Derepressed levels of glutamate synthase and glutamine synthetase in Escherichia coli mutants altered in glutamyl-transfer ribonucleic acid synthetase.

The levels of glutamate synthase and of glutamine synthetase are both derepressed 10-fold in strain JP1449 of Escherichia coli carrying a thermosensitive mutation in the glutamyl-transfer ribonucleic acid (tRNA) synthetase and growing exponentially but at a reduced rate at a partially restrictive temperature, compared with the levels in strain AB347 isogenic with strain JP1449 except for this thermosensitive mutation and the marker aro. These two enzymes catalyze one of the two pathways for glutamate biosynthesis in E. coli, the other being defined by the glutamate dehydrogenase. We observed a correlation between the percentage of charged tRNAGlu and the level of glutamate synthase in various mutants reported to have an altered glutamyl-tRNA synthetase activity. These results suggest that a glutamyl-tRNA might be involved in the repression of the biosynthesis of the glutamate synthase and of the glutamine synthetase and would couple the regulation of the biosynthesis of these two enzymes, which can work in tandem to synthesize glutamate when the ammonia concentration is low in E. coli but whose structural genes are quite distant from each other. No derepression of the level of the glutamate dehydrogenase was observed in mutant strain JP1449 under the conditions where the levels of the glutamine synthetase and of the glutamate synthase were derepressed. This result indicates that the two pathways for glutamate biosynthesis in E. coli are under different regulatory controls. The glutamate has been reported to be probably the key regulatory element of the biosynthesis of the glutamate dehydrogenase. Our results indicate that the cell has chosen the level of glutamyl-tRNA as a more sensitive probe to regulate the biosynthesis of the enzymes of the other pathway, which must be energized at a low ammonia concentration.


L-asparagine uptake in Escherichia coli.

The uptake of L-asparagine by Escherichia coli K-12 is characterized by two kinetic components with apparent Km values of 3.5 muM and 80 muM. The 3.5 muM Km system displays a maximum velocity of 1.1 nmol/min per mg of protein, which is a low value when compared with derepressed levels of other amino acid transport systems but is relatively specific for L-asparagine. Compounds providing effective competition for L-asparagine uptake were 4-carbon analogues of the L-isomer with alterations at the beta-amide position, i.e., 5-diazo-4-oxo-L-norvaline (Ki = 4.6 muM), beta-hydroxyamyl-L-aspartic acid (Ki = 10 muM), and L-aspartic acid (Ki = 50 muM). Asparagine uptake is energy dependent and is inhibited by a number of metabolic inhibitors. In a derived strain of E. coli deficient in cytoplasmic asparaginase activity asparagine can be accumulated several-fold above the apparent biosynthetic pool of the amino acid and 100-fold above the external medium. The high affinity system is repressed by culture of cells with L-asparagine supplements in excess of 1 mM and is suggested to be necessary for growth of E. coli asparagine auxotrophs with lower supplement concentrations.


Properties of a membrane-bound cardiolipin synthetase from Lactobacillus plantarum.

Cardiolipin (CL) synthetase of Lactobacillus plantarum 17-5 catalyzed the stoichiometric conversion of 2 mol of phosphatidylglycerol to 1 mol of CL. The enzyme activity was linear with time for 30 min at 37 C and with protein concentration between 20 and 200 mug of protein per ml. The enzyme was membrane associated, had a pH optimum of 5.1 in phosphate buffer, and was not stimulated by Mg2+, and the activity was not affected by the addition of ethylenediaminetetraacetic acid, cytidine diphosphate diglyceride, or cytidine triphosphate. The reaction was inhibited about 95% by Triton X-100 (0.5% final concentration) and by CL, the end product of the reaction. The activity of this enzyme was studied as a function of growth. The CL synthetase specific activity was highest during the early and midexponential growth phases, as was the cellular content of CL. The results demonstrate a correlation between enzyme-specific activity and lipid content of the cells.


Regulation of glutaminase levels in Escherichia coli.

Nitrogenous metabolites, cyclic adenosine 3':5'-monophosphate (cAMP), and the stage of culture growth all influence the levels of glutaminase A in Escherichia coli, but no variables in culture conditions alter the levels of glutaminase B. Growth of E. coli on culture media containing glucose and excess ammonia results in a rise in the level of glutaminase A as the cultures enter stationary phase; this rise is abolished by ammonia limitation. cAMP or glycerol reduce the level of glutaminase A. In mutants deficient in cAMP receptor protein, glutaminase A levels are unchanged by cAMP, but they are still susceptible to regulation by ammonia. We consider glutaminase B to be a constitutive enzyme, since its levels appear independent of nutritional conditions.


Glucose-forming amylase in human urine.

This paper describes the isolation and study of glucose-forming amylase existing in human urine as a normal component. After removing alpha-amylase [EC 3.2.1.1] by adsorption onto raw starch, urine was treated with DEAE-cellulose and Bio Gel P-150, and three fractionated proteins (F-1, F-2, and F-3), isolated in a homogeneous state by gel filtration, were shown to display glucose-formine amylase activity. They all hydrolyzed starch and glycogen, releasing glucose as the sole product, and also hydrolyzed maltose. However, their molecular weights, as estimated by gel filtration, isoelectric points, stabilities, and several enzymatic properties were different. The implications of the results are discussed.


O-acetylserine and O-acetylhomoserine sulfhydrylase of yeast; studies with methionine auxotrophs.

The nutritional requirements of three yeast mutants, previously shown to possess low O-acetyl-L-serine (OAS) and O-acetyl-L-homoserine (OAH) sulfhydrylase activities, were reinvestigated. It was thus found that one mutant (strain No. 16), previously identified as a homocysteine auxotroph, is in fact a double mutant requiring both cysteine and OAH. In agreement with the previous assignment, the other two strains (strains No. 13 and 17) were shown to be true cysteine auxotrophs. These results can best be explained by assuming the cystathionine pathway to be the main route of homocysteine synthesis in this organism. It was further found that extracts of the three mutants contain genetically modified OAS-OAH sulfhydrylases with much reduced catalytic activities. Modified sulfhydrylase was partially purified from strain No. 16 by the same procedure as for the wild-type enzyme. Both OAS and OAH sulfhydrylase activities of the mutant enzyme were copurified and behaved identically on polyacrylamide gel electrophoresis. The enzymatic and physicochemical properties of the purified mutant enzyme were shown to be very similar to those of the wild-type enzyme, except that the catalytic activities of the former were only 3-5% of those of the latter, and that the ratio of OAH sulfhydrylase to OAS sulfhydrylase activity was somewhat lower in the former than in the latter.


Reduced nicotinamide adenine dinucleotide phosphate-dependent lipid peroxidation by beef heart submitochondrial particles.

Electron transport particles (ETP) prepared from beef heart mitochondria formed malondialdehyde by NADPH-dependent lipid peroixidation in the presence of ferric ions and ADP or ATP. The reaction was inhibited by MnCl2, EDTA, or radical scavengers, but was not inhibited by p-hydroxymercuribenzoate (PHMB) or respiratory chain inhibitors. The oxidation of NADPH and oxygen consumption by ETP were activated by the addition of ferric ions and APT, and inhibited by inhibitors of lipid peroxidation. This peroxidation system was apparently different from those of liver microsomes and mitochondria as regards the effect of PHMB, optimal pH and the concentration of NADPH for half-maximal reaction velocity.


Studies on the state of tyrosyl residues in a ribonuclease from seminal vesicles.

In order to study the state of tyrosyl residues in a ribouuclease from bovine semina vesicles [EC 3.1.4.22, RNase Vs1] several lines of experiments were carried out. Spectrophotometric titration of RNase Vs1 indicated that two out of 8 tyrosine residues were titrated very easily and their apparent pKa values were about 9.8. Next, about 4 residues were titrated at pH up to 13.5. The remaining 2 residues were titrated time-dependently at pH 13.5. In 8 M urea, about 6 tyrosine residues were titrated with apparent pK4 values of about 11.2 and about 2 residues were titrated time-dependently at pH 13.5. Acetylation of RNase Vs1 with N-acetylimidazole was studied at pH 7.5. In aqueous solution, about 1.1-3.5 tyrosine residues were acetylated, depending on the experimental conditions, and in 8 M urea, 5.3 tyrosine residues were modified. RNase Vs1 was nitrated with tetranitromethane at pH 7.5. In aqueous solution, about 2.5 tyrosine residues were nitrated very easily; the enzymatic activity of the modified enzymes was 130-200% of that of the native enzyme. In 8 M urea, the reactivity of the tyrosine residues increased and about 4-5.5 residues were modified. The results of chemical modification and spectrophotometric titration indicated that about two tyrosine residues in RNase Vs1 were exposed to the solvent and were more reactive to various reagents, and 3-4 tyrosine residues were less reactive. The final 2 residues were not accessible to the reagent even in the presence of urea, but were titraten at pH 13.5. The solvent perturbation difference spectrum using ethylene glycol as a perturbant indicated that about 4 tyrosine residues were perturbed. When the pH of the enzyme solution was changed from 7.0 to 1.0, the change in optical density of RNase Vs1 due to denaturation blue shift was about 1,600 at 287nm. The optical density change at 287 nm of native RNase Vs1 on exposure to 8 M urea and 6 M guanidine-HCl indicated that the environments of 2-3 and 4 tyrosine residues were changed by the addition of the denaturants, urea and guanidine-HCl, respectively. In RNase Vs1 having about four nitrotyrosine residues, the two most inaccessible tyrosine residues remained resistant to titration with alkali. On adding nucleotide, nitrated RNase Vs1 gave a difference spectrum in the ultraviolet region but not in 320-460 nm region, where nitrotyrosine residues absorb light. This may indicate that tyrosine residues located relatively near the surface of the molecule are not perturbed directly by nucleotide binding.


Purification and properties of a new ribonuclease from Aspergillus saitoi.

From a commercial digestive produced from Aspergillus saitoi, a ribonuclease [EC 3.1.4.23] having a molecular weight of 12,500 has been isolated in addition to the RNase reported previously, which had a molecular weight of 38,000. The enzyme was found to be homogeneous by chromatography on DEAE-cellulose, disc electrophoresis on polyacrylamide gel, and ultracentrifugation. The NH2-terminal amino acid was identified as glutamic acid. The amino acid composition indicated the presence of about 13 tyrosyl residues, 3 histidyl residues, and 2 half-cystine residues. The pH optimum of the RNase was 4.5, using RNA as a substrate. The enzyme was stable on heating at 70 degrees for 5 min from pH 2 to 10. It hydrolysed RNA completely to mononucleotides via 2', 3'-cyclic nucleotides. The rates of release of nucleotides and 2', 3'-cyclic nucleotides were in the order: guanylic acid is greater than adenylic acid is greater than cytidylic acid is greater than uridylic acid.


