The kinetics of the reduction of cytochrome c by the superoxide anion radical. 1. At neutral pH ferricytochrome c is reduced by the superoxide anion radical (O2-), without loss of enzymatic activity, by a second order process in which no intermediates are observed. The yield of ferrocytochrome c (82-104%), as related to the amount of O2- produced, is slightly dependent on the concentration of sodium formate in the matrix solution. 2. The reaction (k1 equals (1.1+/-0.1) - 10(6) M-1 - s-1 at pH 7.2, I equals 4 mM and 21 degrees C) can be inhibited by superoxide dismutase and trace amounts of copper ions. The inhibition by copper ions is removed by EDTA without interference in the O2- reduction reaction. 3. The second-order rate constant for the reaction of O2- with ferricytochrome c depends on the pH of the matrix solution, decreasing rapidly at pH greater than 8. The dependence of the rate constant on the pH can be explained by assuming that only the neutral form of ferricytochrome c reacts with O2- and that the alkaline form of the hemoprotein is unreactive. From studies at pH 8.9, the rate for the transition from the alkaline to the neutral form of ferricytochrome c can be estimated to be 0.3 s-1 (at 21 degrees C and I equals 4 mM). 4. The second-order rate constant for the reaction of O2- with ferricytochrome c is also dependent on the ionic strength of the medium. From a plot of log k1 versus I1/2-(I + alphaI1/2)-1 we determined the effective charge on the ferricytochrome c molecule as +6.3 and the rate constant at I equals 0 as (3.1+/-0.1) - 10(6) M-1 - s-1 (pH 7.1, 21 degrees C). 5. The possibility that singlet oxygen is formed as a product of the reaction of O2- with ferricytochrome c can be ruled out on thermodynamic grounds.


Algal glyceraldehyde-3-phosphate dehydrogenases. Conversion of the NADH-linked enzyme of Scenedesmus obliquus into a form which preferentially uses NADPH as coenzyme. Scenedesmus obliquus contains two glyceraldehyde-3-phosphate dehydrogenases (EC 1.2.1.-) one of which uses NADH as its preferred coenzyme (D-enzyme) and the other NADPH (T-enzyme). On incubation of the D-enzyme with cysteine and a 1,3-diphosphoglycerate-generating system the specific activity with NADH as coenzyme decreased whilst that with NADPH increased by a factor of 10. The components of the generating system had no effect on the D-enzyme individually and it is concluded that 1,3-diphosphoglycerate was probably responsible for the change in nucleotide specificity. The coenzyme specificity of the T-enzyme was not affected by such treatment. A similar type of activation occurred to a lesser extent on incubation of the D-enzyme with 2,3-diphosphoglycerate. The NADPH-dependent activity of the D-enzyme could also be promoted by incubation with NADPH. However, in this case the activation was less than that seen with either 1,3- or 2,3-diphosphoglycerate. The change in coenzyme specificity of the D-enzyme occurred in parallel with changes in sedimentation behaviour. Initially, a single boundary of S20,w equals 14.5 S was present, but on conversion to NADPH-dependent activity by incubation with the 1,3-diphosphoglycerate-generating system, new boundaries of 7.5 S and 5.5 S appeared. The first of these corresponds in sedimentation coefficient to the native T-enzyme. On removal of 1,3-diphosphoglycerate the 7.5 S boundary disappeared accompanied by an increase in that of 14.5 S, whilst the 5.5 S boundary persisted. These changes are consistent with the reversible conversion of the D-enzyme into a form similar to the native T-enzyme in response to cysteine and 1,3-diphosphoglycerate. These effects may be explained if acylation of the active site of the D-enzyme by 1,3-diphosphoglycerate results in displacement of the bound nucleotide, thus promoting nucleotide exchange. These findings are consistent with the kinetic mechanism established for other glyceraldehyde-3-phosphate dehydrogenases. Similar activation was seen in extracts of other species of the Chlorophyta but not in other photosynthetic organisms. The significance of this type of activation of enzyme activity to the metabolism of these species of algae is discussed.


The uptake of choline by rat liver mitochondria. 1. Rat liver mitochondria can accumulate choline against a concentration gradient. Maximally about 30 nmol choline per mg mitochondrial protein are found in the matrix space. 2. The process of choline uptake is biphasic. After a rapid uptake of 1.5-15 nmol per mg protein, a slower uptake occurs if an energy supply is present. In the absence of energy, only the rapid uptake is found. 3. The inhibition of uncoupler-stimulated choline oxidation by cations is the result of an inhibition of choline uptake.


3alpha-, 7alpha- and 12alpha-hydroxysteroid dehydrogenase activities from Clostridium perfringens. 25 strains of Clostridium perfringens were screened for hydroxysteroid dehydrogenase activity; 19 contained NADP-dependent 3alpha-hydroxysteroid dehydrogenase and eight contained NAD-dependent 12alpha-hydroxysteroid dehydrogenase active against conjugated and unconjugated bile salts. All strains containing 12alpha-hydroxysteroid dehydrogenase also contained 3alpha-hydroxysteroid dehydrogenase although 12alpha-hydroxysteroid dehydrogenase was invariably in lesser quantity than the 3alpha-hydroxysteroid dehydrogenase. In addition, 7alpha-hydroxysteroid dehydrogenase activity was evident only when 3alpha, 7alpha, 12alpha-trihydroxy-5beta-cholanoate was substrate but notably absent when 3alpha, 7alpha-dihydroxy-5beta-cholanoate was substrate. The oxidation product 12alpha-hydroxy-3, 7-diketo-5beta-cholanoate is rapidly further degraded to an unknown compound devoid of either 3alpha- or 7alpha-OH groups. Group specificity of these enzymes was confirmed by thin-layer chromatography studies of the oxidation products. These enzyme systems appear to be constitutive rather than inducible. In contrast to C. perfringens. Clostridium paraputrificum (five strains tested) contained no measurable hydroxysteroid dehydrogenase activity. pH studies of the C. perfringens enzymes revealed a sharp pH optimum at pH 11.3 and 10.5 for the 3alpha-OH- and 12alpha-OH-oriented activities, respectively. Kinetic studies gave Km estimates of approx. 5 X 10(-5) and 8 X 10(-4) M with 3alpha, 7a-dihydroxy-5beta-cholanoate and 3alpha, 12alpha-dihydroxy-5beta-cholanoate as substrates for two respective enzymes. 3alpha-hydroxysteroid dehydrogenase was active against 3alpha-OH-containing steroids such as androsterone regardless of the sterochemistry of the 5H (Both A/B cis and A/B trans steroides were substrates). There was no activity against 3beta-OH-containing steroids. The 3alpha- and 12alpha-hydroxysteroid dehydrogenase activities, although differing in cofactor requirements cannot be distinguished by their appearance in the growth curve, their mobility on disc gel electrophoresis, elution volume on passage through Sephadex G-200 or heat inactivation studies.


Studies on phosphatidylinositol phosphodiesterase (phospholipase C type) of Bacillus cereus. I. purification, properties and phosphatase-releasing activity. A phosphatidylinositol phosphodiesterase from the culture broth of Bacillus cereus, was purified to a homogeneous state as indicated by polyacrylamide gel electrophoresis, by ammonium sulfate precipitation and chromatography with DEAE-cellulose and CM-Sephadex. The enzyme (molecular weight: 29000 +/- 1000) was maximally active at pH 7.2-7.5, AND NOT INFLUENCED BY EDTA, ophenanthroline, monoiodoacetate, p-chloromercuribenzoate or reduced glutathione. The enzyme specifically hydrolyzed phosphatidylinositol, but did not act on phosphatidylcholine, phosphatidylethanolamine and sphingomyelin, under the conditions examined. The products from phosphatidylinositol of enzyme reaction were diacylglycerols and a mixture of myoinositol 1- and 1, 2-cyclic phosphates, suggesting that the enzyme was a phosphatidylinositol-specific phospholipase C. The enzyme released alkaline phosphatase quantitatively from rat kidney slices. A kinetic analysis was made on the release of alkaline phosphatase. The results suggest that phosphatidylinositol-specific phospholipase C can specifically act on plasma membrane of rat kidney slices.


Properties of triacylglycerol lipase in a mitochondrial fraction from baker's yeast (Saccharomyces cerevisiae). A triacylglycerol lipase in a mitochondrial fraction isolated from yeast (Saccharomyces cerevisiae) has been characterized and the hydrolysis studied kinetically using an insoluble artificial triacylglycerol suspension. 1. The triacylglycerol was hydrolyzed almost completely to fatty acids and glycerol. The lipase activity was inhibited by potassium fluoride and the sodium salts of -chloride, -glycocholate and -pyrophosphate as well as by protamine sulfate but at concentrations much too high to indicate that the lipase is a non specific esterase or a lipoprotein lipase. Also parachloromercuribenzoate inhibited the lipase activity. Inhibitory effect of fatty acid was observed at concentrations above 1mM. This inhibition may provide a regulatory mechanism of the lipase in vivo. 2. On the day of isolation the lipase activity of intact mitochondria at pH 7.5 and 30 degrees C was 400 nmol free fatty acid -h-1 - mg-1 at a triacylglycerol concentration of 9.0 mM. Sonication of the mitochondria increased the activity 2-3 fold. Freezing of the mitochondria also activated the lipase and this activation was dependent upon the freezing method, the concentration of mitochondrial protein and the presence of bovine serum albumin. 3. The particulate nature of the assay system was illustrated by the observation that the apparent Km value of the lipase increased with the concentration of mitochondrial protein. For each protein concentration the lipase had two apparent Km values when the activity was assayed with intact mitochondria, but only one when  assayed with submitochondrial particles. At the same protein concentration the Km value for the latter was identical with the "low affinity" Km for the lipase in intact mitochondria.


Properties of microsomal phospholipases in rat liver and hepatoma. Phospholipase A1, A2 and lysophospholipase activities in microsomes of Novikoff hepatoma host rat liver and regenerating rat liver were compared using 1-[9', 10'-3H2]palmitoyl-2-[1'-14C] linoleoyl-sn-glycero-3-phosphoethanolamine, 1-[1' -3H-]hexadecyl-2-acyl-sn-glycero-3-phosphoethanolamine, and 1-[9', 10'-3H2]palmitoyl-sn-glycero-3-phosphoethanolamine as substrates. 1. Microsomes of all three tissues showed two pH dependent peaks of hydrolytic activity, one at pH 7.5 and another at pH 9.5. 2. Phospholipid hydrolytic activity in microsomes from host liver and regenerating liver require Ca2+ for hydrolysis at pH 9.5, but not at pH 7.5. Hepatoma microsomes require Ca2+ for activity at both pH values. 3. Phospholipase A1 activity, stimulated by addition of Triton X-100 to the incubation mixtures, was detected in both host liver and regenerating liver microsomes. There was no evidence of phospholipase A1 activity in hepatoma microsomes. 4. Phospholipase A2 was detected in microsomes of all three tissues using 1-[1'-3H] hexadecyl-2-acyl-sn-glycero-3-phosphoethanolamine as a substrate. The activity required calcium and was inhibited by Triton X-100. 5. Lysophospholipase activity was evident in the microsomes from all three tissues. The activity was inhibited by both Ca2+ and Triton X-100. 6. Differences were also detected between host liver and hepatoma microsomal phospholipid hydrolase activities with respect to the effect of increasing protein concentration, apparent Michaelis-Menten constants, and time course of the reaction.


