged 85% for the free insulin and 87%for the total insulin.


Enzyme activities of NADPH-forming metabolic pathways in normal and leukemic leukocytes.

With respect to the enzymes of NADPH-forming metabolic pathways in human leukocytes: (a) Glucose-6-phosphate dehydrogenase and phosphogluconate dehydrogenase (decarboxylating) were less active in leukocytes (mostly myeloblasts) from eight patients with acute myeloblastic leukemia (I) than in leukocytes (mostly granulocytes) from 16 normal subjects (II). (b) Of the enzymes of the citrate cleavage pathway, ATP citrate lyase and malate dehydrogenase (decarboxylating) (NADP+) were virtually absent in the cells studied. (c) Isocitrate dehydrogenase (NADP+), aspartate aminotransferase, and alanine aminotransferase, which, together with the much more active malate dehydrogenase, constitute a newly proposed NADPH-forming metabolic cycle, showed a higher activity in I than in II or III, and therefore could compensate, as concerns NADPH-generation, for the low activity of pentose cycle dehydrogenases. We are not sure whether the enzymatic characteristic of I cells is attributable to their immaturity or to their leukemic nature.


Colorimetric enzymatic determination of serum total carbon dioxide, as applied to the Vickers Multichannel 300 discrete analyzer.

Phosphoenolpyruvate carbosylase (EC 4.1.1.31) has been used in developing a simple, inexpensive colorimetric assay for serum total carbon dioxide in an open system. The oxaloacetate formed by the action of the enzyme on bicarbonate and phosphoenolpyruvate is measured by use of the diazonium salt of Fast Violet B. Interferences from billirubin, pyruvate, and drugs are negligible. Acetoacetate interference is significant only in highly ketotic samples, and a serum blank corrects for it. Serum protein interference is equivalent to 3.3 plus or minus 1.25 mmol of CO2 per liter and hence is sufficiently constant to be corrected for by use of a serum standard or serum blank. The method has been applied to the Vickers M-300 and D-300 systems and within-batch standard deviations of plus or minus 0.1 to plus or minus 0.6 mmol/liter have been observed. Excellent correlation with orthodox techniques has been obtained.


A kinetic study on the influence of the parameters in the determination of inorganic phosphate by the molybdenum blue reaction.

Parameters which influence the initial rate of formation of phosphomolybdenum blue from phosphate, molybdate, polyvinylpyrrolidone and o-phenylenediamine were investigated. From kinetic data it is concluded that polyvinylpyrrolidone not only acts as a protective colloid, but that it takes part in the reaction and a reaction mechanism is proposed dealing with the stoichiometry of polyvinylpyrrolidone. A rate procedure for the determination of inorganic phosphate in serum and urine, based on this study, is given. Deproteinization of samples is not necessary. The method is rapid requiring one minute for quantitative readings. Because of this short period there is hardly any hydrolysis of organic phosphate esters. As the rate of color development is determined blanks can be omitted. Turbid samples may be analysed with little error.


Cathodic oxygen consumption and electrically induced osteogenesis.

Small amounts of electric current stimulate bone formation in the region of a cathode. The purpose of this experiment is to compare changes in oxygen and hydroxyl ion concentration that occur at the cathode at current levels known to be capable of inducing osteogenesis (10-20 muamps) with those changes that occur at current levels known to be toxic to bone (100 muamps). An oxygen consumption chamber containing an oxygen electrode is fitted with two stainless steel electrodes which are connected to a constant current source. At the cathode, with a current of 100 muamps, oxygen is consumed at nearly stoichiometric rates. At higher current (100 muamps) levels, cathodic oxygen consumption gives way to hydrogen evolution. Cathodic hydroxyl ion production is directly proportional to current. It is concluded from these in vitro experiments that at 10-20 muamps the oxygen tension in the vicinity of the cathode is lowered and the pH is moderately increased. At 100 muamps the oxygen tension is not lowered, but the pH is increased dramatically. If these same changes occur in the vicinity of a cathode in vivo, then lowering the local tissue oxygen tension and raising the local pH may be mechanisms operative in electrically induced bone formation.


[Differences in the quality of vaccinia viruses with normal and abnormal post-vaccination course (with neurological complications) (author's transl)].