Change in effective pH of salt solutions on freezing, as evidenced by altered reactivities of heme alpha towards carbonyl reagents.

Addition of NaHSO3 or HCN to the formyl group of heme alpha was greatly accelerated by freezing reaction mixtures prepared in aq. Na2CO3, and freezing resulted in characteristic color and spectral changes of the solutions. Similar changes were observed on decreasing the pH of alkaline reaction mixtures with HCl at room temperature, indicating that the effective pH of certain salt solutions is greatly lowered by freezing. The reactivity of the formyl group changed depending on the redox state of the heme iron and the species of ligand.


Proteolytic degradation of hemoglobin-haptoglobin complex by lysosomal enzymes from rat liver.

The catabolic degradation of hemoglobin and of its complex with haptoglobin by lysosomal enzymes from rat liver was studied with special emphasis on the action of cathepsins D and E. The digestion of free hemoglobin can be mainly attributed to the action of cathepsin D [EC 3.4.23.5], while the digestion of the complex in the pH rand 2-3 is due more to the action of cathepsin E than that of cathepsin D. The enzymic activities of both cathepsins were strongly inhibited by pepstatin, and 4M urea inactivated cathepsin E. Measurements of the peroxidase activity and optical rotatory dispersion of the hemoglobin-haptoglobin complex showed that the complex suffered rapid denaturation below pH 2.9.


Photooxidation of histidine and tryptophan residues of papain in the presence of methylene blue.

Papain [EC 3.4.22.2] was photooxidized using methylene blue as a sensitizer. The photooxidzed enzyme lost its caseinolytic activity and had significantly decreased histidine and tryptophan contents. The tyrosine content was the same before and after the photooxidation. The SH content of the photooxidized enzyme, as determined after reduction with dithiothreitol, was also unchanged. The loss of histidine was always slower than the loss of enzymatic activity, being less than one residue per molecule even when the enzymatic activity was completely lost. However, the inactivation and the oxidation of a histidine residue were pH-dependent in a similar fashion in the pH range of 5.0-8.0, the pH profiles conforming to theoretical titration curves with apparent pKa values of 6.6 and 6.7, respectively. The fact that the ionization of a histidine residue in papain has a normal imidazole pKa value is entirely in accord with the finding for stem bromelain [EC 3.4.22.4] (Murachi, T., Tsudzuki, T., & Okumura, K. (1975) Biochemistry 14, 249-255), and is of great significance in relation to the mechanism of catalysis by these enzymes.


The pH jump study of enzyme proteins. I. Liquefying alpha-amylase from Bacillus subtilis.

Rapid conformational changes due to pH jump were studied kinetically at 25 degrees mainly by the stopped-flow method using liquefying alpha-amylase from Bacillus subtilis [EC 3.2.1-.1, liquefying]. First, the conformational change due to a pH jump produced by mixing with alkali was monitored as a function of time at 245 nm through the ionization of phenolic hydroxyl groups of tyrosine residues which were originally buried and finally become exposed due to the pH jump. Three distinct phases of conformational change were clearly recognized by this method by varying the final pH values. Each phase involved the exposure of an essentially definite number of tyrosine residues, whose rate constant was crucially dependent on pH. Second, these phases of conformational change were subjected to examination in terms of the optical rotation change at 411 nm and the reversibility upon reverse pH jump with respect to conformational reconstitution, as observed through the protonation ofphenolic hydroxyl groups of ionized tyrosine residues and the enzyme activity. The first phase, which occurs above pH 12.5, involves no change in the optical rotation and is reversible as observed by the above two monitoring methods. In contrast, the other two phases, which are observed above pH 12.7, are accompanied by an optical rotation change and no appreciable reversibility was detected by these methods.


Myosin from molluscan abalone, Haliotis discus. Isolation and enzymatic properties.

Actomyosin was extracted from smooth muscle of molluscan abalone with 0.1 M PPit pH 6.4. Myosin was separated from the actomyosin by centrifugation at 100,000 X g in the presence of 5 mM ATP and 10 mM MgCl2. Myosin in the supernatant was further purified by gel filtration on a Sepharose 4B column. Paramyosin contamination of the actomyosin preparation interfered with the isolation of myosin and complete removal of actin and paramyosin from the myosin has not been accomplished. The myosin appeared to consist of a single f-chain and a single g-chain, as examined by SDS-disc electrophoresis in 8 or 13.7% acrylamide gel. The ATPase [EC 3.6.1.3] activity of this myosin in 0.5 M KCL at neutral pH and at 0 degrees was rather unstable and decreased by 10-20% per day. The effects of rho-chloromercuribenzoate and EDTA on the ATPase activity were similar to those observed with other smooth muscle myosin but the dependence upon pH or KCL concentration was different.


Binding of substrate analogues to hen egg-white lysozyme with 2-nitrophenylsulfenylated tryptophan 62.

The pH dependence of the extrinsic circular dichroic (CD) band at 375 nm of hen egg-white lysozyme [EC 3.2.1.17] in which Trp 62 had selectively 2-nitrophenylsulfenylated (NPS-lysozyme) was studied. This pH dependence was interpreted in terms of the participation of Glu 35 (pK 6.2), Asp 101 (pK 4.6), and Asp 66 (pK1.5). The fact that the ionization of Glu 35 affects the extrinsic CD band of the NPS chromophore attached to Trp 62 confirms the presence of a relation between the state of Trp 62 and the ionization state of one of the catalytic groups, Glu 35, in hen lysozyme, as proposed by Ikeda and Hamaguchi (J. Biochem., 74, 221-230 (1973)). The pH dependence of the binding constants of the dimer and trimer of N-acetylglucosamine (GlcNAc) and the beta-methyl glycoside of GlcNAc (beta-methyl-GlcNAc) to NPS-lysozyme were studied by measuring the changes in the extrinsic CD band. The changes in the CD spectrum on the binding of (GlcNAc)3 and (GlcNAc)2 were very similar to each other but were different from that on the binding of beta-methyl-GlcNAc. However, beta-methyl-GlcNAc competitively inhibited the binding of (GlcNAc)2 to NPS-lysozyme. The binding constants of the three saccharides to NPS-lysozyme were much smaller than those for intact lysozyme. The pH dependence of the binding constants of (GlcNAc)2 and (GlcNAc)3 were interpreted in terms of the participation of Glu 35 (pK 6.2), Asp 52 (pK 3.3), Asp 101 (pK 4.6), and Asp 66 (pK 1.5). These pK values are very similar to those for intact lysozyme, as determined by Kuramitsu et al. (J. Biochem., 76, 671-683 (1974); 77, 291-301 (1975). Comparison of the binding constants of Mn2qnd Co2'ons to the catalytic carboxyls of NPS-lysozyme with those to intact lysozyme also indicated that the catalytic site of NPS-lysozyme is scarcely affected by this modification. When (GlcNAc)2 or (GlcNAc)3 is bound to NPS-lysozyme, pK shifts of Glu 35, Asp 101, and Asp 66 occurred in the same directions as for intact lysozyme. In addition, a pK shift of Asp 52, which has not been observed for intact lysozyme, occurred. The participation of Asp 52 was also observed in the binding of beta-methyl-GlcNAc. However, the binding of the monomer to NPS-lysozyme produced no significant pK shifts of Glu 35 and Asp 101, in contrast to the situation for intact lysozyme. These facts indicate a small difference in the binding orientation of the saccharides between the modified and intact lysozymes.


L-fucose metabolism in mammals. Kinetic studies on pork liver 2-keto-3-deoxy-L-fuconate:NAD+ oxidoreductase.

Pork liver 2-keto-3-deoxy-L-fuconate:NAD+ oxidoreductase has been shown to convert 2-keto-3-deoxy-L-fuconate to a 6-carbon acid tentatively identified as 2,4(or 5)-diketo-5(or 4)-monohydroxyhexanoate. The enzyme has a pH optimum of 10. 5 or higher. It is stabilized by dithiothereitol and inhibited by p-hydroxymercuribenzoate and heavy metals (Ag+, Hg2+, Co2+, Cd2+, Pb2+, Zn2+, and Cu2+), suggesting the presence of a functionally essential sulfhydryl group; pre-treatment of enzyme with NAD+ prevents inhibition by p-hydrocymercuribenzoate and heavy metals indicating that this sulfhydryl group may be near the NAD+ binding site. The enzyme has an absolute requirement for NAD+; NADP+ is an ineffective coenzyme. Several lines of evidence indicate that the same enzyme acts on both 2-keto-3-deocy-L-fuconate and 2-keto-3-deoxy-D-arabonate; thus, the pure enzyme acts on both substrates, the two substrates have very similar kinetic parameters (Km values are: 2-keto-3-deocy-L-fuconate, 0.20 mM; 2-keto-3-deoxy-D-arabonate, 0.25 mM; NAD+ for either substrate, 0.22 to 0.25 mM), the two substrates show identical pH and temperature profiles and the two substrates compete for common enzyme active sites. A large number of other sugars and sugar acids, including several 2-keto-3-deoxyaldonates, were ineffective as substrates. The dehydrogenase was also found in calf, beef, lamb, mouse, and rat liver. These studies when considered together with previous studies on the metabolism of L-fucose in pork liver indicate the presence of a soluble enzyme pathway capable of converting L-fucose to 2,4(or 5)-diketo-5(or 4)-monohydroxyhexanoate; this pathway can also convert D-arabinose, and probably L-galactose, to the analogous derivatives (diketomonohydroxypentanoate and diketodihydroxyhexanoate, respectively.


pH dependence of the cooperative interactions and conformation of tryptophan oxygenase.

Allosteric interactions in the cupro-heme enzyme tryptophan oxygenase (EC 1.13.11.11) of Pseudomonas acidovorans are shown to be pH-dependent. Increasing the assay pH from 6.0 to 8.0 progressively desensitizes the enzyme from both homotropic and heterotropic ligand interactions. This pH-dependent reversible transition has a pK of 6.2. Hill coefficients for the substrate L-tryptophan of 2.0 and 1.4 were measured at pH 6.0 and pH 7.0, respectively. In attempting to identify the enzymatic residue (or residues) responsible for these pH-dependent effects, the enzyme was observed to be irreversibly inactivated by photoinduced oxidation in the presence of the sensitizer, methylene blue. The photoinactivated enzyme showed a loss of one-half its Soret (405 nm) absorption which accompanied the loss of one-half its heme and histidine contents. This first order photoinduced inactivation was pH-dependent and corresponded to a requirement for a protonated species with a pK of 6.2. These results suggest that histidine residues may be involved in the catalytic function and in mediating cooperative interactions of tryptophan oxygenase. Absolute and difference sedimentation velocity analyses indicate that the molecule undergoes a conformational transition when the pH is decreased from pH 8.0 to pH 6.0. This conformational alteration, measured as a 3.9% increase in S20, w can be regarded as an equivalent decrease in the frictional coefficient. If, a more or less spherical shape to the molecule is assumed, then, the 3.9% decrease in the frictional coefficient between pH 8.0 and 6.0 corresponds to a 12% decrease in apparent hydrodynamic volume of the enzyme. Thus, protonation of an enzymatic moiety, possibly histidine, determines both the conformational and functional interactions between enzymatic sites.