Purification and positional specificity of sn-glycerol-3-phosphate acyltransferase from Escherichia coli membranes. SN-Glycerol-3-phosphate acyltransferase was solubilized from membranes of Escherichia coli B and K-12 and purified on an affinity column of Sepharose 4B coupled with 6-phosphogluconic acid. Phosphatidylglycerol was required for activation and stabilization of the purified enzyme. The acyl residues were exclusively transferred to the position 1 of sn-glycerol 3-phosphate by the enzyme, regardless of whether the acyl-CoA was saturated or unsaturated.


Isolation and characterization of aldose reductase from calf brain. Aldose reductase activity (alditol: NADP+ 1-oxidoreductase, EC 1.1.1.21) from calf brain was separated into two protein fractions by DEAE chromatography. Further purifcation by molecular sieve chromotography and electrofocusing yielding two distinctive enzymes, which were designated AR I and AR II. AR I was purified 646-fold and found to have an isoelectric point of 6.18. AR I was most active as a monomer with a molecular weight of 29 000 and appeared to be in equilibrium with a less active dimer. AR II was purified 425-fold and found to have an isoelectric point of 4.88. The molecular weight of this enzyme was 30 000. Although both enzymes had specificity for aldoses as substrates, AR I had two to three times larger turnover numbers with aromatic aldehydes and hexonates than did AR II. AR I was activated by sulfhydryl compounds and exhibited biphasic double reciprocal plots. AR I was more sensitive to inhibition by high substrate and phenobarbital concentrations than was AR II. AR I and AR II did not have antigenic similarity as tested by Ouchterlony immunodiffusion and counter immunoelectrophoresis. An immunochemical cross-reaction was observed  between AR II and lens aldose reductase.


Studies on the role of methionine in porcine pancreatic phospholipase A2. The unique methionine-15 residue located at the N-terminal site of iso- or beta-phospholipase A2 from porcine pancrease has been specifically carboxymethylated with iodoacetic acid. The modification results in a complete inactivation of the enzymatic activity toward micellar and monomeric substrates. Spectroscopic measurements reveled that the carboxymethylated protein still binds Ca2+ and monomeric substrates with comparable affinities as the native enzyeme. The active site histidine-54 residue in the modified enzyme shows a reactivity toward the active site-directed irreversible inhibitor p-bromophenacylbromide which is identical to that of the native enzyme. The alkylated protein, however, has lost its ability to bind to lipid-water interfaces. Although circular dichroic spectra of the carboxymethylated enzyme display some changes in the tertiary structure as compared with the native enzyme, the alpha-helix content remains rather constant. It is concluded that carboxymethylation of methionine-15 destroys the interface recognition site but has only limited influence on the active site of the molecule. Therefore, it seems that methionine-15 is not involved in the catalytic events but that this residue is part of the interface recognition site which embraces the N-terminal hydrophobic part of the enzyme: Ala-Leu-Trp-Gln-Phe-Arg-Ser-Met.


The effect of calcium ions on the hydrolysis of benzoylarginine ethyl ester by porcine enteropeptidase. Calcium ions are shown to have a marked pH-dependent effect on the kinetics of benzoyllarginine ethyl ester hydrolysis by porcine enteropeptidase (EC 3.4.21.9). Below pH 6.0, calcium ions stimulate benzoylarginine ethyl ester hydrolysis but inhibit this activity above pH 6.0. This effect is mainly on the Km for benzoylarginine ethyl ester. At pH 5.3, 2mM calcium ions reduce the Km for benzoylarginine ethyl ester from 0.31 mM to 0.26 mM while at pH 6.5 the Km increases four-fold from 0.035 mM to 0.12 mM in the presence of calcium ions. Enteropeptidase activity is not inhibited by ethylenediaminetetra-acetate indicating that calcium ions are a non-essential cofactor for benzoylarginine ethyl ester hydrolysis.


Purification and characterization of bile salt hydrolase from Bacteroides fragilis subsp. fragilis. A high-molecular-weight (250 000) bile salt hydrolase (cholylglycine hydrolase, EC 3.5.-.-) was isolated and purified 128-fold from the "spheroplast lysate" fraction prepared from Bacteroids fragilis subsp. fragilis ATCC 25285. The intact enzyme had a molecular weight of approx. 250 000 as determined by gel infiltration chromatography. One major protein band, corresponding to a molecular weight of 32 500, was observed on 7% sodium dodecyl sulfate polyacrylamide gel electrophoresis of pooled fractions from DEAE-cellulose column chromatography (128-fold purified). The pH optimum for the 64-fold purified enzyme isolated from Bio-Gel A 1.5 M chromatography was 4.2 and bile salt hydrolase activity measured in intact cell suspensions had a pH optimum of 4.5. Substrate specificity studies indicated that taurine and glycine conjugates of cholic acid, chenodeoxycholic acid and deoxycholic acid were readily hydrolyzed; however, lithocholic acid conjugates were not hydrolyzed. Substrate saturation kinetics were biphasic with an intermediate plateau (0.2--0.3 mM) and a complete loss of enzymatic activity was observed at high concentration for certain substrates. The presence or absence of 7-alpha-hydroxysteroid dehydrogenase was absolutely correlated with that of bile salt hydrolase activity in six to ten strains and subspecies of B. fragilis.


On the partial reactivation of inactivated pantothenase from Pseudomonas fluorescens. Partial reactivation of inactivated pantothenase (pantothenate amidohydrolase, EC 3.5.1.22) from Pseudomonas fluorescens was studied. After partial inactivation during storing, pantothenase activity is increased by 10-40% when incubated with, for instance, oxalate, oxaloacetate or pyruvate. Reactivation proceedes slowly; with oxaloacetate the stable level of enzyme activity is attained in 20-30 min. The same compounds also cause reactivation of thermally inactivated pantothenase when partial inactivation has occurred at 28-37 degrees C. The amount of the reactivating enzyme form is relatively greater the lower the temperature during inactivation, but it never exceeds 20% of the original amount of active enzyme. Also another, unstable form of pantothenase is formed in thermal inactivation. This form becomes inactivated in a few minutes after the heat treatment, at pH 6-8 and at temperatures between 0 and 10 degrees C. Reactivation causes special problems in enzyme kinetic measurements; for instance, curvature is found in the lines of Ki determination by the Dixon plot.


Studies on aspartase. III. Alteration of enzymatic properties upon trypsin-mediated activation. Highly purified aspartase (L-aspartate ammonia-lyase, EC 4.3.1.1) from Escherichia coli, already of full activity, is further activated 3.3-fold by limited treatment with trypsin. The activation requires a few minutes to attain maximum level, and hereafter the activity gradually decreases to complete inactivation. Prior or intermediate addition of soybean trypsin inhibitor results in an immediate cessation of any further change in the enzyme activity. Upon trypsin-mediated activation no appreciable change is detected in the molecular weight of the enzyme subunits as judged from sodium dodecyl sulfate polyacrylamide gel electrophoresis, nor in the pH vs. activity profile in the presence of added metal ions. However, S0.5 and hill coefficient for L-aspartate considerably increase upon activation. As the trypsin-mediated activation proceeds, a marked absorbance difference spectrum of the trypsin-treated aspartase vs. untreated aspartase appears with negative absorbance maxima at 278 and 285 nm. When the trypsin-activated enzyme is denatured in 4 M guanidine-HCl, followed by removal of the denaturant by dilution, the enzyme activity is readily restored to as much as 1.5 times that of the native enzyme, indicating that the trypsin-activated enzyme is rather a stable molecule.


Investigations on the kinetic properties of estrone glucuronyltransferase from pig kidney. The microsomal fraction of the pig kidney catalyzes the glucuronidation of estrone in the presence of UDP-glucuronic acid. This bireactant system exhibits a sequential type of reaction mechanism. Increasing concentrations of either substrate increase the affinity of the enzyme for the other substrate. The Hill coefficient, n, was calculated to be 1.0 for both estrone and UDP-glucuronic acid. The Kestrone and KUDP-glucuronic acid are 6.6 muM and 254 muM, respectively. The estrone glucuronyltransferase (UDP-glucuronate: 17 beta-oestradiol 3-glucuronosyltransferase, EC 2.4.1.59) exhibits high substrate specificity in that it is inhibited noncompetetively by estradiol-17 beta, estradiol-17 alpha, estriol, testosterone, phenolphthalein and bilirubin; p-nitrophenol and o-aminophenol do not inhibit the glucuronidation of estrone. Mg2+ and Ca2+ were found to be nonessential activators. One of the two products of the reaction, estrone glucuronide, inhibits the enzyme competitively in the presence of increasing concentrations of UDP-glucuronic acid. The other product of the reaction, UDP, inhibits the enzyme noncompetitively with varying estrone concentrations and uncompetitively with varying UDP-glucuronic acid concentrations. Under incubation conditions for the glucuronidation of estrone, the enzyme catalyzes the reverse reaction with estrone glucuronide and UDP as reactants to an extent of about 0.4% of the forward reaction; this reverse reaction is also of a sequential type.


Interactions between phospholipids and barbiturates. The effects of a number of barbiturates on the temperature of the lipid phase transition have been studied using chlorophyll a as a fluorescence probe. The barbiturates cause a reduction in the temperature of the phase transitions of dipalmitoyl phosphatidylcholine and dipalmitoyl phosphatidylethanolamine, the effects being greatest at lower pH values where more of the barbiturate is present in the uncharged form. There was no significant interaction between the barbiturates and dipalmitoyl phosphatidylserine. These and other observations on the actions of local anaesthetics are used to develop a model for local anaesthesia. It is suggested that the sodium channel is surrounded by an annulus of lipid in the gel state, this rigid microenvironment preventing the sodium channel relaxing from its active configuration to an inactive one. Local anaesthetics, which reduce the temperature of lipid phase transitions, trigger a change of the annular lipid from the gel to the liquid-crystalline state, with a consequent relaxation of the sodium channel to an inactive configuration, in which the sodium current is reduced or blocked.


Transport and phosphorylation of D-galactose in renal cortical cells. An improved analytical procedure for the extraction and determination of total, free and phosphorylated tissue sugar is described. This method, employing ZnSO4 plus Ba(OH)2 for the precipitation of sugar phosphates, yields values identical with those obtained by the more laborious separation of free and phosphorylated sugar by ion-exchange chromatography. Erroneous values for free sugar due to the action of a Zn2+ -activated phosphatase and/or the lability to acids of some sugar phosphates, are avoided. Using this technique for the sudy of transport and phosphorylation of D-galactose in rabbit renal cortical slices and tissue extracts, it was found: 1. The cellular uptake of D-galactose was associated with the appearance of both free and phosphorylated sugar whether or not external Na+ was present. At 1 mM sugar, galactose was accumulated in the cells against a modest concentration gradient of 1.445 +/- 0.097 (n = 17). Galactose phosphate appeared in the cells considerably faster than free sugar under conditions of net uptake as well as of steady-state exchange (pulse-labelling). 2. Increasing saline pH (6-8) increased the cellular levels of sugar phosphate without affecting the steady-state values of free sugar. With tissue extracts, increasing pH also stimulated the activity of galactokinase and the dephosphorylation of galactose 1-phosphate by a Zn2+ -activated phosphatase. 3. 0.5 mM phlorizin inhibited the tissue uptake of galactose and its subsequent oxidation to CO2 only to a minor degree (30 and 10%, respectively). The absence of external Na+ further depressed the phlorizin effect. Preincubation of the tissue with phlorizin and subsequent washing in part abolished the inhibitory effect. The data suggest that a major portion of the galactose uptake by the tissue proceeds by a mechanism with a low affinity for phlorizin. 4. Efflux studies showed that the wash-out of free galactose from slices was associated with a net decrease of both free and phosphorylated tissue sugar. 5. The above results suggest the possibility that phosphorylation may represent a step in the Na+ -independent, phloretin-sensitive transfer of D-galactose across the antiluminal cell membrane. The participation of intracellular galactokinase and a Zn2+ -activated alkaline phosphatase in the maintenance of the steady state of free and phosphorylated galactose in the cells has been demonstrated.