Conventional vaccinia strains were cultured at different temperatures, because it is known that vaccinia viruses which are human pathogens grow even at higher temperatures (41 degrees C). Neither the vaccination strains nor the vaccinia viruses which were isolated from vaccination vesicles after normal (n equals 7) or abnormal primary vaccinations (n equals 2) multiplied at higher incubation temperatures; Vaccinia viruses isolated from CSF of four patients with neurological complications behaved differently. Uninhibited growth at 41 degrees C (with high infection titres) suggest that the viruses isolated from CSF were vaccinia variants with abnormal characteristics. Accordingly, these variants morphologically formed plaques in Vero-cell cultures which were two to three times larger than those of vaccinia viruses obtained from CSF of primary vaccinated cases after only moderately severe general reactions, and which on temperature test responded like the viruses used in the original vaccination.


The oxidation of arsenite in seawater.

The oxidation rate of arsenite in seawater is very slow. Of the parameters investigated which might affect this rate the most important were temperature, initial arsenite concentration, salinity and pH. An empirical rate expression was developed from experimental data which led to a prediction that the natural rate of oxidation in the ocean is about 0.023 micromoles of As(III) per liter each year. This value is about twice the natural concentration of arsenite; the only mechanism which seems able to account for the continued presence of arsenite is biological reduction of arsenate.


Enzymatic degradation of protein mixtures.

The rate of protein enzymatic degradation in a mixture is slower than that of single proteins. In a mixture of haemoglobin or casein with protamine, the release of tyrosine by trypsin and chymotrypsin A is slower. A slower rate in the release of arginine from protamine occurs only in mixtures with haemoglobin. The inhibition of enzymatic degradation of proteins in a mixture is due to formation of intermolecular associations and to changes in their spacial structure.


Deoxyribonuclease in human parotid saliva.

Deoxyribonuclease (EC 3.1.4.5, EC 3.1.4.6) activities in human parotid saliva were examined by microdisc electrophoresis. Three fractions, differing in their electrophoretic mobility and in their optimal incubation conditions, were characterized. The various enzyme activities were tested at pH 5.0 in 100 mmol with l-minus 1 Na-acetate buffer or at pH 7.04 in 100 mmol with l-minus 1 Tris-HCl-buffer in the presence of different combinations of MgCl(2), CaCl(2), EDTA, and Na(2)SO(4).


Influence of possible in situ ionic environment on kinetics of purified citrate synthase from an osmoconformer sea anemone, Bunedosoma cavernata.

Kinetics of utilization of acetyl coenzyme A by citrate synthase of a sea anemone, an osmoconformer, were compared with those of citrate synthases of various osmoregulators. The Kms of the latter enzymes were substantially increased by higher concentrations of salt and the enzyme exhibited hyperbolic substrate saturation curves. Citrate synthase from sea anemone, on the other hand, exhibited allosteric kinetics and minimal effects of salt on its Km. We suggest that the adaptive advantage of this enzymic property to a sedentary osmoconforming organism such as sea anemone is obvious since the osmoregulating creatures are apparently unable to maintain an appropriate low ionic environment in situ and thus probably the Km of their citrate synthases at a low level.


Separation of two L-glutamine-hydroxylamine glutamyltransferases from rat liver.

The purification of glutamine synthetase (GS) from rat liver demonstrates that a small portion of glutamine-hydroxylamine-glutamyltransferase activity (GT) remains associated with GS activity (GT(S)). As GS is purified from the water extract, the ratio between GT(S) activity (GT(T)) is left to be extracted by KC1 from the pellet and, on further purification, appears to be independent of GS activity. Subtle differences in pH optimum, substrate requirement and reaction rates on addition of cofactors and amino acids in vitro and in responses to hormonal stimuli in vivo indicate that the glutamine transfer reaction may be catalyzed by two distinguishable proteins; only the minor component may be identical to GS.


Effect of free and conjugated bile salts on alpha-amylase activity.

The effect of individual bile salts on alpha-amylase hydrolysis of Cibachron Blue starch was studied at pH 6.0. With sodium cholate, taurocholate and taurodeoxycholate, enzyme activity was increased to 150-160 percent of the control value, at a concentration of similar to 1 mmol/l bile salt. The increased activity extended up to 4 mmol/l. The bile salts sodium deoxycholate and taurochenodeoxycholate exerted activation and inhibition depending on the concentration. With deoxycholate (0.75 mmol/l), activation (150 percent) was evident, while inhibition was apparent above 2.5 mmol/l. With taurochenodeoxycholate maximum activity (135 percent) was observed at 0.25 mmol/l, while inhibition was evident above 1.5 mmol/l. Chenodeoxycholate and lithocholate exerted marked inhibition at concentrations as low as 0.5 mmol/l. Inhibition of alpha-amylase by chenodeoxycholate was competitive with both soluble and insoluble starch substrates. Since the pH of the jejunum is in the region of 6.0 the phenomenon of activation and inhibition of alpha-amylase by bile salts at this pH could be of physiological significance.