Guanylate cyclase in Escherichia coli. Purification and properties.

Guanylate cyclase has been purified from extracts of Escherichia coli. After a 1000-fold purification, the enzyme contains only minor contaminants as judged by disc gel electrophoresis. The Km for GTP is approximately 7 times 10(-5) M and the optimal pH is 8.0. More activity is observed with Mn2+ than with Mg2+, and maximal activity is observed at 0.14 mM Mn2+ and 1.4 mM Mg2+. Based on its behavior on Sephadex G-100, the molecular weight of E. coli guanylate cyclase is about 30,000. Disc gel electrophoretic analysis indicates that the enzyme consists of a single polypeptide chain. Guanylate cyclase does not form 3':5'-AMP from ATP, and therefore, is distinct from adenylate cyclase.


Cascade control of Escherichia coli glutamine synthetase. Properties of the PII regulatory protein and the uridylyltransferase-uridylyl-removing enzyme.

The PII regulatory protein of Escherichia coli glutamine synthetase exists in two interconvertible forms: a uridylylated form (PIID) which promotes the deadenylylation of glutamine synthetase and an unmodified form (PIIA) which promotes the adenylylation of glutamine synthetase (Mangum, J.H., Magni, G., and Stadtman, E.R. (1973) Arch. Biochem. Biophys. 158, 514-525). PII has been purified to homogeneity. Its molecular weight is 44,000. The protein is composed of four subunits, each with a molecular weight of approximately 11,000. The subunits are identical as judged by: (a) the homogeneity of the subunits in sodium dodecyl sulfate, 8 M urea, and 6 M guanidine HCl; (b) the minimal molecular weight calculated from the amino acid composition; and (c) the isolation of only two tryptic peptides containing tyrosine (there are 8 tyrosyl residues per 44,000 molecular species). Following iodination of PIIA and PIID with 125I in the presence of chloramine-T, tryptic digestion yields two radioactive peptides from PIIA and only one from PIID. Since a tyrosine with a substituted hydroxyl group cannot be iodinated, this result indicates that 1 tyrosyl residue in each subunit is modified by the covalent attachment of UMP. This conclusion is supported also by the fact that treatment of PIID with snake venom phosphodiesterase results in the release of covalently bound UMP and the stoichiometric appearance of phenolate ion (pH 13) as measured by ultraviolet absorption spectroscopy. The enzyme activities (uridylyl-removing) responsible for removal and (uridylytransferase) responsible for attachment of UMP to PII have been partially purified. These activities co-purify through a variety of procedures, including hydrophobic chromatography, and are stabilized by high ionic strength buffers. Whereas Mn2+ alone supports only uridylyl-removing activity, ATP, alpha-ketoglutarate, and Mg2+ support both uridylyl-removing and uridylyltransferase activities.


Hemoglobin Abruzzo (beta143 (H21) His replaced by Arg). Consequences of altering the 2,3-diphosphoglycerate binding site.

Hemoglobin Abruzzo is an abnormal human hemoglobin with a substitution at a residue known to be involved in the binding of 2,3-diphosphoglyceric acid. It has increased oxygen affinity and reduced heme-heme interaction in the absence of organic or inorganic phosphate cofactors. In inorganic phosphate buffers the Bohr effect and heme-heme interaction are normal, but the oxygen affinity remains higher than that of hemoglobin A. CO combination in inorganic phosphate is more strongly autocatalytic than in normal hemoglobin and a slower rate of oxygen dissociation is observed. Although many of the functional differences of this variant may be attributed to the high oxygen affinity of the mutant beta chains, the interactions between subunits are also affected by the histidine to arginine substitution at beta143. Stripped hemoglobin Abruzzo appears to be significantly more dissociated than hemoglobin A. Kinetic studies indicate that interaction with organic or inorganic phosphates decreases its subunit dissociation. In all of the functional properties examined, hemoglobin Abruzzo is more sensitive to the allosteric influence of organic and inorganic anions than is hemoglobin A.


Transient state kinetic studies of proton liberation by myosin and subfragment 1.

Myosin and subfragment 1 give a maximum burst size of 0.25 to 0.30 protons per active site at pH 8 with ATP, alpha,beta-methylene-ATP, ADP, and adenylyl imidodiphosphate as substrates. The proton is derived from a change in conformation of the enzyme-substrate complex since it is produced by substrates which are not hydrolyzed. The rate constants for the binding of ATP and the proton release step in 0.1 M, 0.5 M, and 1.0 M KCl have been determined by analysis of the concentration dependence of the apparent rate. (see article)


Alpha-methylisocitrate. A selective inhibitor of TPN-linked isocitrate dehydrogenase from bovine heart and rat liver.

Alpha-Methylisocitrate (3-hydroxy-1,2,3-butanetricarboxylate) is a potent inhibitor, competitive with isocitrate (1-hydroxy-1,2,3-propanetricarboxylate), of the TPN-linked isocitrate dehydrogenase from bovine heart and rat liver; it does not inhibit the DPN-specific enzyme from these tissues. In the presence of magnesium ion, values of Kis for DL-alpha-methylisocitrate for purified bovine heart enzyme, rat liver cytosol, and rat liver mitochondrial extract were in the range of 0.1 muM to 0.3 muM. This compared to values of apparent Km for DL-isocitrate for the same tissue preparations of 14 muM to 20 muM. One of the DL isomer pairs of alpha-methylisocitrate was inactive; the observations suggest that it is threo-alpha-methylisocitrate which inhibits TPN-linked isocitrate dehydrogenase. A method of synthesis of DL-threo-alpha-methylisocitric lactone (2-methyl-5-oxo-2,3-furandicarboxylic acid) from dimethyl trans-epoxymethylsuccinate and dimethylmalonate is described.


Nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase of Neurospora. III. Inactivation by nitration of a tyrosine residue involved in coenzyme binding.

Neurospora glutamate dehydrogenase (NADP-specific) is rapidly inactivated upon reaction with tetranitromethane. This inactivation is completely prevented by the presence of coenzyme (NADP) or nicotinamide mononucleotide (NMN) but not by substrate. NADH, or 2'-monophosphoadenosine-5'-diphosphoribose. Amino acid analysis indicates that the primary effect of modification is nitration of a single residue of tyrosine per polypeptide chain. We have identified the reactive tyrosine by isolation of a single, uniquely labeled peptide after hydrolysis with trypsin followed by cleavage with cyanogen bromide. The modified residue proved to be tyrosine-168 in the linear sequence. This residue is not present in the part of the sequence that had been previously implicated as involved in the binding of the adenylate portion of the coenzyme. Both NMN and 2-monophosphoadenosine-5'-diphosphoribose act as competitive inhibitors of NADP in the oxidation of glutamate with Ki values of 4.65 x 10(-4) M and 4.30 x 10(-4) M, respectively. Thus, the specific protection afforded by NADP and NMN, but not by 2'-monophosphoadenosine-5'-diphosphoribose, indicates that tyrosine-168 is involved in binding the nicotinamide portion of the coenzyme.


Molecular aspects of the inactivation of tryptophanyl transfer ribonucleic acid synthetase by N-ethylmaleimide.

The tryptic maps of tryptophanyl-tRNA synthetase from beef pancreas show that the 8 cysteinyl residues of the enzyme subunit are located, 2 by 2, on four different peptides. The kinetics of the incorporation of radioactivity from N-[ethyl-14C]ethylmaleimide into these peptides are compared in this paper with the kinetics of the changes of the catalytic properties of the enzyme occurring during alkylation. This comparison allows the identification of (a) the peptide carrying the cysteinyl residues located on the surface of the molecule, (b) the peptide carrying the deeply buried residues unmasked by the dissociation of the subunits, and (c) the peptide carrying the --SH group located in the vicinity of the binding site of tryptophan. The fourth peptide is shown to have a great sensitivity to pH with respect to the reactivity of its cysteinyl residues toward N-ethylmaleimide. The same unusual pH dependence is found for the rate of quenching of the intrinsic fluorescence of the protein during the alkylation, suggesting a strong sensitivity of the conformation of tryptophanyl-tRNA synthetase to pH in the range of 7 to 9.


5-Oxo-L-prolinase (L-pyroglutamate hydrolase). Purification and catalytic properties.

5-Oxo-L-prolinase, an enzyme that catalyzes the conversion of 5-oxo-L-proline (L-pyroglutamate; L-2-pyrrolidone-5-carboxylate) to L-glutamate coupled with the cleavage of ATP to ADP and Pi, has been purified about 1600-fold from rat kidney. Purification was carried out in the presence of 5-oxo-L-proline which protects the enzyme under a variety of conditions. An estimate of the molecular weight (about 325,000) was made by gel filtration on Sephadex G-200. K+ (or NH4+) and Mg2+ were required for activity. GTP, ITP, CTP, and UTP were much less active than ATP; dATP was 43% as active as ATP. ADP inhibited and addition of pyruvate kinase and phosphoenolpyruvate activated the reaction. The enzyme, which is protected during storage by dithiothreitol, is inhibited by p-hydroxymercuribenzoate, N-ethylmaleimide, and iodoacetamide. The apparent Km values for 5-oxo-L-proline and ATP are, respectively, 0.05 and 0.17 mM. The pH profile indicates a broad range of activity from about pH 5.5 to pH 11.2 with apparent maxima at about pH 7 and pH 9.7. The formation of Pi and glutamate was equimolar over a wide pH range. When the enzyme was incubated with ATP, Mg2+, K+, and L-2-imidazolidone-4-carboxylate or L-dihydroorotate, cleavage of ATP to ADP and Pi occurred, but no cleavage of the imino acid substrates was observed; when the enzyme was incubated under these conditions with 2-piperidone-6-carboxylate, 4-oxy-5-oxoproline, and 3-oxy-5-oxoproline, the corresponding dicarboxylic amino acids were formed, but the molar ratio of Pi to amino acid formation was significantly greater than unity.


Isolation and characterization of jack bean beta-galactosidase.