Transport and phosphorylation of 2-deoxy-D-galactase in renal cortical cells. The transport and phosphorylation of 2-deoxy-D-[3H]galactose in rabbit renal cortical cells was studied. 1. The uptake of 2-deoxy-galactose by cortical slices is associated with an appearance of both free and phosphorylated sugar in the cells. At 1 mM external sugar the cells establish a steady-state gradient of free 2-deoxy-galactose of 3.97 +/- 0.15 (23 animals). 2. The acid-labile sugar phosphate accumulated in the tissue has been identified by a combination of paper and radio-chromatography, as well as on the basis of some of its chemical properties, as 2-deoxy-D-galactose 1-phosphate. Ice-cold trichloroacetic acid produces a decomposition of this compound. 3. Increasing external pH (6-8) brings about a decrease in the steady-state levels of both free and phosphorylated sugar in slices. On the other hand, increasing pH activates the phosphorylation of 2-deoxy-D-galactose by a crude kinase in a tissue extract. 4. Sugar phosphate accumulated in the cells is dephosphorylated by the action of a Zn2+ -activated phosphatase. 5. The efflux of 2-deoxy-D-galactose from the cells is rather slow compared with that found for D-galactose. The efflux is associated with some dephosphorylation of cellular sugar phosphate, and some loss of 2-deoxy-galactose phosphate into the wash-out medium takes place. 6. An inhibition analysis of the uptake of 2-deoxy-D-galactose by the slices indicates that the transport site is shared by D-galactose. The following points of interaction between the sugar molecule and the carrier are identified: C1-OH, C3-OH and C4-OH (both axial) and C6-OH. A (pyranose) ring structure is also essential. A close packing between the substrate and the carrier in the vicinity of C2 is indicated. 7. The data suggest that the above transport system is localized predominantly at the antiluminal (basolateral) face of the renal tubular cells. While the detailed mechanism of the actual transport step (i.e. active transport of the free sugar, or by the action of a phosphotransferase) is still unclear, the data present evidence that both galactokinase and a Zn2+ -activated phosphatase participate in the maintenance of an intracellular steady state of the transported sugar.


Heterogeneity of histidine transport in the Ehrlich cell. We have reexamined the heterogeneity shown by histidine in its uptake by the Ehrlich ascites tumor cell, in the face of a contradiction of our earlier interpretation. We again find the fraction of histidine uptake at neutral pH inhibitable by the model substrate for System A, 2-(methylamino)-isobutyric acid, to be fully dependent on the presence of Na+ or Li+. The small Na+ -independent component not attributable to System L can be identified with System Ly+ through its inhibitability by homoarginine. This component increases as the pH is lowered with an apparent pK' a of 6.1. The simultaneous decrease in the uptake by the neutral systems could be identified, for System L, with the same titration of histidine to its cationic form, but for System A the sharp decrease is identified with the protonation of a structure on the membrane rather than one on the substrate. The action of H+ in the latter case proved approximately non-competitive with Na+ when tested with ordinary substrates.


Calcium incorporation by smooth muscle microsomes. The purpose of the present work was to study the factors influencing calcium incorporation into a microsomal fraction prepared from the longitudinal smooth muscle of the guinea-pig ileum. Calcium incorporation required the presence of both ATP and Mg2+ and was unaffected by azide. It was enhanced by oxalate; this effect was pH dependent and it was maximal at pH 6.6. The relation between calcium uptake with oxalate and free Ca2+ concentration in the medium was represented by a curve with an optimum for Ca2+ equal to 3-10-5 M. The threshold concentration was comprised between 5-10-7 and 10-6 7. The optimum calcium uptake rate was 4.5 nmol Ca2+/mg protein per min. In the absence of oxalate, two distinct groups of binding sites were identified. Low affinity sites had a binding constant of 7-104 M-1 and a maximum binding capacity of 0.6-106 M-1 and a binding capacity of 33 nmol Ca2+/mg protein; their capacity was sensitive to pH changes. In the absence of oxalate, Ca2+ binding was depressed by Na+ with respect to K+ or choline. When the medium was supplemented with oxalate, the stimulation of 45Ca incorporation was barely detectable in the presence of choline+ and it was lower in a medium containing Na+ instead of K+. The subcellular distribution profiles of calcium incorporation with and without oxalate indicate the microsomal location of both activities. However, the oxalate-stimulated calcium uptake activity sedimented faster than the calcium binding activity. The subcellular distribution of marker enzyme actvities has been examined. The present results indicate that Ca2+ incorporations with and without oxalate are the result of two processes likely related to two different structures. The role of microsomal calcium uptake in excitation-contraction coupling and its modification by the activity of the sodium pump is discussed.


Alternating current studies of charge carrier transport in lipid bilayers. Pentachlorophenol in lecithin-cholesterol membranes. Surface and interior electrical properties of lecithin-cholesterol bilayer membranes treated with the uncoupler pentachlorophenol have been determined on the basis of a.c. measurements over a wide range of frequencies (0.02 to 1000 kHZ). The method used depends on accurately determining the resistance of the aqueous solution in series with each individual membrane by extrapolating admittance data into infinite frequency. Loss tangent vs. frequency curves are corrected by subtracting out a loss contribution which is present in untreated membranes and is due, presumably, to dielectric relaxation. The results, which are useful below 100 kHZ, can be fitted to loss tangent curves computed for a three-element equivalent circuit consisting of frequency independent conductance-capacitance pairs, arranged in series to represent surface and interior properties of membranes. Interior conductances agree with net conductances obtained from d.c. measurements. The pH and concentration dependence of surface conductance is consistent with a scheme of transport in which a fixed number of surface binding sites are filled preferentially with neutral pentachlorophenol molecules, which in turn dissociate to supply protons to the aqueous phase. Surface capacitances range from 15 to 90 times that of interior capacitance and show a systematic increase with pentachlorophenol concentration at high pH, and a decrease with concentration at low pH.


The lasat mouse: a new model for transplantation of human tissues. A new animal model characterized by hereditary athymia and asplenia was developed by breeding congenitally athymic with hereditarily asplenic mice. The new hybrids have no cellular immunity and produce significantly less antibody than nude mice. The lasat mouse has been found useful for the transplantation of normal and malignant hemopoietic cells and other neoplastic tissues of human origin.


The processing of human bone marrow for cryopreservation and reinfusion. Bone marrow was aspirated from 22 patients with solid tumors. A median of 1.2 x 10(10) net bone marrow cells were obtained during a 1 hour 45 minute operative procedure. Three methods were used to concentrate the bone marrow for cryopreservation. The greatest concentration of mononuclear cells was obtained by the Haemonetics pheresis procedure. Bone marrow was cryopreserved in 10 % DMSO in volumes ranging from 150 to 700 ml. The thawed marrow was further processed to remove both DMSO and free hemoglobin prior to reinfusion. Cell clumping was observed in the first 6 of the 10 marrows thawed. Acidification of the thawed marrow to pH 6.65 was found to retard cell clumping. The immediate recovery of cells frozen was 66 % with a viability of 50 %. The mean number of viable mononuclear bone marrow cells per kilogram available for reinfusion was therefore 5.5 x 10(7).


Physical characteristics of porous cellulose beads as supporting material for immobilized enzymes. Cellulose beads prepared in this report have high porosity (75-80%) and evenly distributed pores. The pore size is about 1000 A. The cellulose beads are physically strong and contain large amounts of reactive groups, making them suitable for use as carriers for immobilized enzymes.


[Induction of tyrosine aminotransferase by blastomogenic metabolites of tryptophan and tyrosine]. A comparitive study was made of the effect produced by endogenous blastomogenic agents (3-oxyanthranylic and paraoxphenyl-lactic acids) and their nonblastomogenic anaogues (anthranylic and phenyl-lactic acids) on the activity of tyrosine-aminotranspherase in the rat liver. Blastomogenic metabolites proved to be capable of inducing sharply the enzyme activity. This phenomenon and data on the role played by the increase in the activity of tyrosine-aminotranspherase and tryptophane-oxygenase in tyrosine and tryptophane catabolism on the way of a possible formation of blastomogenic metabolites permitted to put forward a suggestion on the "chain reaction" of accumulation of the endogenous blastomogenic agents in the organism.


[Effect of cocaine on the tyrosine hydroxylase of rat hypothalamus]. The influence of cocaine on tyrosine hydroxilase of rat brain hypothalamus was investigated in vivo (0.5 mg/kg) and in vitro (10(--6)--10(--5)M). Cocaine was used as a substance with a known adrenergic type of action. It was shown that under standard conditions cocaine in vitro increased the enzyme activity and decreased the Km for DMPH4 cofactor without changing Vmax of the reaction analyzed by the membrane enzyme. Cocaine in vitro decreased the tyrosine hydroxylase activity, especially that of the membrane enzyme. In this case there occurred a decrease of Km for DMPH4 and a decrease of Vmax of the reaction. The decrease of Vmax is considered to be the result of the secondary effect of cocaine.


[Analysis of the structure of the components of the convulsive action of corazole following administration of sulazepam and its metabolites to mice]. A study was made of the interrelationship between the minimal effective doses of pseudoclonic and clonico-tonic convulsions, and also tonic extension caused by the intravenous injection of corazol to mice and the effect of anticonvulsive action of sulazepam and its metabolites (diazepam, desmethyldiazepam and oxadiazepam) on this process. It was shown that all the compounds under study increased the values of the minimal effective doses by the recorded indices of the convulsive seizure, whereas the maximum of the anticonvulsive activity was reached 15 minutes after the sulazepam and oxazepam, and 5 to 30 min after diazepam administration. There proved to be a distinct correlation between the minimal effective doses values of the recorded indices of the confulsive seizure in the control animals which also persisted after the administration of the agents under study. It is supposed that sulazepam and its metabolites increased the minimal effective doses of corazol for the recorded effects, but failed to alter the general picture of the convulsive attack and did not influence the dispersion corazol dose-effect dependence.


[Participation of phytohemagglutinin-transformed mouse lymphocytes in development of the "graft-versus-host" reaction]. Transformed lymphocytes obtained as a result of phytohemagglutinin-stimulation of the cells of the lymph nodes of the CBA mice were incapable of realizing the graft-versus-host reaction in administration to the sublethally irradiated (CBAXC57BL/6) F1 hybrids. In this case the graft-versus-host reaction is induced by small untransformed lymphocytes with the same concentration of surface antigens detected by the antilymphocytic serum as in the intact lymphocytes.


[Suppression of interferon synthesis during the "graft-versus-host" reaction in F1(CBAXC57BL/6) mice]. The development of the graft-versus-host reaction (GVHR) in the F1(1CBA X C57BL/6 hybrid mice after the transplantation of spleen cells from the C57BL/6 parent donor resulted in a strong inhibition of the serum interferon production induced by the intraperitoneal injection of the Newcastle disease virus. In vitro with the mouse bone marrow cells during the development of the GVHR the interferon response was first reduced and then disappeared completely. The described phenomenon could therefore serve as an index of the development of the GVHR.


Analgesia produced by morphine when acting from the liquor space. 1 In cats analgesia was produced by morphine sulphate introduced into different parts of the liquor space in doses too small to be effective on intravenous injection. Analgesia was measured with the tail pinch method of Russell & Tate (1975). 2 On infusion into the fourth ventricle or into the subarachnoid space beneath the ventral surface of the brain stem caudal to the pons, doses of 100 to 200 mug of morphine sulphate were sufficient to produce strong long-lasting analgesia. On injection into the cisterna magna somewhat larger doses (400 to 800mug) were required. 3 It is concluded that the site where morphine acts when producing analgesia in all three circumstances is at the ventral surface of the brain stem. 4 The possibility is discussed that the structures acted upon are tryptaminergic nerve fibres. They arise from the raphe nuclei, belong to a descending inhibitory pathway, and on their way to the spinal cord, reach the ventral surface of the brain stem lateral to each pyramid, where they could be reached and acted upon by the morphine. This theory postulates a morphine sensitivity of tryptaminergic nerve fibres.


Behavioural treatment of orgasmic dysfunction: a controlled study. Twenty-two anorgasmic women received 20 sessions of a multiple-technique behavioural therapy. The design included blind ratings by two independent assessors, multiple assessment instruments, and a waiting list control group. Treatment was significantly better than no treatment in terms of: (1) the percentage of patients experiencing orgasm during at least 50 per cent of sexual relations; (2) the percentage of women reporting satisfactory sexual relations at least 50 per cent of the time; (3) patients' ratings of positive reactions to various sexual behaviours; and (4) assessors' global clinical ratings. Significant improvement was also noted on the MMPI, IPAT, and Symptom Check List. Improvement was maintained at a follow-up average 9 months later. These results support the impression that a behavioural approach offers much promise in treating female orgasmic dysfunction.


The relationship of the serum gastrin and calcium concentrations in patients with multiple endocrine neoplasia type I. The relationship between the serum gastrin and calcium concentrations has been examined in patients with multiple endocrine neoplasia type I. Variations in gastrin concentrations were induced by metiamide and secretin; calcium concentrations were altered by parathyroidectomy and by calcium infusion. Changes in the serum gastrin concentrations were not accompanied by changes in the serum calcium concentration. However, alteration of the serum calcium was accompanied by significant parallel changes in the serum gastrin. It is concluded that acute changes in the serum calcium may induce changes in the serum gastrin. Parathyroidectomy in these patients produced a fall in the serum gastrin, but the ability to produce large quantities of gastrin remains. It is postulated that the thyro-parathyroid hormones may modulate the relationship between calcium and gastrin.


Inhibitors of gastric secretion: current progress. Several new compounds have become available recently which are potent inhibitors of gastric secretion. The therapeutic potential of these inhibitors in the peptic ulcer diathesis is reviewed and it is concluded that the histamine H2-receptor antagonists show most promise at present. The prostaglandins and gastro-intestinal polypeptides are of considerable physiological interest but are unlikely to have clinical importance in the immediate future.


Effect of surgical isolation of the hypothalamus on its neurotransmitter content. The concentrations of norepinephrine, dopamine-beta-hydroxylase, dopamine, tyrosine hydroxylase, phenylethanolamine-N-methyltransferase, serotonin, tryptophan hydroxylase, histamine, glutamic acid decarboxylase, and choline acetyltransferase were determined in selected hypothalamic nuclei and in the median eminence after deafferentation of the medial basal hypothalamus. Norepinephrine and dopamine-beta-hydroxylase fell markedly while dopamine and tyrosine hydroxylase did not. Serotonin also decreased in all regions studied; histamine decreased in none. Choline acetyltransferase, phenylethanolamine-N-methyltransferase, and glutamic acid decarboxylase declined in some areas, but not in others.


Cardiac responsiveness to beta-adrenergic stimulation in experimental anemia. Cardiac reactivity of beta-adrenergic stimulation was assessed by isoproterenol dose-reponse curves (dose range 0.025-0.4 mug/kg) before and 1 h after the rapid induction of anemia in dogs anesthetized with halothane:N2O:O2. Anemai (hematocrit = 16 +/- 4%) was induced by an isovolumic exchange transfusion with Dextran 70, and was followed by significant increments in cardiac output (+57 +/- 9%), max dP/dt of the left ventricle (+37 +/- 7%), and in peak acceleration of blood flow in the ascending aorta (+46 +/- 13%). Anemia was associated with a significant reduction of the chronotropic responses to all but the lowest dose of isoproterenol. The simultaneously determined inotropic responses (max dP/dt) where the same before and after the induction of anemia. The responses in terms of peak acceleration of aortic blood flow tended to be greater in the anemic than in the control phase, at all dose levels used. These findings indicate that in rapidly induced experimental anemia the heart is capable of responding to marked degrees of beta-adrenergic stimulation, representing a more than two-fold increase in the dP/dt.


Propranolol induces acute natriuresis by beta blockade and dopaminergic stimulation. dl-Propranolol (0.8-1.6 mg/kg - h for 1 h) produced a transient two- to three-fold increase in sodium excretion in nondiuretic rats infused with Pitressin and aldosterone and in water diuretic rats. Sodium excretion increased more in rats depleted of renin by chronic Doca and salt administration than in rats maintained on a low salt diet. An angiotensin inhibitor (1,sarcosine-8,valine angiotensin II) decreased sodium excretion. Therefore the natriuresis was not mediated by antidiuretic hormone, aldosterone, or renin-angiotensin. d-Propranolol did not produce a natriuresis. Prior treatment with phenoxybenzamine did not prevent the natriuretic response but chlorisondamine pretreatment did. The natriuresis is produced by beta blockade and requires post ganglionic nerve function but is independent of alpha receptors. dl-Propranolol decreased heart rate and cardiac output but systemic pressure did not fall and renal blood flow increased. This suggests a dopamine-mediated renal vasodilation and natriuresis. Haloperidol and pimozide, both dopamine blocking agents with minimal beta blocking effects, prevented the natriuretic response. We conclude that propranolol may increase sodium excretion directly by blocking beta receptors in the distal nephron and indirectly by dopamine-mediated renal vasodilation.


Differences in the altered energy metabolism of hemorrhagic shock and hypoxemia. The effect of hemorrhagic shock, hypoxemia, and anoxia on the levels of adenine and pyridine nucleotides of liver and kidney was assessed. ATP levels in liver and kidney of animals in shock or animals subjected to 7 min of anoxia decreased by 85 and 73%, respectively. Under hypoxic conditions (arterial PO2 AT 18 MMHg), the decrease was only 62 and 48% in liver and kidney, respectively. Tissue NAD levels decreased and NADH levels increased during shock but were found to be essentially unaltered during experimental hypoxemia. Thus, shock produced greater alterations in adenine and pyridine nucleotides than did hypoxemia alone, indicating that stagnant hypoxemia due to shock is more deleterious to energy metabolism than is severe hypoxemia with an otherwise normal circulation. The results also suggest that if an anterial PO2 OF 18 MMHg represents the initial stages of tissue hypoxia, then tissue ATP levels are a more sensitive indicator of this than NAD levels.


Reduction in brain tyrosine hydroxylase activity following acetylcholinesterase blockade in rats. Activation of cholinergic neurons in the brain is produced by administration of the acetylcholinesterase inhibitors physostigmine and diisopropylfluorophosphate (DFP). This activation has a biphasic effect on tyrosine hydroxylase (EC 4.14.3-) activity. The acute effect of DFP, 1 mg/kg, intraperitoneally, or physostigmine, 0.2 mg/kg, intravenously, or 10 mug, intraventricularly, was a rapid reduction in tyrosine hydroxylase activity in the hypothalamus. The activities of DOPA decarboxylase (EC 4.1.1.28) and dopamine-beta-hydroxylase (EC 1.14.17.1) were not changed. In contrast to the acute effect, chronic administration of physostigmine, 0.2 mg/kg, intravenously, twice daily for 7 days produced an increase in tyrosine hydroxylase activity in the hypothalamus. The rapid acute effects may be due to an allosteric inactivation of tyrosine hydroxylase, while the chronic effects may reflect enzyme induction.


Reduced hemoglobin affinity for oxygen in venous blood following hemodilution, independent of changes in pH or PCO2. A rapidly induced and readily reversible shift in the affinity of hemoglobin for oxygen has been demonstrated. The shift, similar to the Bohr effect, is independent of PCO2 or pH changes. It occurred within 30 min of hemodilution and was seen in portal venous blood but not arterial blood. A hypothesis is suggested involving a phasic alteration in levels of 2,3-diphosphoglycerate (DPG) or ATP binding to hemoglobin. It is proposed that, following hemodilution, the degree of these phosphates to hemoglobin increases on passage through the intestinal vascular bed. The increased DPG binding to hemoglobin results in displacement of additional oxygen. As the blood becomes reoxygenated, the levels of DPG-hemoglobin binding decline and DPG is displaced from the hemoglobin by oxygen.


Microdose mammography.

Microdose mammography. Tens of thousands of mammograms are performed daily in the United States. Accumulated radiation biologic data suggest that approximately 90-rad is the level of radition to the breast above which the incidence of cancer may be increase. A microdose system of roentgenography is described that provides better images at one-third to one-fifth the exposure of the commonly used low-dose system and about one-eight to one-tenth the dose of Xeromammography. This system should allow for repeated x-ray study in a patient as required, without exceeding the critical level.


An ESR study of the anchoring of spin-labeled stearic acid in lecithin multilayers. In egg lecithin-water lamellar phases, spin-labeled stearic acid gives two superimposed ESR spectra which are only well resolved when the temperature is greater than 30 degrees C. These two spectral components are attributed to the dissociated and non-dissociated forms of the fatty acid carboxylic group, anchored at two different positions in the polar interface constituted by the hydrated lipid polar heads. Results on such interactions of other functional groups (spin-labeled fatty ester and fatty alcohol) are also presented.


[Guinea pig liver cytosol has an L-threonine deaminase activity distinct from L-serine deaminase]. Using sodium sulfate precipitation, "Sephadex G200" gel filtration and polyacrylamide gel electrophoresis, a L-threonine desaminase was demonstrated in the Guinea-Pig liver cytosol. This enzyme was separated from the guinea pig liver L-serine desaminase possessing an auxiliary activity on L-threonine substrate described by us in a previous work. The optimals for pH (7,1) and temperature (+ 55 degrees C) and the apparent molecular weight (134,000 + 20,000) were established.