A simple procedure has been devised to isolate beta-galactosidase from jack bean meal. The final preparation gives one major protein banc in disc gel electrophoresis. The substrate specificity of this enzyme toward some natural oligosaccharides, glycoproteins, and sphingoglycolipids has been examined in detail. Among three isomers of N-acetyllactosamine, Galbeta1leads to4GlcNAc; while Galbeta1leads to3GlcNAc was hydrolyzed very slowly. This property can be used to distinguish the galactose linkage in asialo-GM1 (Galbeta1leads to3GalNAcbeta1leads to4Galbeta1leads to4Glcleads toCer) and that in lacto-N-neotetraosylceramide (Galbeta1leads to4GlcNAcbeta1leads to 3Galbeta1leads to4Glcleads toCer). For hydrolyzing glycolipids, the effect of sodium taurodeoxycholate and sodium taurochenodeoxycholate on the rate of hydrolysis was carefully examined. This enzyme hydrolyzes lactosylceramide and asialo-GM1 faster than GM1. These results suggest that in addition to the type and linkage of the penultimate sugar unit, the sugar unit at the distal position of the saccharide chain also affects the hydrolysis rate. It also readily liberates 80% D-galactosyl units from asialo alpha1-acid glycoprotein. Escherichia coli beta-galactosidase on the other hand cannot hydrolyze asialo-alpha1-acid glycoprotein, lactosylceramide, GM1, asialo-GM1, and lacto-N-neotetraosylceramide. The molecular weight of this enzyme is about 75,000 and the isoelectric point is pH 8.0. With p-nitrophenyl beta-D-galactopyranoside as substrate, optimal activity occurs at pH 2.8 with glycine-HCl buffer and at pH 3.5 with citrate-phosphate buffer. With lactose as substrate, the pH optimum in these two buffers are 2.8 and 4.0, respectively. Km values for p-nitrophenyl beta-D-galactopyranoside, o-nitrophenyl beta-D-galactopyranoside and lactose are 0.51 mM, 0.63 mM, and 12.23 mM, respectively. Many inhibitors for this enzyme including inorganic ions, monosaccharides, and glycosides are investigated. In contrast to E. coli beta-galactosidase, jack bean beta-galactosidase is not inhibited by p-aminophenyl thio-beta-D-galactopyranoside.


Liver cytosol corticosteroid binder IB, a new binding protein.

A new binding protein named corticosteroid Binder IB elutes just after ligandin in DEAE-Sephadex chromatograms. It has been partially purified to about 2500-fold over cytosol proteins. Calculation of the number of steroid binding sites, assuming one site per molecule of Binder IB fraction after DEAE-Sephadex chromatography, suggests a concentration of the binding protein of about 0.0004% of cytosol proteins. Its pI value is judged to be 7.5 to 8 from it elution position on DEAE-Sephadex chromatograms. IB has an apparent molecular weight of 30,500 +/- 10% by gel filtration and a Stokes radius of 20 A. Binder IB binds radioactive dexamethasone, cortisol, and corticosterone in vitro with estimated KD values of 1, 13, and 25 nM, respectively. Saturation curves are abnormal, showing two phases. The saturation curves within the physiological range of concentrations of steroid are abnormal and suggestive of cooperativity. The second phase, at concentrations of glucocortidoids above saturation and physiological levels, shows extensive binding. After fractionation from other steroid binding proteins, the specificity of binding from competition studies in vitro is dexamethasone greater than or equal to cortisol = corticosterone = estradiol-17beta greater than or equal to deoxycorticosterone = dihydrotestosterone greater than aldosterone = cortexolone greater than testosterone. Other steroids tested are less efficient ligands. The binding is probably noncovalent, but strong; and the complex becomes more dissociable as purification proceeds, suggesting a conformational change in the protein. Storage and rebinding with steroid are possible throughout the purification process, although extensive ligand dissociation and denaturation of the protein occur after the final purification step. Binding in vitro is temperature-sensitive and binding is sharply pH dependent with an optimum at 7.5. The ligand is the unmetabolized steroid as judged by extraction of steroid-IB complex with methylene chloride  and subsequent thin layer chromatography. The physiological function of this protein is unknown at present and purification fo the major corticosteroid hormone receptor to homogeneity may be required before the function of Binder IB is fully understood.


Pyridoxal 5'-phosphate, a fluorescent probe in the active site of aspartate transcarbamylase.

Pyridoxal-P reacts specifically with a single lysine residue at the active site of Escherichia coli aspartate transcarbamylase (Greenwell, P., Jewett, S. L., and Stark, G. R. (1973) J. Biol. Chem. 248, 5994-6001). Reduction of the Schiff base with sodium borohydride, succinylation of the remaining lysine residues, and digestion with trypsin result in formation of a single pyridoxyl peptide, which was purified to homogeneity after chromatography on DEAE-cellulose, treatment with alkaline phosphatase, and rechromatography. Amino acid composition and the results of limited sequential degradation showed that this peptide corresponds to residues 62 to 98 in the sequence of Konigsberg and co-workers, and contains 2 residues of lysine (Henderson, L., Roy, D., Martin, D., and Konigsberg, W., personal communication). By similar isolation, a second peptide was obtained from unsuccinylated catalytic subunit, containing only the pyridoxylated lysine, which corresponds to Lys-80. Derivatives of catalytic subunit containing an average of either one, two, or three pyridoxamine-P moieties per trimer have been prepared by reduction. These species, which retain catalytic activity in proportion to their unmodified active sites, were recombined with regulatory subunit to prepare partially modified derivatives of native aspartate transcarbamylase. At pH 8, fluorescence emission bands were observed at 340 nm, due to aromatic amino acids in the protein, and at 395 nm, due to the pyridoxamine-P moiety. Upon excitation at 280 nm energy transfer from protein to pyridoxamine-P was approximately 15%. The properties of the probe were used to study changes accompanying the binding of substrates and inhibitors. The effects of CTP and ATP were small. With the transition state analog N-(phosphonacetyl)-L-aspartate (PALA) or the substrate carbamyl-P, two types of response were observed. Derivatives of catalytic subunit and native enzyme which contain some unmodified sites and hence retain partial catalytic activity gave large increases in fluorescence at 395 nm. However, fully modified inactive derivatives gave much smaller increases. A derivative of native enzyme containing one triply modified and one unmodified catalytic subunit behaved like the other partially modified species. These results indicate that there is communication among the active sites of different catalytic trimers in modified native enzyme, as well as among active sites within the same modified catalytic trimer. The increases in fluorescence result from a red shift of the absorption maximum of the pyridoxamine-P moiety from 315 to 325 nm, which increases the absorbance at the excitation wavelength for fluorescence. At pH 7, the absorption spectrum is already shifted and, consequently, the binding of PALA and carbamyl-P has little effect on the fluorescence. Therefore, the binding of these compounds at pH 8.0 must cause a structural change in the protein, which in turn causes protonation of a group in the modified active sites, altering the spectral properties.


Hemoglobin Richmond. Subunit dissociation and oxygen equilibrium properties.

In hemoglobin Richmond (beta102 leads to Lys), amino acid substitution has occurred at the same site as the mutation in hemoglobin Kansas (beta102 Asn leads to Thr), a variant with very low oxygen affinity. Although hemoglobin Richmond has been shown to have increased tetramer-dimer dissociation, its oxygen affinity has been inferred to be normal from studies on hemolysates of carriers. We have isolated hemoglobin Richmond and have further studied its properties. We confirm that the oxygen affinity of pure hemoglobin Richmond under conditions similar to those found in vivo is normal. However, the Bohr effect of the variant hemoglobin is markedly abnormal. Its oxygen affinity is low at high pH and high at low pH, relative to hemoglobin A. The tetramer-dimer equilibrium displays a strong pH dependence such that protons promote dissociation. A model is presented in which the structural change in hemoglobin Richmond results in low oxygen affinity, like hemoglobin Kansas. However, the close linkage between tetramer-dimer dissociation and proton concentration seen with hemoglobin Richmond results in normal oxygen affinity at intracellular pH and hemoglobin concentration, and carriers display no hematological abnormalities.


Proton magnetic resonance spectra or porcine muscle adenylate kinase and substrate complexes.

Porcine muscle adenylate kinase with a molecular weight of 22,000 has 2 histidine, 5 phenylalanine, 7 tyrosine, and no tryptophan residues. The effect of pH, substrate, and the paramagnetic manganous ion on the proton magnetic resonance spectrum of the enzyme, particularly the aromatic region, has been investigated at 220 MHz. The well resolved C2 proton peaks of the 2 histidine residues have been individually assigned to His-36 and His-189 by comparison with the spectrum of the carp muscle enzyme which has only one C2 proton peak and only 1 histidine residue, 36. The chemical shift of the peak designated C2-H of His-36 in the porcine enzyme has a normal titration curve with a pKalpha = 6.3 but the peak for His-189 is not titratable in the pH range 5.8 to 8.1. The pKalpha of the single His-36 of the carp enzyme is similar to that of His-36 of the porcine enzyme. Changes in pH, particularly at low pH, also affect the chemical shifts of the tyrosine residues. Occupation of either the monophosphate site by AMP or the triphosphate site by ATP or GTP causes a downfield shift of the C2-H of His-36, and the equilibrium mixture causes an even greater shift, but no shift in the C2-H of His-189. The substrates also induce changes in the chemical shifts in the phenylalanine-tyrosine region of the spectrum. Tentative assignments of the highest and lowest field peaks in this region have been made based on the three-dimensional structure determined by x-ray crystallography. On the basis of these assignments, it is concluded that Phe-183 is unperturbed by substrate binding but that Tyr-153 or -154 at the hinge of the molecule, are perturbed. The C2-H of adenine and C8-H of adenine or guanine of the bound substrates were also observed; those of AMP are unperturbed but C2-H of ATO is shifted downfield and the C8-H of ATP and GTP are shifted upfield. The paramagnetic manganous ion had no effect on the spectrum at Mn(II) to enzyme ratios below 1:10; above this ratio, a general broadening was observed...


A gas-liquid chromatographic method for the determination of naltrexone and beta-naltrexol in human urine.

A rapid quantitative method was developed to assay in urine naltrexone and its major urinary metabolite, beta-naltrexol. Following solvent extraction and elimination of interfering materials, the weakly basic drug, its metabolite and the internal standard (etorphine) were silylated and analyzed by gas-liquid chromatography. As little as 0.02 mug/ml of naltrexone and beta-naltrexol was detectable using a hydrogen flame ionization detector. Twenty-four-hour urine samples were analyzed from three subjects taking 180 mg naltrexone daily. The major urinary excretion product was beta-naltrexol which accounted for 48.6% of the administered dose. Only 5% of the administered dose excreted in the urine was naltrexone. Beta-naltrexol was excreted 70% as free drug and 30% conjugated while naltrexone was 90% conjugated.