Comparison of internal mammary artery and saphenous vein bypass grafts for myocardial revascularization: exercise test and angiographic correlations. The effectiveness of myocardial perfusion with internal mammary artery (IMA) bypass revascularization has been questioned. We compared 37 patients with single patent IMA bypass grafts with 26 patients who had single patent saphenous vein graft (SVG) bypass. Serial computer assisted graded maximal stress testing was used for quantitation of palliation. Patients were classified and compared according to the completeness of revascularization by postoperative catheterization. All patient subgroups, had statistically significant improvements in work capacity, maximal heart rate, maximal rate-pressure products, abnormal exercise electrocardiograms, and exercise-induced angina pectoris. The quantitative improvements in these measurements of patient palliation were the same during postoperative stress tests after either SVG or IMA revascularization. Considering the overall higher patency rate of the IMA bypass, the data indicate that this procedure, when surgically applicable, is preferable to SVG bypass.


Renal acidification in hypothyroid man.

Renal acidification in hypothyroid man. The role of thyroid hormone in renal hydrogen ion secretion remains largely unknown, and there is only limited information on renal acidification in hypothyroid patients. In the present study two of five adult male patients with untreated primary hypothyroidism and without clinical evidence of systemic autoimmune disease were unable to lower their urine pH appropriately after short duration acid-loading. Since, prior to acid-loading, their arterial blood gas values were within the normal range and urinary bicarbonate excretion was trival, the findings are consistent with the incomplete syndrome of distal renal tubular acidosis. Although the mechanism of this abnormality remains unknown, thyroxine deficiency per se may in part be responsible.


Group desensitization of dissimilar anxieties. Group desensitization is an effective, efficient anxiety-reduction therapy that, nonetheless, is not applicable in most mental health settings. This paper outlines several procedural modifications that make possible the desensitization of diverse anxieties within a single group and thus make group desensitization a more practical strategy.


Effect of ascorbic acid on arylsulfatase A and B activities in human chondrocyte cultures. Cultured normal human articular cartilage chondrocytes exhibited decreasing levels of arylsulfatase A and B activities when grown in the presence of increasing levels of ascorbic acid (0 to 90 mug/ml) in the media. That this was not a general effect on all lysosomal enzymes was supported by the increase in acid phosphatase activity and no change in beta-glucuronidase activity observed with increasing levels of vitamin C under identical culture conditions. No decrease in either arylsulfatase activity was observed when ascorbic acid was replaced by ascorbate-2-sulfate. Ascorbic acid did not inhibit either arylsulfatase activity when added directly to the assay mixture. These data, combined with results of mixing experiments, suggest that the effect of vitamin C is mediated through cellular factors produced in response to its inclusion in the growth media.


Enzymes from human articular cartilage: isolation of arylsulfatase B and its comparison with arylsulfatase A. This study describes the isolation of arylsulfatases A and B (arylsulfate sulfohydrolase EC 3.1.6.1) from human articular cartilage. These enzymes were extracted from collagenase digests of tissue homogenates. After fractionation with ammonium sulfate the enzymes were separated from each other by DEAE-cellulose chromatography and further purified by gel filtration on Sephadex G-200. Sulfatase B, subsequently chromatographed on CM-cellulose was apparently homogenous as judged by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. The enzyme has a pH optimum of 5.6, a molecular weight of 51,000 and Km of 2.6 mM for 4-nitrocatechol sulfate. Sulfatase A was found to be a glycoprotein with a pH optimum of 4.8, a molecular weight of 105,000 and a Km of 0.16 mM for 4-nitrocatechol sulfate. The competitive inhibition of both enzymes by inorganic sulfate, sulfite and phosphate support the likelihood of a common reaction mechanism. In contrast to sulfatase B which showed minimal inhibition, sulfatase A was totally inhibited by 5 mM N-ethylmaleimide.


Lorazepam as a sedative-amnesic in an intensive care unit. A clinical study was carried out to evaluate the usefulness of intravenous lorazepam, given for sedation instead of opiate narcotics or diazepam, in 25 seriously-ill patients being treated in a respiratory and intensive care unit. All but 3 patients were on assisted ventilation. Standard doses of 4 mg lorazepam were given at 4 or 6-hourly intervals for periods up to 25 days. ECG, haemodynamic stability and biological determinations were monitored constantly. Apart from some delay in onset of action, lorazepam proved to be a useful sedative with diminished recall on the part of the patients. No side-effects were reported, nor was there any local reaction to the injection. Cardiac output was measured in 9 patients following intravenous administration of a single-dose of either 4 mg or 8 mg lorazepam. No significant changes were recorded.


[Effective treatment of duodenal ulcer with cimetidine (author's transl)]. Twelve male duodenal ulcer in-patients received in a double-blind trial either the histamine H2-receptor antagonist cimetidine (4 X 200 mg/d p.o.) or placebo capsules. Ulcer sizes were assessed endoscopically before therapy followed by repeat endoscopy at weekly intervals. Duodenal ulcer healing was significantly more rapid in cimetidine-treated patients than in those receiving the placebo (chi2 test; P less than 0.0005). Plotting of log ulcer sizes (mm2) against time (days) resulted in regression lines the slopes of which indicated the respective half-time of ulcer healing: about 6 days on cimetidine therapy and about 20 days on placebo treatment. Gastric secretion of acid, protein, pepsin, and N-acetylneuraminic acid-containing glycoproteins was not altered by a 4-week course of daily cimetidine or placebo, nor pancreatic secretion of bicarbonate and enzymes. No statistically significant changes in laboratory findings (haemoglobin, white blood-cells, neutrophils, platelets, alkaline phosphatase, blood-urea, serum-creatinine, GOT, GPT) were associated with treatment.


[Treatment of diabetic coma and precoma with continuous low-dose insulin infusions (author's transl)]. 13 patients, nine women and four men, aged 22 to 83 years, were treated for diabetic coma or precoma between September 1974 and January 1976. Ten patients were known diabetics and six of them had been treated with insulin. On admission blood sugar was 32.4 +/- 3.3 mmol/l (5.84 +/- 0.6 g/l). The capillary blood pH was 7.15 +/- 0.06 (n = 13). Treatment consisted of continuous insulin infusion (6 IU soluble insulin/hour), physiological saline, potassium substitution and sodium bicarbonate (if the pH was below 7.15). In the first hours of treatment 98 +/- 12IU of insulin, 6.5 +/- 0.5 litres of fluid, 168 +/- 22 mmol of potassium and 237 +/- 55 mmol NaHCO3 were required. During the first 4 hours of the insulin infusion the blood sugar decrease per hour was 3.55 mmol/l (0.64 g/l). Hypokalaemia during treatment occurred in one case, hypoglycaemia was not observed. A preceding treatment with insulin and severe acidosis did not influence therapeutic success. Twelve patients were treated successfully, one patient died 6 hours after admission following mesenteric arterial embolism.


[Tranquilizers. Pharmacologic aspects].

[Tranquilizers. Pharmacologic aspects]. An inventory was made of the various substances utilized as minor tranquillizers in man. Throughout the various methods of study of these drugs in animals the main pharmacological properties of tranquillizing drugs were considered. Attention is drawn on the various secondary or adverse effects of minor tranquillizers.


[The sector and the walls of the asylum]. The author has been able to strengthen his position of self confident, active practitioner by means of a noteworthy case: patient suffering of a chronic delusion whose internement has been avoided by the prescription of a long acting drug.


Interaction of uteroglobin with progesterone, 5alphapregnane-3,20-dione and estrogens. Uteroglobin was obtained from 5 day pregnant rabbits and purified to homogeneity by Sephadex G 75 and DEAE-cellulose chromatographies. Progesterone binding to uteroglobin was decreased by lyophilization and enhanced by SH-reducing agents. Dithiothreitol was more effective than dithioerythritol, and beta-mercaptoethanol was only active at 25 to 100 mM concentrations. SH-blocking agents (iodoacetate, iodoacetamide, phydroxymercuribenzoate and, dithiobisnitrobenzoic acid) inhibited binding. In the absence of SH-reducing agents only one in every 500 uteroglobin molecules bound the hormone, whereas under optimal conditions (20 mM dithiothreitol) one in every two molecules bound progesterone. There was no significant difference in equilibrium dissociation constants under these two conditions. Uteroglobin had a relatively high affinity for progesterone (KD=4.1 X 10(-7)M) but a threefold higher affinity for 5alpha-pregnane-3,20-dione (KD=1.3 X 10(-7)M). Estradiol was bound but non-specifically with a very low affinity, and its binding was not enhanced by SH-reducing agents. Hormonal specificity of binding to uteroglobin was different from that of binding to rabbit uterine progesterone receptor. Various synthetic progestagens (chlormadinone acetate, norethisterone, R5020) were bound to the latter but not to the former protein. Diethylstilbestrol had some affinity (15% of that of progesterone) for uteroglobin and no affinity for the progesterone receptor. Uteroglobin incubated in the presence or absence of cofactors (NADH and NADPH) with or without dithiothreitol did not metabolize progesterone.


Progesterone metabolism in vitro by rabbit testes at different stages of development. Testicular homogenates from white rabbits of 0.4, 1, 3, 4, 5, 8, 18, and 24 months of age were incubated with [3H]progesterone and NADPH. At 12 days of age, major C21-17-OH-and C19- steroids formed from progesterone were 17alpha-hydroxyprogesterone and testosterone. However, at 4-24 months of age, 17alpha-hydroxyprogesterone, 3beta,17alpha-dihydroxy-5alpha-pregnan-20-one, and 5alpha-reduced C19-steroids such as 17beta-hydroxy-5alpha-androstan-3-one, 3beta-hydroxy-5alpha-androstan-17-one, and 5alpha-androstane-3beta,17beta-diol were the major products. The formation of significant quantities of 5alpha-reduced C19-steroids, which had been demonstrated previously only in prepubertal testes of rats and mice, was present in prepubertal as well as adult testes of rabbits. These findings clearly indicate that the metabolic patterns of progesterone in the adult rabbit differ from those in the adult rat and mouse. The major C19-steroids formed by testes are testosterone in the adult rat and mouse, but 5alpha-reduced C19-steroids in the adult rabbit.


Direct determination of ligand interactions with beta-adrenergic receptors on intact turkey erythrocytes: correlation of binding with biological activity. Previous studies on the interaction of labeled beta-adrenergic blockers with beta-adrenergic receptors have employed broken cell or membrane preparations. We have now carried out direct binding analysis on intact turkey erythrocytes employing the potent, high specific activity blocker [125I]-hydroxbenzylpindolol (HYP). [125I]HYP binds to a single class of receptor sites with a K of 5.3 X 10(10)M-1 and a binding capacity of 400-500 sites/cell. These results as well as the kinetics of association and dissociation and lack of evidence for negative cooperativity all agree well with studies reported earlier on membrane preparations from the same cells. True dissociation constants (Kd) for agonists and antagonists determined by inhibition of binding of [125I]HYP are in good agreement with results in membrane preparations. These Kd's have been compared directly with activation or inhibition constants for effects on adenylate cyclase using generation of [14C]cAMP from [14C]adenine in intact cells. The close correlation between effects on binding and adenylate cyclase activity in whole cells are similar to results obtained in membrane preparations in the presence of guanine nucleotides, suggesting the presence of an analogous regulatory substance in vivo.