High-pressure liquid chromatography of drugs. An evaluation of an octadecylsilane stationary phase.

The performance of a commonly used high-pressure liquid chromatographic stationary phase, octadecylsilane, has been evaluated for 30 compounds selected as representative of a wide variety of drug substances. Chromatographic behavior is found to be highly predictable on the basis of pKa and partition coefficient, and the stationary phase should be especially valuable for the separation of acidic and neutral drugs. For basic drugs, however, the column efficiency is poor, detracting from the overall usefulness.


Determination of nanogram amounts of primary aromatic amines and nitro compounds in blood and plasma.

A rapid and highly sensitive method, based on the direct fluorimetric scanning of thin-layer chromatograms, is described for the quantitative determination of flunitrazepam and its main metabolites in human blood or plasma. This method is generally applicable to aromatic nitro compounds that are reducible with tin(II) chloride. In particular, it is possible to determine primary amines in nanogram amounts. Fluorescamine is used as a regent to produce fluorescent derivatives. The method is suitable for pharmacokinetic studies of flunitrazepam and its main metabolites in human blood and plasma.


Suppression by thyrotropin-releasing hormone (TRH) of human growth hormone release induced by L-dopa.

Oral administration of L-dopa (600 mg) significantly raised plasma human growth hormone (hGH) in 6 of 7 normal subjects examined. This L-dopa induced hGH release was significantly suppressed by the intravenous infusion of 1 mg of thyrotropin-releasing hormone (TRH). TRH administration alone had no significant effect on plasma hGH. In contrast, plasma human prolactin (hPRL) consistently increased following TRH infusion. L-dopa significantly lowered basal plasma hPRL levels and also significantly blunted TRH-induced hPRL release. These results suggest that TRH plays an inhibitory role in the regulation of hGH secretion in normal subjects, whereas it stimulates hPRL release.


Studies of the human testis. VII. Conversion of pregnenolone to progesterone.

Delta5-3beta-Hydroxysteroid dehydrogenase (EC.1.1.1.145) and steroid delta-isomerase (EC.5.3.3.1) were extracted from frozen human testicular tissue and co-precipitated by addition of ammonium sulfate. The activities of both enzymes were localized in the 0-40% (NH4)2SO4 fraction. The enzyme preparation catalyzed conversion of pregnenolone, 17alpha-hydroxypregnenolone, dehydroepiandrosterone, and androstenediol to the corresponding delta4-3-oxosteroid. Since isomerization appeared not to be the rate-limiting step of the overall reaction, measurement of activity of delta5-3beta-hydroxysteroid dehydrogenase was related to the amount of delta4-3-oxosteroid produced from the corresponding delta5-3beta-hydroxysteroid. Delta5-3beta-Hydroxysteroid dehydrogenase required NAD for maximal activity. The Michaelis constants (Km) for NAD were 50 muM, 33 muM and 14 muM, respectively for the dehydrogenation of pregnenolone, 17alpha-hydroxypregnenolone, androstenediol and dehydroepiandrosterone. Km values for each substrate were: pregnenolone 10 muM, 17alpha-hydroxypregnenolone and dehydroepiandrosterone 2.5 muM and androstenediol 3.0 muM. Human testicular delta5-3beta-hydroxysteroid dehydrogenase was inhibited by most of the steroids procued by the testis. The following steroids acted as competitive inhibitors with pregnenolone: 17alpha-hydroxypregenolone (Ki = 1.3 muM), androstenediol (Ki = 2.4 muM), dehydroepiandrosterone (Ki = 0.74 muM), 20alpha-dihydroprogesterone (Ki = 1.1 muM) estrone (Ki = 0.33 muM) and estradiol-17beta (Ki = 0.87 muM). 17alpha-Hydroxyprogesterone, testosterone and androstenedione showed mixed-type inhibition of the enzyme for pregnenolone. Progesterone and NADH were noncompetitive inhibitors of the enzyme for pregnenolone. Ki values, with respect to prenenolone, were 7.4 muM for progesterone and 150 muM for NADH. NADH, however, acted competitively with NAD and Ki value was 30 muM.


Antileukotactic properties of tumor cells.

A chemotactic factor inactivator (CFI) has been found in extracts of Walker and Novikoff tumor cells maintained in rats. The CFI directly inactivates the bacterial chemotactic factor as well as the leukotactic activity (fro both neutrophils and monocytes) associated with C3 and C5 fragments and with culture fluids of lectin-stimulated lymphoid cells. The inactivation of the bacterial chemotactic factor is temperature and pH dependent. Subcellular fractionation procedures indicate that CFI is largely associated with the microsomal and cytosol fractions of tumor cells. CFI activity is also found in rat neutrophils, alveolar macrophages, and in extracts of liver, spleen, and kidney from normal animals. CFI derived from normal tissues also directly inactivates the bacterial chemotactic factor and has the ability to inactivate chemotactic activity associated with C3 and C5 fragments. A feature of the tumor-associated CFI is its presence in ascitic fluids of animals bearing tumor cells and the relative absence of any CFI activity in acute inflammatory exudates. The finding of the tumor-associated CFI may explain, at least in part, the tendency of malignant tumor cells to suppress cellular inflammatory reactions.


Selective beta-1 receptor blockade with oral practolol in man. A dose-related phenomenon.

The purpose of this study was to test the hypothesis that oral administration of a low dose of practolol in man produces selective beta-1 receptor blockade, whereas oral administration of a high dose blocks both beta-1 and beta-2 receptors. Normal men were studied 2-4 h after a single oral dose of practolol (1.5 or 12 mg/kg) and after placebo. Effects on beta-1 receptors were studied by measuring heart rate responses to exercise. Effects on beta-2 receptors were tested by measuring forearm vascular responses to brachial arterial infusions of isoproterenol. Neither dose of practolol altered base-line heart rate, forearm vascular resistance, and arterial pressure, Both low and high doses significantly attenuated heart rate responses to exercise. Forearm vasodilator responses to isoproterenol were attenuated by the high dose, but not the low dose, of practolol. Serum concentrations of practolol 2 h after administration of the drug and at the time of the studies of forearm vascular responses averaged 0.5+/-0.1 (SE) and 5.9+/-1.0 mug/ml for low and high doses of practolol, respectively. The results indicate that the phenomenon of selective beta-1 receptor blockade in man is related to the dose and serum concentration of practolol selectively block beta-1 receptors; a high dose and serum concentrations block both beta-1 and beta-2 receptors.


Sickling times of individual erythrocytes at zero Po2.

A rapid-reaction parallel-plate flow channel was used to study the kinetics of erythrocyte sickling upon sudden deoxygenation with sodium dithionite. The erythrocytes were recorded on 16-mm film or video tape and visually tracked in time. Sickling was identified by morphologic criteria. At the flow rate used in these studies, the rate of sickling was a reaction-limited process. There was no loss of cellular deformability or membrane flicker before the onset of sickling. Typical sickling times for sickle (SS) cells and trait (AS) cells at room temperature in isotonic buffer were 2.0 and 70 s, respectively. Increasing the buffer osmolality resulted in shorter sickling times and under hypotonic conditions the time required for sickling was prolonged. Between pH 6.4 and 7.0 there was little change in the time required for 50% of the originally discoidal cells to sickle (t50); whereas a marked increase in t50 occurred between pH 7.4 and 7.6. Whole populations of AS and SS erythrocytes were separated into three fractions after centrifugation. The t50 of the fractions progressively decreased from top to bottom, which paralleled an increase in mean corpuscular hemoglobin concentration (MCHC). The t50 decreased as the temperature was increased from 13 degrees to 34 degrees C. This temperature effect was more pronounced for cells that had osmotically induced reductions in MCHC. A two-step process for erythrocyte sickling is proposed: an initial lag phase, during which there is little or no change in internal viscosity, followed by a rapid phase of cellular deformation. The lag phase is altered by changes in MCHC, pH, and temperature.


Pyridine nucleotide-dependent superoxide production by a cell-free system from human granulocytes.

Using an assay that measured superoxide dismutase-inhibitable nitro blue tetrazolium reduction, we studied superoxide (O2-) production by a cell-free system from human granulocytes. At 40 muM NADPH and a protein concentration of 0.12 mg/ml, lysates prepared from human granulocytes formed O2- at a rate of 18. 4 +/- 4.6 SE nmol/ml reaction mixture per donor, but not with glucose-6-phosphate, 6-phosphogluconate, glyceraldehyde-3-phosphate, sodium lactate, glutathione, or ascorbic acid. The Km's for NADPH and NADH were 8.6 +/- 4.6 muM and 0.83 +/- 0.30 mM, respectively, suggesting that NADPH is the physiological electron donor in this system. O2- production was not inhibited by 1mM KCN. The rate of O2- production by the cell-free system was comparable to the rate of O2-production by an equivalent quantity of intact granulocytes incubated under similar conditions. O2- production by lysates from granulocytes preincubated with serum under conditions previously shown to stimulate O2- production in the intact cells was no different than its production by lysates from unstimulated cells. O2- production at 0.2 mM and 0.02 mM NADPH by lysates from the granulocytes of two patients with chronic granulomatous disease was similar to O2- production by control lysates. This finding was interpreted in terms of the possibility that the metabolic lesion in chronic granulomatous disease may lie outside the oxygen-metabolizing enzyme system of the granulocyte, or alternatively, that the granulocytes may contain two O2- - forming enzymes, one of which is inactive in chronic granulomatous disease.


Corticosteroids increase superoxide anion production by rat liver microsomes.

Superoxide anion production by liver microsomes from intact, adrenalectomized, and cortisoltreated adrenalectomized rats has been determined. The amount formed was roughly proportionate to the amount of cortisol given, and a similar response was seen in the activity of NADPH-cytochrome c reductase. The amount of measurable superoxide anion was markedly reduced by the addition of superoxide dismutase. The increased production of this potent free radical with cortisol therapy suggests that its formation may contribute to some of the harmful effects of corticosteroids given in more than physiologic amounts.


Pneumococcus-induced serotonin release from human platelets. Identification of the participating plasma/serum factor as immunoglobulin.

Pneumococcus-induced serotonin release from human platelets is greatly facilitated by a factor present in normal human plasma and serum. We have identified this factor as immunoglobulin by: (a) removing if from plasma and serum with solid phase antiFab antibody; (b) demonstrating its absence from the serum of an individual with severe immunoglobulin deficiency; and, (c) showing its presence in IgG preparations isolated from normal individuals. Evidence suggesting that the release reaction is triggered by pneumococcal antigen-antibody complexes rather than by nonimmune interaction of immunoglobulin with pneumococcus includes: (a) the failure of isolated IgG myeloma proteins to support release; (b) a lack of correlation between IgG concentration and "releasing factor activity" in normal human sera; (c) the identification of a normal serum that supports release by types II and III pneumococci but not type VII; and, (d) the ability of most normal sera to support release by pneumococca serotypes II and VII, though these types have not shown nonimmune reactivity with the Fc portion of the IgG molecule. The ability of antibodies present in normal serum to support pneumococcus-induced serotonin release suggests that the thrombocytopenia seen in pneumococcal infection may at least in part be caused by pneumococcal antigen-antibody complexes.