The fate of ACTH released from rat anterior pituitary into the incubation medium in vitro: enzymatic degradation and acid activation. The bioactivity of ACTH released from isolated rat anterior pituitary glands into the incubation medium was determined. After the pituitaries were removed, ACTH activity in the medium decreased exponentially during further incubation at 37degreesC. The loss of ACTH activity was temperature- and pH-dependent and inhibited both by protease inhibitor (trasylol) and by preheating. Crude tissue extracts from median eminence, cerebral cortex and liver similarly inhibited the loss of ACTH activity. These results indicate that ACTH released into the medium may be destroyed by proteolytic enzyme(s) from the rat anterior pituitary. ACTH activity in the incubation medium was increased promptly by acidification of the medium to pH 1.5-2.5 with HC1, and reduced to the initial level by NaOH reneutralization of the medium (pH 6.8-7.8). These phenomena were not observed after the incubation medium had been heated at 100degreesC for 5 min.


Ferredoxin from a red alga, Porphyra umbilicalis. A plant-algal type ferredoxin was isolated from the red alga, Porphyra umbilicalis. In its oxidised form the ferredoxin had absorption maxima at 277, (281), 323, 420 and 462 nm. Two atoms each of non-haem iron and labile sulphur were present per molecule protein. The midpoint potential of the protein was -400 mV and it effectively mediated electron transport in the NADP-photoreduction system of barley. The amino acid composition of Porphyra umbilicalis ferredoxin was determined as (Lys4, His2, Arg1, Asx10, Thr8, Ser7, Glx16-17, Pro3, Gly7, Ala8, Cys5, Val6, Met1, Ile5, Leu8, Tyr5, Phe2). The minimum molecular weight of approximately 11000 was confirmed by sedimentation-equilibrium studies in the analytical ultracentrifuge. Approaching half of the total amino acid sequence was determined by means of an automatic sequencer.


The antigen-inexperienced thymic suppressor cells: a class of lymphocytes in the young chicken thymus that inhibits antibody production and cell-mediated immune responses. Transfer of thymus cells from young chickens in combination with a light whole body irradiation (360 R) was found to suppress the rejection of skin grafts across strong histocompatibility (B) differences. On the average, the suppressed animals also showed decreased serum hemagglutinin titers against erythrocytes of the skin donor strain and a decreased graft-versus-host (GvH) reactivity against embryos of this strain. The thymic suppressor cells can be obtained from animals that have not experienced the antigen under test. However, after transfer and contact to the antigen (skin graft) they can lead to the formation of specific ("activated") suppressor cells and can mediate in the long run a specific inhibition of the response to this antigen. The suppressive activity is associated with a bursa-dependent cellular subpopulation in the thymus that is different from B lymphocytes, B precursor cells or GvH-reactive T cells. The bursa dependency of the thymic suppressor cell suggests that functionally different lineages of thymic and thymus-derived lymphocytes are derived from different sources of prethymic stem cells. The suppressor cells are predominantly found in the young chicken thymus and already detectable in the 16-day-old embryo, while poor suppressive activity is found in the adult thymus. The suppressive effect can be obtained with thymus cells from either syngeneic or allogeneic donors. Embryonic allogeneic donors provide suppressive cell preparations free of GvH reactivity. The possibility that the thymus suppressor cells mediate self tolerance and "neonatal tolerance" is discussed.


Inhibition of T cell activity in vivo: a test model for quantitative evaluation. A test model is presented which, in comparison with the conventional models of skin transplantation or graft-versus-host (GvH) reaction in mice, permits a more sensitive quantitative evaluation of T cell inhibition in vivo. Prospective donors (type AA) are immunized with prospective recipient material (type AB); the resulting T cell reaction of A versus B is inhibited by consecutive treatment. Extent of inhibition can be evaluated after transfer of the pretreated AA material onto AB recipients by calculation of remaining GvH reactivity, if compared to adequate control tranfers. In this model the target animal for T cell reactivity (the AB recipient) remains untouched from immunosuppressive regimen.


Antibody response to phosphorylcholine in vitro. II. Analysis of T-dependent and T-independent responses. Spleen cells from BALB/c mice primed with keyhole limpet hemocyanin (KLH), were stimulated with heat-killed vaccine of rough Pneumococcus pneumoniae R36A (Pn) and/or phosphorylcholine (PC)-coupled KLH to induce an anti-PC response in vitro. The response to PC-KLH was found to be T-dependent while it is T-independent to Pn. The antibodies induced with either antigen had similar avidity and expressed the TEPC 15 idiotype exclusively; thus T cell involvement in the response to PC-KLH failed to alter these parameters of the anti-PC response. At the precursor cell level, Pn induced small clones with an average size of 10 plaque-forming cells (PFC), whereas PC-KLH gave rise to larger clones of 40-50 PFC. This difference in the proliferative potential of PC precursor B cells hinted at the possibility that Pn and PC-KLH were stimulating different precursors. This was corroborated by the observation that a) when Pn and PC-KLH were added to the same cultures a synergistic effect was seen, i.e. the number of plaques was greater than the sum of the responses induced by each antigen, and b) in microcultures, under conditions limiting B cells only, Pn plus PC-KLH induced a higher fraction of responding wells than either antigen on its own. We postulate that Pn and PC-KLH stimulate subpopulations of PC precursor cells which are T-independent and T-dependent, respectively.


Mechanisms by which hapten conjugates of pneumococcal polysaccharide interfere with the challenge of anti-hapten memory cells. Incubation of trinitrophenylated hemocyanin (TNP-KLH)-primed spleen cells with microgram amounts of 2,4-dinitrophenyl (DNP) or 2,4,6-trinitrophenyl (TNP) conjugates of pneumococcal polysaccharide type 3 (SIII) for as little as 5 min at 4 degrees C results in a specific "block" of the 19 S and 7 S adoptive memory response to TNP-KLH. This hapten-SIII-induced block of anti-hapten memory B cell responsiveness seems to be an example of specific receptor blockade. The block is specific and can be prevented by simultaneous incubation of the primed cells with hapten-protein conjugates which presumably compete with the hapten-polysaccharide for attachment to the B cell surface via anti-hapten Ig receptors. Removal via capping of these Ig receptors by exposure of TNP-KLH-primed memory cells to rabbit anti-mouse Fab serum for 45 min at 37 degrees C renders these cells refractory to the blocking effect of hapten-SIII. Once the hapten-SIII has attached to the memory cells, these blocked cells can be "rescued" (i.e. returned to a state of responsiveness) by incubating these cells with either mouse anti-SIII at 37 degrees C or rabbit anti-DNP serum at 4 degrees C. Since a papain digest of the IgG fraction of rabbit anti-DNP did not rescue the cells while the intact IgG did, a capping off of the TNP-SIII was proposed as the mechanims for this return to responsiveness of the hitherto blocked cells. A rescue was not seen by treatment of recipient mice with such B cell mitogens as dextran sulfate, endotoxin or purified protein derivative of tuberculin.


Suprression of local graft-versus-host reactions by mouse fetal and newborn spleen cells. Fetal splenic but not thymic lymphocytes significantly reduce the ability of parenteral adult spleen cells to elicit local graft-versus-host reactions in F1 recipients. This suppressive activity wanes early after birth. There is no requirement for histo compatibility between reacting and suppressor cells.


In vitro studies on smooth muscle of the human renal pelvis. Isolated segments of human renal pelvis were studied by an isometric technique. Increases in tension following the addition of adrenaline, noradrenaline and phenylephrine were shown to be mediated via alpha-adrenoceptors. Similar responses to acetylcholine were demonstrated to be due to muscarinic receptor stimulation. Specimens responded to transmural electrical stimulation only when the pulse width was greater than 4 msec, and such responses were unaffected by pretreatment with tetrodotoxin, phentolamine and atropine. These experiments suggest that there is no effective innervation of the receptor sites identified, and hence that renal pelvis motility in vivo is not amenable to regulation by the autonomic nervous system.


Differential response control by isopropamide: a peripherally induced discriminative cue. The peripherally acting anticholinergic drug isopropamide (0.02 mg/kg s.c.) is shown to produce a discriminative stimulus complex in rats. In rats trained to discriminate isopropamide from saline, dexetimide and methylscopolamine were generalized with isopropamide treatment. The results indicate that drug discrimination learning does not necessarily require a central drug action.


Antiarrhythmic action of a new beta-adrenergic blocking agent, 6-(2-hydroxy-3-isopropylaminopropyloxy)-benzothiazole succinate (KF-577), compared with that of propranolol. The antiarrhythmic activity of a new beta-adrenergic blocking agent, 6-(2-hydroxy-3-isopropylaminopropyloxy)-benzothiazole succinate (KF-577), was compared with that of propranolol. KF-577 antagonized ouabain-induced arrhythmias in normal and bilaterally vagotomized guinea pigs; its antagonistic activity was equal to that of propranolol. Reserpinization greatly reduced ouabain intoxication and neither of the two beta-blockers produced further reduction. Aconitine-induced arrhythmias in rats were not antagonized by the two agents. In intact guinea pigs, the reduction of ouabain intoxication by both beta-blockers could not exceed that produced by simulataneous infusion of KCl, and vice versa. In isolated guinea pig atria, propranolol was about 10 times more effective than KF-577 in reducing the ouabain intoxication. The antiaarhythmic activity of KF-577 paralleled its beta-blocking activity in the isolated preparations but not in the intact animals.


Effects of drugs on the formation of homovanillic acid in the rat retina. Homovanillic acid (HVA) levels were measured in the eye and the corpus striatum of rats under normal conditions and after different drug treatments. Neuroleptic agents such as clozapine, cis-flupenthixol and haloperidol induced comparable increases in HVA levels, whereas the non-neuroleptic trans-isomer of flupenthixol was inactive in both structures. Apomorphine decreased HVA levels in the retina and the corpus striatum, while amphetamine induced a decreased HVA formation in the retina and did not change levels of HVA in the corpus striatum. Probenecid caused a similar percentage rise of HVA in both structures. Morphine and oxotremorine induced a rise in HVA levels in the corpus striatum but not in retinal samples.


Relationship between the prevention of rat gastric erosions and the inhibition of acid secretion by prostaglandins. The formation of gastric mucosal erosions induced by indomethacin in the rat was inhibited in a time- and dose-dependent manner by antisecretory prostaglandins, the methyl analogues of PGE2 being 400 times as active as the parent prostaglandin. PGA2, a methyl analogue of PGF2alpha and the H2-receptor antagonist metiamide, also inhibited erosion formation. There was a variable relationship between the doses required to inhibit erosions and to inhibit gastric acid secretion. In the anaesthetised rat, the low incidence of erosions with indomethacin was markedly increased by concurrent gastric perfusion with acid saline and taurocholate. This mucosal damage was inhibited by the methyl analogues of PGE2, suggesting protective actions on the mucosa other than inhibition of acid secretion.