Thermodynamic solubility product of human tooth enamel: powdered sample.

Solubility of human dental enamel in H3PO4 was studied in the pH range of 4.5 to 7.6. Thermodynamic solubility of the enamel mineral was calculated in terms of the ion activity product, (Ca2+)5(PO43-)3(OH-), for hydroxyapatite. The solubility product varied from 7.2 times 10(-53) to 6.4 times 10(-58) mol9 liter-9 depending on the cumulative amount of the dissolution of the solid in a series of repetitive sequences of solubility experiments.


Effect of alpha, alpha'-dipyridyl on the basement membrane of tooth germs in vitro.

Dipyridyl inhibits the morphogenesis of tooth germs, but the germs recover when transferred to control medium. In this study, the effect of dipyridyl on basement membrane was investigated in vitro. The basal lamina was always present, but the subjacent collagen fibrils disappeared in the presence of dipyridyl and reappeared during recovery in control medium.


Some properties of uridine 5'-diphosphogalactose: N-acetylglucosamine galactosyltransferase in human parotid saliva.

Uridine 5'-diphospho (UDP)-galactose: N-acetylglucosamine galactosyltransferase was separated from human parotid saliva and partially purified. The optimum pH of the enzyme was 7.5. The enzyme required a specific acceptor, and its activity was activated by manganese and magnesium ions.


Purification and biochemical characterization of rat skin cathepsin D.

The hemoglobin-hydrolyzing, acidic proteinase activity of rat skin was purified by using ammonium sulfate precipitation. Sephadex G-100 gel column chromatography, acid treatment, and DEAE-cellulose column chromatography, giving a purification coefficient of 182. The pH optimum, molecular size, substrate specificity, as well as inhibitor and activator sensitivity of the enzyme preparation, corresponded closely to those of cathepsin D. The enzyme activity was separated from cathepsin B1. The present status of the knowledge of skin cathespins is reviewed.


Enterotoxigenic intestinal bacteria in tropical sprue. III. Preliminary characterization of Klebsiella pneumoniae enterotoxin.

Cell-free broth filtrates of a strain of Klebsiella pneumoniae serotype 5 retained their capacity to induce fluid secretion in the rabbit ileal loop model after heating (100 C for 30 min), acid treatment (pH 4.4), and dialysis through viscose tubing. Sequential passage of an acetone precipitate of the broth filtrate through Diaflo membranes yielded an active fraction in the UM 2, but not the UM 10, retentate; this observation indicates that the molecular weight of the enterotoxin is in the range of 1,000-10,000. Klebsiella pneumoniae enterotoxin thus resembles the heat-stable enterotoxin of Escherichia coli in a number of respects.


The effects of Staphylococcus aureus and Klebsiella pneumoniae peritonitis in mice exposed to normal and hypoxic conditions on red cell oxygen transport function.

Normal mice exposed to 10% oxygen concentration developed a slight but statistically significant decrease in blood pH and a slight statistically insignificant decrease in red cell 2,3-DPG. Mice that were infected intraperitoneally with Staphylococcus aureus or Klebsiella pneumoniae and exposed to 20% oxygen developed acidosis, hemoconcentration, and decreased red cell 2,3-DPG levels. When mice with acute bacterial peritonitis were exposed to 10% oxygen concentration they likewise developed significant acidosis and hemoconcentration, but their reduction in red cell 2,3-DPG was not as great as that in the similarly infected mice exposed to 20% oxygen concentration.


Renin and arterial pressure in perfused dog kidneys.

20 isolated dog kidneys were perfused in vitro at 37 degrees C during 6h in a closed circuit system with a Roller pump maintaining a pulsatile flow with a constant output. The perfusate electrolyte concentrations were kept constant. In 12 experiments where a hemodiluted standardized perfusion fluid was used, a significant (0.01 greater than p greater than 0.001) correlation (r = 0.798) was found in the beginning of the experiments between the mean arterial pressure expressed in mm Hg (y) and the renin level, expressed in log U/1 (x): y = 57.0+76.8x. In 4 other experiments, phenoxybenzamine added to the perfusion fluid decreased the arterial pressure but did not prevent the rise of the renin level. In 4 other experiments where the perfusion fluid did not contain blood, renin level and arterial pressure increased, whereas in the experiments using a circuit without a kidney, the renin level did not rise. The possible role of the renin-angiotensin system in causing increased renal vascular resistance during in vitro kidney perfusions is discussed.


Use of L-rhamnose to study irreversible adsorption of bacteriophage PL-1 to a strain of Lactobacillus casei.

L-rhamnose has been found to be useful to investigate the process of irreversible adsorption of PL-I phage to its host bacterium, Lactobacillus casei ATCC27092. L-rhamnose inhibited phage adsorption to cells without inactivating free phages. Adsorption inhibition was correlated with the concentrations of L-rhamnose. The inhibitory effect of L-rhamnose on phage adsorption was of a competitive nature against host cells. Among other saccharides tested, L-fucose, L-mannose and D-ribose showed a slight degree of adsorption-inhibiting activity. In early stages of phage adsorption in a tris-maleate buffer, where the binding of phages to cells was still reversible, addition of L-rhamnose resulted in the partial desorption of phages from the cells to which they had adsorbed. However, the number of infective phages desorbed by L-rhamnose treatment gradually fell off as incubation continued, showing that the phages became firmly bound to the cells. Therefore, it is possible to determine the number of phages irreversibly adsorbed to cells by using this desorption technique with L-rhamnose. The process of irreversible phage adsorption, that is, the formation of phage-cell complexes from which no more infective phages could be desorbed, was dependent on temperature and strongly inhibited at 0 degrees C.


Manipulation of neurotransmitters by acupuncture (?) (A preliminary communication).

Varying reactions of the vegetative nerve system to various point combinations (for example: vomiting, dizziness, diarrhea, urge to urinate, fatigue or drowsiness, headache), especially to the needling of Tai Chong (Li 3), induced us to perform biochemical studies before and after acupuncture treatment. A group of children and a group of adults were studied. The material studied was urine and blood; from the children, urine only. The following were determined in the urine: indolacetic acid, 5-hydroxy-indol-3-acetic acid, homovanillic acid, and vanillic-mandelic acid; in the blood, tyrosine and tryptophan (free and bound). Individual points with wide influence (He Gu = LI 4; Zu San Li = St 36; Tai Chong = Li 3) and their combination with generally effective points were tested. The needling of Tai Chong especially showed a clear increase in indolamine metabolism. Isolated increases in metabolites of catecholamine metabolism could be correlated with the patient's increased physical activity after acupuncture. Noteworthy is the observation that no significant chemical reactions were evident if local reactions to the needling no longer appeared at the end of a series of acupuncture treatments.


Sites of organic acid production and pattern of digesta movement in the gastrointestinal tract of geese.

Sixteen geese were used to assess the movement of fluid and particulate digesta through their gastrointestinal tracts and to determine the diurnal variation in organic acid levels for the various segments of the tract. Fluid (polyethylene glycol and chronium-labeled ethylenediaminetetraacetic acid) and particulate markers (2 and 5 mm long) were administered with the meal. Animals were killed at given intervals after the administration of markers. The gastrointestinal tract was divided into nine segments for measurement of markers, pH, volatile fatty acids (VFA), and lactic acid contents. The data indicated a rapid evacuation of fluid marker from the foregut (crop, ventriculus, and proventriculus), while particulate markers were retained for extended periods of time. Retention of fluid marker was observed only within the cecum. Retrograde movement of particulate marker was demonstrated from the duodenum to ventriculus and proventriculus. Retrograde movement of fluid marker was observed from the cloaca to the colon, cecum, and distal third of the small intestine. However, particulate marker showed no retrograde movement in these segments of tract. Highest VFA levels were observed in the cecum. Retention of digesta and production of VFA within the colon were less than those noted for the dog, pig and pony. Lactic acid comprised less than 10% of the organic acids present in the gastrointestinal tract and were at their highest levels in the proximal and mid small intestine.


Effects of undernutrition and protein malnutrition on brain chemistry of rats.

The present study was undertaken to assess the effects of different degrees of nutritional restrictions during fetal life, suckling, and after weaning on the chemical composition of the brain. At 42 days of age, the rats were killed, and the brains were processed for analyses. The rats exposed to severe protein malnutrition after weaning had lower brain weights than those on controls. The brain seems to be resistant to the effects of moderate protein malnutrition imposed during suckling or after weaning. Thus, the brain is either resistant to the effects of mild nutritional deficiency imposed during suckling, or brain composition is very responsive to nutritional rehabilitation initiated after weaning. The effects of severe undernutrition during suckling were not, however, reversed when adequate nutrition was initiated after weaning. The suckling period seems to be critical during development, as the process of myelination was lowered and the levels of electrolytes were irreversibly disturbed. The brains of the rats born to the mothers protein malnourished during gestation were not significantly different from those of controls. The brain seems to be either preferentially protected from the effects of malnutrition imposed during fetal development, or the brain component are very responsive to nutritional rehabilitation initiated immediately after birth. It is suggested that the mother's nutritional status during gestation does not significantly affect the development of the brain. When the young were born to and nursed by protein-malnourished mothers, the growth and the maturation of the brain in such animals were similar to those in rats moderately undernourished during suckling. Growth and maturation of the brain are affected by a lowered level of protein in the diet. Moderate undernutrition imposed during suckling is not important, but the effect is maximum when undernutrition is severe during this period. The suckling period is therefore, comparatively more critical during development.


Effects of early undernutrition and subsequent refeeding on alkaline ribonuclease activity of rat cerebrum and liver.

The effect of undernutrition and subsequent rehabilitation on free and total alkaline ribonuclease activity (RNase) of cerebrum and liver was studied in 14- and 21-day-old rats. Free RNase activity was higher in both organs at 14 and 21 days of age. However, at day 21 the difference was statistically significant only in liver. Total RNase activity was not changed by undernutrition in cerebrum and was reduced in liver. Nutritional rehabilitation returned both free and total RNase activities to control levels in liver but had no apparent effect on cerebrum. The elevation of free RNase seems to be secondary to a reduced concentration of inhibitor protein and not to de novo synthesis of RNase.


Demonstration of a specific metabolic effect of dietary disaccharides in the rat.

Male Wistar rats were starved and refed diets containing either 40% carbohydrate as monosaccharides (glucose, fructose, invert sugar) or disaccharides (maltose, sucrose), or 42.2% carbohydrate as glucose. Induction of various liver enzymes and changes in total liver lipid levels by the different dietary sugars were studied. Liver enzymes measured included glucose-6-phosphate dehydrogenase (g6pd), 6-phosphogluconate dehydrogenase (6PGD), malic enzyme (ME), phosphofructokinase (PFK), L-alpha-glycerol phosphate dehydrogenase (LalphaGPD), pyruvate kinase (PK), citrate cleavage enzyme (CCE), acetyl CoA carboxylase (AcCoAC), and fatty acid synthetase (FAS). The responses in enzyme activity to diets containing glucose or invert sugar were used as the basal response. Enzyme responses to refeeding the carbohydrate diets fell into three categories: (1) enzyme activity increased both by the disaccharide configuration of the carbohydrate and by fructose (G6PD, PK, CCE, AcCoAC, FAS); (2) enzyme activity increased only by the disaccharide configuration of the carbohydrate (6PGD, ME); and (3) enzyme activity increased only by fructose (PFK, LalphaGPD). Total liver lipid level was increased both by the disaccharide configuration of the carbohydrate and by fructose. Refeeding diets containing equal molar amounts of glucose or maltose did not abolish the disaccharide effect. The data indicate that the disaccharide configuration of maltose and sucrose may have an effect at the gastrointestinal level, which causes an increased induction of certain enzymes in the liver.


Early versus delayed initiation of continuous negative pressure in infants with hyaline membrane disease.

Twenty-three infants with HMD of similar severity, who were less than 24 hours of age and who were breathing spontaneously, were divided by random numbers into early and delayed CNP groups. The infants who were treated with CNP before their PaO2 was less than 50 mm Hg while breathing 70% oxygen experienced a significantly greater increase in PaO2 in response to the initiation of CNP, required less time with O2 therapy, required no mechanical ventilation, and had fewer complications. Based on these results, it is suggested that CNP be initiated in infants with HMD, who are less than 24 hours of age and are breathing spontaneously, before the PaO2 becomes less than 50 mm Hg on 70% O2.


Enhancement of the antitumor effect of 1,3-bis(2-chloroethyl)-1-nitrosourea by various psychotropic drugs in combination with caffeine.

Certain psychotropic drugs when combined with caffeine significantly enhanced the antitumor effect of 1,3-bis(2-chloroethyl)-1-nitrosourea in murine leukemia L1210. Enhancement required that all three drugs be given together and optimal results were obtained when the psychotropic drug was given 6 hours before 1,3-bis(2-chloroethyl)-1-nitrosourea and caffeine. Thus, 1,3-bis(2-chloroethyl)-1-nitrosourea alone or with caffeine resulted in 5% cures. Addition of chlorpromazine increased the cure rate to 51% while prochlorperazine gave 30% cures. Chlordiazepoxide produced 39% cures while the dibenzazepine compounds studied were ineffective. For the phenothiazine, benzodiazepine and dibenzazepine compounds studied, tentative conclusions could be drawn on the relationship of chemical structure to enhancing activity. For phenothiazines, the substituent in the R2 position of the phenothiazine ring determined activity to a greater extent than did the substituent in the R1 position. For the benzodiazepine compounds, chlordiazepoxide was superior to diazepam. Although the mechanism of action of psychotropic drugs in this system is unknown, these preliminary results suggest the possibility of a change transfer reaction between the free radical form of the psychotropic drug and one or more intracellular constituents.


Site of action and active form of procaine in squid giant axons.

The active form of procaine and its, site of action on the nerve membrane have been studied in intact and internally perfused squid giant axons. Voltage clamp techniques were employed to measure the masimum values for peak sodium conductance and for steady-state potassium conductance as an index of activity. Changes in internal pH between 7 and 8 do not influence the blocking potency of procaine applied internally. This result, when compared to theoretical curves, is only compatible with the notion that the charged form of procaine present inside is the active form. If one momentarily arrests internal perfusion during an experiment in which procaine is being applied externally, the conductance block is significantly potentiated. When procaine is applied simultaneously to both external and internal phases, most of the block can be reversed by washing out the inside at a time when the outside is still being perfused with procaine. If one reverses this procedure, very little recovery is noted with removal of procaine from the external phase. The rate of recovery from the procaine blockage is much faster in internally perfused axons. These three observations support the notion that procaine acts from inside the nerve membranes. It is concluded that procaine, as other lidocaine derivatives studied previously, acts from the internal nerve membrane surface in the charged form.


On the use of functional antagonism to estimate dissociation constants for beta adrenergic receptor agonists in isolated guinea-pig trachea.

In guinea-pig trachea, the maximum degree of relaxation that can be elicited by beta adrenergic receptor agonists depends upon the degree of contraction of the smooth muscle induced by cholinergic agonists. In these studies, it is shown that increasing concentrations of carbamylcholine (carbachol) result in a shift to the right of the dose-response curves to (-)-isoproterenol and (-)-soterenol and a reduction of the maximum degree of relaxation produced by these agonists relative to that produced by papaverine. Soterenol is demonstrated to be a partial agonist relative to isoproterenol since its maximum response is reduced to a greater extent by carbachol and it displaces the carbachol dose-response curves to the right less than does isoproterenol. The data were used to calculate, by three different theoretical models of drug-receptor interactions, a dissociation constant (KA) for soterenol. All values obtained were within 2-fold differences. The range of KA values for soterenol was from 5.4 to 9.6 times 10(-8) M. These values are about 100 times larger than the ED50 value for soterenol obtained in the absence of carbachol. The KA value estimated for (-)-isoproterenol (about 3 times 10(-8) M) by one of the models is also around 100 times larger than its ED50 value. This demonstrates further that ED50 values are unreliable indicators of the affinities of agonists for receptors.


Distribution of chlorpromazine in the gastrointestinal tract of the rat and its effect on absorptive function.

The distribution of [14C]chlorpromazine was studied in segments of the gastrointestinal tract of the rat after an oral dose of 20 mg/kg (1-2 muc). The total radioactivity in the gastrointestinal tract was, respectively, 43.2 +/- 3.6, 28.9 +/- 2.2 and 14.1 +/- 1.1 (mean +/- S.E. percent administered dose) at 2, 6 and 24 hours after dosing. The maximum observed total radioactivity in the different segments occurred at different times after dosing. The percentage of total radioactivity present in each tissue as unchanged [14C]chlorpromazine decreased with time in all the tissues. At all time points, this parameter also showed a consistent pattern: % stomach greater than % duodenum greater than % jejunum greater than % ileum. The effect of chronic administration of [14C]chlorpromazine on intestinal absorptive function was studied by the everted sac technique. Mucosal transport of L-[14C]methionine was significantly inhibited (24.4% of control), but no effect on transport of D-[14C]xylose was observed.


Estimation of testosterone and androstenedione in the plasma and testes of cryptorchid offspring of mice treated with oestradiol during pregnancy.

The injection of pregnant mice with 5 mg oestradiol dipropionate induced a high proportion of bilateral cryptorchidism in the male offspring. The ectopic testes were reduced in size and occupied one of three different positions in the abdomen: pararenal, paravesicular or inguinal. The seminal vesicles were found to be similarly reduced in size whereas the adrenals and pituitaries appeared to be unaffected. The total content of testosterone was reduced in the pararenally placed gonads but was not significantly different from that of the controls when the testes were in the inguinal region. The content of androstenedione was not significantly altered. The concentration of both hormones, however, was increased in all cases. Plasma testosterone levels were lower in all the offspring of treated mothers, especially in those with high ectopia.


Potential histamine H2-receptor antagonists. 2. N-alpha-Guanylhistamine.

The agonist molecule, histamine, has been used as a starting point for the design of potential H2-receptor antagonists. Converting the side-chain amino group into a guanidine yielded the first histamine H2-receptor antagonist, Nalpha-guanylhistamine. Antagonism of H2 receptors was demonstrated by the inhibition of histamine-stimulated gastric acid secretion in the rat at high dose levels (approximate ID50 800 MUmol/kg, iv) and by the inhibition of histamine-stimulated tachycardia of guinea-pig right atrium (pA2 equals 3.9). Guanylhistamine behaves as a partial agonist at histamine H2 receptors.


Potential inhibitors of L-asparagine biosynthesis. 3. Aromatic sulfonyl fluoride analogs of L-asparagine and L-glutamine.

The N-[p-(fluorosulfonyl)benzyl] derivatives of L-asparagine and L-glutamine (1a,b) were synthesized as potential inhibitors of L-asparagine synthetase (ASase). Condensation of p-(fluorosulfonyl)benzylamine (2) with the suitably protected amino acid in the presence of dicyclohexylcarbodiimide, followed by deblocking, afforded 1a and 1b. Derivatives 1a and 1b at 10 mM inhibit ASase isolated from Novikoff hepatoma (rats) by 60 and 46%, respectively. Preliminary results on inhibition of Jensen sarcoma (L-asparaginase sensitive) and JA-1 sarcoma (L-asparaginase resistant) tissue cultures by 0.3 mM 1a (139,90%) and 1b (101, 103%), respectively, are discussed.


Chemical differentiation of histamine H1- and H2-receptor agonists.

Histamine exists predominantly as the NT-H tautomer of the monocation (IIa) at a physiological pH of 7.4 and structure-activity studies indicate that this tautomer is likely to be the pharmacologically active species for both H1 and H2 receptors. Effective H2-receptor agonists appear to require a prototropic tautomeric system whereas H1-receptor agonists do not need to be tautomeric. This identifies a chemical difference in the receptor requirements which provides the basis for obtaining selective histamine H1-receptor agonists. Thus 2-(2-aminoethyl)thiazole and 2-(2-aminoethyl)pyridine are nontautomeric and are highly selective agonists for histamine H1 receptors (H1:H2 ca. 90:1 and 30:1, respectively). In conjunction with the selective H2-receptor agonist, 4-methylhistamine, they are of great value for studying the pharmacology of histamine receptors.


Calcium ion-dependent phosphorylation of human erythrocyte membranes.