Centrally induced reduction in sympathetic tone-a postsynaptic alpha-adrenoceptor-stimulating action of imidazolines. Naphazoline or oxymetazoline (both 30 mug/kg) were injected into the cisterna magna of anaesthetized cats and reduced blood pressure, heart rate and the electrical discharge rate of small fibre bundles of the preganglionic sympathetic splanchnic nerve. Cats were depleted of endogenous noradrenaline by pretreatment with reserpine (5 mg/kg, 18 h) and alpha-methyl-p-tyrosine (twice 300 mg/kg, 18 and 2 h). In these animals, intracisternal injection of 30 mug/kg oxymetazoline exerted a decrease of sympathetic discharges similar to that described for non-pretreated animals. In noradrenaline-depleted cats intracisternal injection of 1 mug/kg clonidine also decreased the sympathetic discharges. It is concluded that these imidazolines exert their sympathoinhibitory and cardiovascular effects by stimulation of postsynaptic alpha-adrenoceptors in the CNS.


Contribution of granulocytopenia to endotoxin sensitivity of mice irradiated or undergoing graft-versus-host reaction. Animals compromised by irradiation or graft-versus-host reaction (GVHR) are highly sensitive to endotoxin (ET). In order to determine the causes of the increased sensitivity of compromised mice, we studied alterations of hepatic (central) and bloodborne (peripheral) ET clearance processes. We observed that increased sensitivity to ET, as determined by mortality, occurred shortly after irradiation and correlated with granulocytopenia nd thrombocytopenia rather than with impairment of liver function. The involvement of granulocytes in ET clearance was indicated by injection of 51Cr-ET suspended in whole blood. By comparison with clearance of ET injected with saline or plasma, greater amounts of 51Cr-ET were sequestered in peripheral organs than in the liver. Similar results were obtained when Cr-ET in whole blood was perfused through a rat liver. ET clearance was enhanced 50% over that seen in M-199, plasma, or platelet-rich plasma. It was also found that intestinal ET contributes to mortality of granulocytopenic-thrombocytopenic mice. This was supported by the observation that bacteriologically decontaminated, irradiated animals were eight times more resistant to challenge with ET than were conventional animals. Thus, an important aspect of increased sensitivity to ET, in comprised mice, is defective peripheral clearance.


Mitigation of Graft-versus-host disease in mice with xenogeneic antithymocyte serum and complement. In vitro treatment of parental C57BL/6 lymphohematopoietic cell grafts with unabsorbed guinea pig anti-mouse thymocyte serum (ATS) and guinea pig complement (GPC), prior to inoculation into lethally irradiated B6D2F hybrid hosts, has proven to be of value in terms of mitigating graft-versus-host disease (GvHD). However, the beneficial effect of such a pregrafting procedure is limited to the prevention of acute GvHD. The late GvHD remains a continuing problem, and is probably due to the graft-versus-host activity (GvHA) of newly produced nontolerant lymphocytes from lymphoid precursors resistant to ATS. Possible ways to render these precursors sensitive to ATS and complement are discussed. The potential significance of thymic hormones and cyclic AMP in achieving this is emphasized.


Acid mucopolysaccharides in fibroblast cultures. 1. Influence of cell density, pH-value and lactate concentration on the MPS distribution pattern. From cells and culture media of embryonic rat fibroblasts (1st subculture) the acid mucopolysaccharides were isolated and fractionated. The per cent calculation of the 6 fractions was based on the content of glucuronic acid. The cultures were maintained as follows: 0.5 X 10(6) to 3 X 10(6) cells were examined in Demeter flasks at pH 7.4 or 6.6; lactate concentration was enhanced to 100 mg%. The amount of each fraction was correlated with the cell density (linear regression). The pH-value and lactate concentration in connection with cell density proved to be important factors in the modification of the MPS distribution pattern.


Acid mucopolysaccharides in fibroblast cultures. 2. 35S-sulfate incorporation kinetics in dependence on pH-value and cell density. Cultures of embryonic rat fibroblasts were incubated with 35S-sulfate at pH 6.6 and 7.4 (Eagle basal medium plus HEPES buffer) for 12 to 48 hours. The acid mucopolysaccharides were isolated and fractionated after the method of SVEJCAR and ROBERTSON. Sulfate incorporation was determined by liquid scintillation counting.


Allantoinase in the marine polychaete Eudistylia vancouveri. Allantoinase, an enzyme in the purine-urea cycle, was found in Eudistylia vancouveri (Polychaeta). The enzyme had a pH optimum at 7.6. The Km was 0.012 M allantoin, and the Arrhenius energy of activation was 12.6 to 14.6 kcal/mol.


Influence of the trypsin activity by the side chain of arginine homologues. The N-alpha-tosyl-p-nitroanilides of homoarginine and of the two shorter arginine homologues were synthesized. These compounds behave as specific, chromogenic substrates for trypsin.


Beta-adrenergic receptors in rat myocardium: direct detection by a new fluorescent beta-blocker. A new fluorescent beta-blocker, 9-amino-acridin propranolol (9-AAP), was administered i.v. to rats. Multiple fluorescent 9-AAP binding sites were observed on cardiac muscle cells in frozen sections. Intensity and density of cardiac 9-AAP fluorescence were markedly reduced following pretreatment with (+/-)- and (-)-propranolol but not with (+)-propranolol. Our findings suggest that 9-AAP may label beta-adrenergic receptor sites in rat myocardium.


Histamine-induced hypotension modified by H1 and H2 antagonists in the monkey (Macaca mulatta). Blocking H2 receptors with burimamide in the dose used (20 mg/kg) approximately doubles the amount of histamine needed to produce the same effect as seen when H1 antagonists (chlorpheniramine or mepyramine) are used alone. The Kz ratios for chlorpheniramine-chlorpheniramine plus burimamide are 117-204 and for mepyramine-mepyramine phus burimamide are 200-478. H1 and H2 receptors, in the monkey, when stimulated, both cause cardiovascular responses in the same direction.


The oxygen-linked hydrogen ion binding (the Haldane coefficient) of bovine hemoglobin. The Haldane coefficient (the amount of the oxygen-linked hydrogen ion binding of hemoglobin) was determined in bovine erythrolysate (Hb concentration equals 13.5 mM) by means of the differential titration method with varying PCO2 from 0 to 74 mm Hg and pH from 6.0 to 8.5 at 37 degrees C. The maximum value of the coefficient was found to be 0.49 mM per mM Hb at PCO2 equals 0 and pH 7.20. With increasing of PCO2, the coefficient became smaller in all ranges of pH studied. The coefficient under the conditions of pH 7.20 and PCO2 equals 45 mm Hg that are normally prevailing in the interior of bovine erythrocytes was 0.31.


Activation of sustained sympathetic vasodilatation in dog by spinal cord stimulation. Electrical stimulation in lateral sites of the upper cervical spinal cord evoked vasodilatation after adrenergic blockade. Sympathetic fibres mediating sustained vasodilatation were shown to be separate from adrenergic sympathetic fibres since the adrenergic vasoconstrictor response in the paw evoked by vasomotor stimulation in the medulla was not reversed to vasodilatation after bretylium.


Effects of morphine administration on cerebellar guanosine 3',5'-monophosphate. An increase in mouse cerebellar C-GMP levels during acute morphine treatment was observed, which was possibly related to the decrease in C-GMP phosphodiesterase levels also observed in acute treatment. Chronic treatment lowered C-GMP levels as did abrupt withdrawal without naloxone.


[Effect of liver damage by thioacetamide on microsomal aromatization of testosterone in rats (author's transl)]. Rat liver microsomes, NADPH-regenerating system, and 1beta, 2beta-3H-testosterone have been incubated in vitro. The loss of tritium from the steroid, associated with aromatization of testosterone, was linear with time for 20 min and required NADPH. Pre-treatment of the rats with thioacetamide raised the liberation of tritium from 1beta, 2beta-3H-testosterone. The results suggest that liver damage by thioacetamide in rats may give rise to increased aromatization of testosterone.


Reduction of the graft-versus-host reactivity of mouse and rat spleen cells by 5alpha-androstane-3,17-dione. The local graft-versus-host reaction as evaluated by the popliteal lymph node enlargement was used for studying the immunosuppressive potency of placental steroid 5alpha-androstane-3,17-dione. Spleen cells from virgin female mice and rats injected subcutaneously with this steroid compound produced in appropriate F1 recipients a significantly weaker reaction (P less than 0.001) than spleen cells from untreated or medium-treated control animals. On the other hand, the pretreatment of cell donors either with 5beta-androstane-3,17-dione or testosterone, the compounds which are not present in the mouse and rat placenta, did not influence the normal graft-versus-host reactivity.


The sensitivity of chromatin from thymuses and spleens of irradiated mice to alkaline solutions. Increasing amounts of DNA and proteins are released from the suspensions of chromatin from thymuses and spleens of irradiated mice (6 hours after 600R whole-body) by the action of alkaline solutions (pH 8-10) at physiological ionic strengths. The suspension of chromatin from normal tissues releases in this pH range only a small amount of proteins and negligible amount of DNA. The behaviour of liver and kidney chromatin to alkaline solutions shows no difference between normal and irradiated tissues. The time of onset and dose relation of the increased sensitivity of thymus and spleen chromatin from irradiated mice to alkaline solutions show a similar course as earlier described signs of postirradiation damage to chromatin of these tissues.


Some biological properties of mouse spleen cells fractionated by the adherence of Sephadex G 25 and glass bead columns. The possibilities of separation of haemopoietic cells from lymphocytes capable of eliciting the graft-versus-host reaction through column chromatography were investigated. Strain-A mouse spleen cells were fractionated into the adherent and non-adherent fraction on Sephadex G-25, glass bead columns and glass beads coated with antibody against mouse globulin. Increased numbers of cells forming haemopoietic colonies were found in the cell fraction which did not adhere to the antibody-coated glass beads and in cells reversibly adhering to glass beads. No significant decrease in local graft-versus-host reaction was found in any fraction obtained, and the prolonged survival of irradiated semiallogeneic recipients was observed in both fractions obtained on Sephadex G-25 columns.


Dopamine as a possible neurotransmitter in gastric relaxation. In dogs with gastric fistulas, intragastric pressure was measured with a flaccid ballon containing 500 ml of water. Graded doses of dopamine caused graded decreases in intragastric pressure. The effect was blocked by pimozide or by metoclopramide but was not significantly affected by phenoxybenzamine, propranolol, guanethidine, or FLA-63 (a beta-hydroxylase inhibitor). Pretreatment with metoclopramide or with pimozide shifted the volume-pressure diagram of the stomach to the left; that is, at any given volume the pressure was greater after than before these drugs. In dogs with vagally innervated gastric pouches and gastric fistulas, feeding for 1 min (while allowing the food to leave the stomach through the gastric fistula) caused a prompt decrease in pressure in the pouch that lasted for about 5 min. Pretreatment with metoclopramide decreased the magnitude and duration of this receptive relaxation. It is concluded that these findings are consistent with (but do not establish) the hypothesis that dopamine is the neurotransmitter for receptive relaxation of the stomach, because dopamine mimics receptive relaxation, and dopamine antagonists partially block reflexly induced receptive relaxation.


Inhibition of intestinal iron absorption by laundry starch. The pathogenesis of iron deficiency anemia associated with amylophagia is usually attributed to dietary iron lack. However, large quantities of starch may inhibit intestinal iron absorption. Accordingly, studies were carried out to determine the effect of laundry starch on the intestinal absorption of inorganic and hemoglobin iron. In vitro, laundry starch bound 19 to 80% of the available 59FeSO4 and 34 to 68% of the available 59Fe-hemoglobin. Binding of both forms of iron was pH-dependent, with maximal binding at pH 7.0. In vivo, laundry starch significantly inhibited mucosal uptake of 59FeSO4 from isolated duodenal loops. In nonanemic rats, administration of laundry starch (100 mg) 1 hr before a 100-mug dose of 59FeSO4 significantly decreased the absorption of 59FeSO4, as compared to saline or low iron chow controls (6.2 +/- 2.0 versus 14.9 +/- 2.1 and -1.8 +/- 1.7, respectively, P less than 0.001). In anemic rats the absorption of either a 100-mug dose of 59FeSO4 or a 500-mug dose of 59Fe-hemoglobin was also significantly decreased by prior administration of laundry starch. The data obtained indicated that laundry starch (1) binds appreciable quantities of inorganic and hemoglobin iron in vitro; (2) inhibits the mucosal uptake or inorganic iron by isolated intestinal loops; (3) inhibits the intestinal absorption of inorganic iron in normal nonanemic rats, and (4) blunts the compensatory increase in inorganic and organic iron absorption in anemic rats.


Effect of serotonin on water and electrolyte transport in the in vivo rabbit small intestine. The influence of intravenously administered serotonin on water and electrolyte fluxes in the in vivo rabbit jejunum and ileum was examined. Animals were divided into four groups: (1) those receiving saline intravenously while a glucose-free isotonic saline solution perfused the jejunum and ileum; (2) serotonin given intravenously while glucose-free intestinal perfusate was used as in group 1; (3) intravenous saline given while a 10 mM glucose-isotonic saline solution perfused the jejunum and ileum; and (4) intravenous serotonin given while the intestinal perfusate was as in group 3. Serotonin administration resulted in highly significant net secretion of H2O and sodium in both jejunum and ileum in the groups with a glucose-free perfusate. In jejunum, serotonin evoked net water and sodium secretion, whereas controls absorbed water and sodium. In ileum, serotonin significantly enhanced secretion. The addition of glucose to the perfusate completely abolished the serotonin effect. Unidirectional 22Na flux analysis revealed a marked diminution in both mucosal to serosal and serosal to mucosal fluxes in serotonin-treated animals. The decrease in mucosal to serosal flux was greater than the decrease in serosal to mucosal flux, thus explaining the enhanced net secretion observed with serotonin in the groups receiving glucose-free perfusate. In spite of its pronounced effect on water and electrolyte transport, serotonin failed to produce any detectable histological alterations in small bowel mucosa, either by light or electron microscopy. We postulate that serotonin may be an important mediator of the diarrhea so frequently noted in the carcinoid syndrome by virtue of its effects on small intestinal H2O and electrolyte transport.


Lower esophageal sphincter response to oral administration of cimetidine in normal subjects. Anithistamines that specifically block the gastric and secretory action of histamine have recently been developed. One of these H2-receptor blockers, metiamide, has been found to increase lower esophageal sphincter (LES) pressure in the opossum. Because of reported agranulocytosis with metiamide, another H2-receptor blocking agent, cimetidine, was developed. To determine its effect on LES pressure, 8 normal volunteers received placebo or oral doses of cimetidine (50, 100, 200, and 400 mg) in a random, blinded manner. Indicative of adequate absorption, significant serum levels were achieved with all doses of cimetidine (50 mg = 0.17 mug per ml; 100 mg = 0.33 mug per ml; 200 mg = 0.76 mug per ml; and 400 mg = 1.61 mug per ml). Although these serum levels have been found to produce marked inhibition of gastric acid secretion, no discernible effect was found on LES pressure when compared to placebo. Thus cimetidine does not increase LES pressure. It does not decrease sphincter pressure either and is therefore not contraindicated in patients with reflux esophagitis.


The role of histamine receptors in the pathophysiology of gastric mucosal damage. In four canine Heidenhain pouches the net fluxes of H+ and Na+ have been examined before, during, and after instillation of sodium taurocholate into the pouch. These experiments were conducted in animals given H1 (mepyramine maleate) and H2 (metiamide) histamine antagonists, alone and in combination. Control experiments without antagonists were also conducted. In control experiments, as well as in those using  the histamine antagonists separately, the usual sequence of events followed exposure to taurocholate-that is, a gain in the volume of the solution in the pouch and an increase in the fluxes of Na+ and H+ across the mucosa. In experiments in which H1 and H2 histamine antagonists were used in combination, taurocholate had very little effect on the ionic fluxes of H+ and Na+, suggesting that changes in the ionic permeability of the gastric mucosal barrier are mediated by histamine through both H1 and H2 receptor sites.


Properties of gastric antrum. III. Selectivity and modification of shunt conductance. The permselectivity of the paracellular pathway of amphibian (Necturus and bullfrog) antrum was investigated with respect to intracationic selectivity and the K+ and Cl- permeability ratio as a function of mucosal pH. The intracationic selectivity sequence was Rb+ greater than K+ greater than Cs+ greater than Na+ greater than Li+. Both antra showed weak cationic selectivity at pH 7.4, and at pH 4.4 for bullfrog and pH 3.0 for Necturus, the ratio Pk+/P Cl- was unity. At lower mucosal pH the tissues were anion selective. Treatment of the tissue with a water-soluble carbodiimide enhanced anion selectivity at higher pH; carbenoxolone, a weak acid, resulted in maintained cation selectivity at lower pH. These data suggest that carboxyl groups play a role in determining shunt selectivity; the increase in anion selectivity below pH 2.0 suggests that phosphate or sulfate groups could also be involved.


Effect of fundusectomy on serum and antral gastrin levels in rats. In adult male rats, fundusectomy decreased acid secretion but significantly increased total antral gastrin and both fasting and food-stimulated serum gastrin levels. The rise in fasting serum gastrin could be inhibited by antral acidification, suggesting that decreased acidity caused postfundusectomy hypergastrinemia. The mechanism for the increase in total gastrin in antral tissue is probably the same. These studies provide a useful experimental model for the increasing of antral gastrin and for the production of hypergastrinemia.


Postprandial gastric, pancreatic, and biliary response to histamine H2-receptor antagonists active duodenal ulcer. Histamine H2-receptor antagonists are potentially useful agents in duodenal ulcer and knowledge of their effect on postprandial digestive events will contribute to their clinical application. We studied the effect of 200- and 300-mg doses of cimetidine, an H2-receptor antagonist, taken with an ordinary meal, on gastric, pancreatic, and biliary function. Both doses significantly reduced acid output and its delivery into the duodenum. Gastric secretory volume and pepsin output were less affected. Acid inhibition was related to blood drug levels and was less than that previously found at night in nocturnal fasting studies. As the stomach emptied the food, the gastric pH rose. The fractional gastric emptying rate, pancreatic enzyme, and bile acid outputs were unaltered. Cimetidine taken orally with meals at these doses is a potent gastric antisecretory agent without affecting other postprandial gastric, pancreatic, or biliary functions.


Isolation and characterization of four peptide hydrolases from the cytosol of rat intestinal mucosa. The high speed supernatant fluid prepared from rat intestinal mucosa was subjected to ion-exchange chromatography on diethlaminoethyl-cellulose eluted with a linear gradient of sodium chloride (0 to 0.27 M). Assay of eluted fractions for Phe-Gly hydrolase activity revealed four distinct peaks of enzyme activity. These cytosol enzymes have been designated I, II, III, and IV in order of their elution from the column. Examination of the substrate specificity of the four enzymes by use of 20 mM peptide concentrations indicated the most discriminating substrates for the four enzymes were Leu-Gly-Gly, His-Met, Ser-Phe, and leucine amide, respectively. The mean distribution of the recovered peptide hydrolase activities against these substrates among the four enzymes I, II, III, and IV was 96.1, 1.4, 1.7, and 0.8%, respectively, for Leu-Gly-Gly; 0.6, 96.4, 2.4, and 0.6% for His-Met; 0, 0, 95.8, and 4.2% for Ser-Phe; and 20.8, 19.8, 5.6, and 53.8% for leucine amide. Ion-exchange chromatography resulted in increases in specific activity of 19-, 19-, 46-, and 3.5-fold for enzymes I, II, III, and IV, respectively. The activity of all four enzymes, but especially III and IV, were stabilized by the presence of 150 muM dithioerythritol. Activity of each of the four enzymes was decreased 79 to 100% by 1mM ethylenediaminetetraacetate, HgCl2, 1, 10-phenanthroline, or 0.5 mM p-hydroxymercuribenzoate, except that the activity of enzyme I was decreased only 15% by ethylenediaminetetraacetate. No significant activation of the partially purified enzymes occurred in the presence of 500 muM Zn++, Co++, or Mg++. The four enzymes exhibited distinct pH profiles with optima at 7.5, 7.5, 8.5, and 8.0 for enzymes I, II, III, and IV, respectively. Molecular weights of the four enzymes determined by gel filtration on Sephadex G-200 were 58,500, 74,000, 97,500, and 113,000, respectively. All four enzymes lost more than 85% of their activity after 1 hr at temperatures of 50 degrees C or higher in sodium phosphate buffer, pH 7.0. The Km values determined with the most specific substrates for each enzyme were 0.76, 0.44, 3.82, and 8.3 mM for enzymes I, II, III, and IV, respectively. Recent evidence suggests that a significant amount of some small peptides are absorbed intact and hydrolyzed by cytosol peptide hydrolases. Adequate understanding of the function and control of these intracellular enzymes requires knowledge of the characteristics and substrates specificity of individual enzymes. The study described here demonstrates the presence of at least four cytosol peptide hydrolases with distinct substrate specificities. Substrates almost exclusively hydrolyzed by each of three of the enzymes, and therefore suitable for assay of each of these enzymes in the presence of the others, have been identified.


[Characteristics of two new mutant forms of erythrocyte glucose-6-phosphate dehydrogenase: "Kirovograd" G6PD and "Zhitomir" G6PD]. The paper comprises the description of properties of three mutant forms of glucoso-6-phosphate dehydrogenase characterized according the WHO program. Preparations of the enzymes were isolated from erythrocytes of patients with G6PD deficiency from three unrelated to one another Ashkenasi families coming from the Ukraine and from Byelorussia. Two new variants of G6PD hitherto never described in the literature were discovered. These variants were designated as "Kirovograd" and "Zhitomir" after the towns the probands came from. The properties of purified enzymes revealed by the methods of the WHO program were as follows: the variant "Kirovograd" has a normal electrophoretic mobility in tris and TEB buffers and 98% of the normal in phosphate buffer. KM for G6P is 6,54; KM for NADP--2,19. It is characterized by a reduced thermostability and by an acute peak of activity at pH 8,5. The variant "Zhitomir" has 90-98% of the normal electrophoretic mobility in TEB buffer and 78-84% in phosphate buffer. KM for G6P is 5,4-8,3. KM for NADP is 1,4-3,1; with deoxyG6P is 50% and with deaminoNADP is 35%. It is also characterized with a reduced thermostability, while the curve of the dependence of its activity on pH has two peaks. Both variants are perfectly inactive with erythrocytes and thus should be assigned to the second group of the mutants variants of G6PD. The comparison of these variants to other variants encountered in the same national group revealed that they resemble certain quantitative variations.