Calcium ions promote the rapid transfer of the terminal phosphate of ATP to a protein of human erythrocyte membranes. The concentration of Ca2+ for half-maximal effect is 7 muM. At nonlimiting ATP concentrations the level of 32P incorporated by the membranes is independent of the presence or absence of Mg2+. The number of phosphorylating sites in a single erythrocyte membrane is about 700. The influence of pH on the rate of hydrolysis of the bound phosphate and its rapid release on exposure to hydroxylamine are both consistent with an acylphosphate bond. The phosphate in the protein undergoes rapid turnover. Enzymatic splitting of the phosphate is stimulated by Mg2+ but not by Ca2+. It is proposed that Mg2+ accelerates the splitting of the phosphate by favoring the conversion of the phosphoprotein from a state of low reactivity to a state of high reactivity towards water. The reactions described probably are intermediate steps in the hydrolysis of ATP catalyzed by the Ca2+-dependent ATPase of human erythrocyte membranes.


Loss of marrow allograft resistance in mice with transplanted methylcholanthrene-induced sarcomas.

To determine if the effector cells responsible for allogeneic marrow stem cell rejections were suppressed in mice with tumors, C57BL/6 (B6) mice were inoculated with 3-methylcholanthrene (MCA)-induced sarcoma cells. When the tumor reached 2.0--2.5 cm in diameter, these mice and control B6 and (BALB/c times A)F1 (CAF1) uninoculated animals were irradiated and given BALB/c marrow cells in the first of a two-step "stem cell rescue" experiment. Four days later, spleen cells of the primary hosts were reinoculated into irradiated CAF1 secondary hosts compatible with BALB/c marrow cells and immunized against B6 antigens. Splenic uptake (percent) of 125I-5-iodo-2'-deoxyuridine 5 days after spleen cell regrafting was used as a measure of cell proliferation and reflected growth of the stem cells in the primary hosts. BALB/c stem cells grew as well in B6 mice with tumors as in CAF1 primary hosts but were rejected by B6 controls. Seeding efficiency of BALB/c stem cells 6 hours after infusion of marrow cells and growth of syngeneic B6 stem cells were enhanced twofold in spleens of tumor-bearing B6 mice. To exclude the possibility that enhanced seeding resulted in greater survival of allogeneic stem cells, more DBA/2 marrow cells were infused into control B6 primary hosts than into tumor-bearing B6 and control DBA/2 mice. Control B6 mice resisted growth of even 7.5 times 10(6) DBA/2 marrow cells, whereas B6 tumor bearers allowed growth of 2.5 times 10(6) cells. No "suppressor cells" capable of inhibiting marrow cell allograft reactions were detected in spleens of tumor-bearing mice. Thus transplanted syngeneic MCA-induced sarcomas abrogated the ability of mice to reject allogeneic marrow stem cells.


Influence of allogeneic effect on 7,12-dimethylbenz(alpha)anthracene carcinogenesis in mice.

Charles River mice either untreated or treated at birth with 7,12-dimethylbenz]alpha[anthracene (DMBA) were given allogeneic spleen cells from adult C57BL/Cas donors. These spleen cells were given as a single injection to mice at birth or at 7 or 14 days of age. Allogeneic treatment of mice at birth significantly increased the incidence of DMBA-induced lymphomas and shortened the latency period of the tumors. The incidence of subcutaneous tumors was moderately increased in DMBA-treated mice given grafts of allogeneic cells at 7 days of age. Lung tumors appeared to be decreased in the group given DMBA at birth and allogeneic cells 14 days later. Treatment with only allogeneic cells gave results essentially similar to those observed in untreated controls.


Utilization of supervised physician's assistants in emergency room coverage in a small rural community hospital.

A study has been made of an initial 2-year experience in a 90-bed rural community hospital where emergency room primary coverage has been provided by Physician's Assistants under supervision of specialists in the field of general surgery, pediatrics, and internal medicine. Emphasis has been placed on a rigidly supervised program as well as physician supervision and availability on a patient-call basis. This supervision may take the form of immediate availability of the physician to the emergency room, telephone consultation or, in minor illnesses and injuries, continued review of the record of each individual patient seen by the Physician's Assistant. A review of the most recent 2-month ER-OPD experience showed that in a small rural hospital, a majority (62%) of the patients had an illness or injury of a minor, nonserious degree which could be handled primarily by a Physician's Assistant. This has led to emergency room care which has been judged by the patients, the emergency-room nurses, and the supervising physicians to be more efficient and prompt than had been previously provided, yet not reduced in quality. The program described was developed by physicians active in private practice working with the Physician's Assistants and RN's in the ER-OPD. The physicians' time spent in teaching, supervision and development of written policy was great, and, at times, threatened the continuation of the program. A fulltime physician in the hospital could better initiate the program of teaching, supervision, formulating written procedures, and establishing policies with different specialist.


Polyacrylamide gel electrophoresis of intact bacteriophage T4D particles.

A method for the electrophoresis of intact bacteriophage T4D particles through polyacrylamide gels has been developed. It was found that phage particles will migrate through dilute polyacrylamide gels (less than 2.1%) in the presence of a low concentration of MgCl2. As few as 5 x 10(9) phage particles can be seen directly as a light-scattering band during the course of electrophoresis. The band can also be detected by scanning gels at 260 to 265 nm or by eluting viable phage particles from gel slices. A new mutant (eph1) has been identified on the basis of its decreased electrophoretic mobility compared with that of the wild type; mutant particles migrated 14% slower than the wild type particles at pH 8.3 and 35% slower at pH 5.0. The isoelectric points of both the wild type and eph1 mutant were found to be between pH 4.0 and 5.0. Particles of T4 with different head lengths were also studied. Petite particles (heads 20% shorter than normal) migrated at the same rate as normal-size particles. Giant particles, heterogenous with respect to head length (two to nine times normal), migrated faster than normal-size particles as a diffuse band. This diffuseness was due to separation within the band of particles having mobilities ranging from 8 to 35% faster than those of normal-size particles. These observations extend the useful range of polyacrylamide gel electrophoresis to include much larger particles than have previously been studied, including most viruses.


Studies of introital colonization in women with recurrent urinary infections. I. The role of vaginal pH.

The pH of the vaginal introitus was compared to quantitative cultures of the introital mucosa (Escherichia coli, Proteus mirabilis and Streptococcus faecalis) in 800 samples. Introital carriage of more than 100 bacteria per ml. was significantly greater at pH of more than 4.4 (p less than 0.005) when compared to mucosal pH of less than or equal to 4.4.


Studies of introital colonization in women with recurrent urinary infections. II. A comparison of growth in normal vaginal fluid of common versus uncommon serogroups of Escherichia coli.

Vaginal fluid collected from volunteers who denied a history of urinary infections was bactericidal to Escherichia coli at pH 4.0. When the pH was increased to 6.5 with sodium hydroxide, the same vaginal fluids supported bacterial growth. If vaginal fluid was adjusted at pH increments between 4.0 and 5.0, and inoculated with common and uncommon serogroups of Escherichia coli, the uncommon O-groups that never cause urinary infections were significantly (p less than 0.005) more inhibited by the lower vaginal pH than were the common O-groups of Escherichia coli.


Studies of introital colonization in women with recurrent urinary infections. III. Vaginal glycogen concentrations.

The concentration of glycogen in vaginal fluid was measured in 37 samples from 6 volunteers without urinary infections and in 96 samples from 12 patients with recurrent urinary infections. The mean glycogen concentrations, 440 mug. per ml. from the controls and 564 mug. per ml. from the patients, were not statistically different (p more than 0.05). Although an introital pH more than or equal to 4.4 was associated with low concentrations of glycogen in vaginal fluid, a pH less than 4.4 was found with high and low glycogen concentration. Sodium concentration was statistically higher in vaginal fluid from patients compared to controls (p less than 0.01) while potassium concentration was the same in both groups.


Tissue invasion by unnamed marine vibrios.

Vibrio parahaemolyticus is an extremely common organism of major importance as a cause of gastroenteritis, but not common as a cause of tissue infection. Of three patients who had serious localized tissue infections, one died because of an unnamed marine Vibrio infection. Vibrios are easily isolated, although their final speciation is quite difficult in view of their unsettled taxonomic position. It is hoped that agreement can be reached as to bacteriologic genealogy; perhaps then the specific pathogenic manifestations will be clarified. The clinician should consider the possibility of this pathogen in any patient with a wound related to saltwater or seafood.


Long-term prognosis of renovascular hypertension: comparative study in operated and non-operated patients.

Long-term prognosis of renovascular hypertension has been studied in 56 patients. Twenty-nine patients underwent surgical treatment and 27 patients were not operated upon for various reason. Initial severity of hypertension in the operated group was nearly comparable to that of the non-operated group. Average of observation periods in each group is 5.8 and 5.9 years respectively, and the longest follow-up period has been up to 12 years. Hypertension cured or improved in 25 cases of the operated group. Antihypertensive therapy was effective in 7 of the medically treated patients. During the entire observation periods, 2 cases of the operated and 10 cases of the non-operated group died. Seven deaths of the non-operated group seemed to be causally related to hypertension. It is concluded that long-term prognosis of operated patients is significantly improved as compared with that of patients whose renovascular hypertension could not be treated surgically. Prevelence of aortitis (Takayasu's disease) as a cause of renovascular hypertension is a particular problem in our series because extensive and complicated renovascular lesions may make surgical intervention difficult in some patients.


[Comparison of the antibacterial activity of amikacin (BB-K8) with other aminoglycosides against pathogens recently isolated from clinical materials (author's transl) ].

We determined the antibacterial activity of amikacin against 1,277 strains of pathogenic bacteria isolated from clinical materials during 1974, including beta hemolytic streptococci, pneumococci, enterococci, Staphylococcus aureus, Staph. epidermidis, Escherichia coli, Klebsiella, Enterobacter, Citrobacter, Serratia, Proteus morganii and Pseudomonas aeruginosa, and compared the minimum inhibitory concentration (MIC) of this drug with gentamicin, dibekacin, tobramycin and kanamycin. 1)Antibacterial activity of amikacin against beta hemolytic streptococci, pneumococci and enterococci was as weak as the other four aminoglycosides, but against Staph. aureus, Staph. epidermidis, various groups of Enterobacteriaceae and Pseudomonas aeruginosa showed amikacin the good antibacterial activity as gentamicin, dibedacin and tobramycin, and also showed the good activity against kanamycin resistant strains. 2) Amikacin has the similar antibacterial spectrum as gentamicin, dibekacin or tobramycin, but its antibacterial activity is generally weakest among these four drugs. 3) On many strains tested the cross resistance is observed between amikacin and one of gentamicin, dibekacin and tobramycin, but several strains of Proteus morganii and Pseudomonas aeruginosa which have rather large MIC against gentamicin, dibekacin or tobramycin showed rather small MIC against amikacin.


[Chemotherapy of purulent meningitis in children (author's transl)].

Purulent meningitis in patients admitted to the pediatric department of Kyoto University Hospital and affiliated institutions from 1951 through 1973 were studied with emphasis on the kinds of the causative organisms and the susceptibility of these organisms to antibiotics. The findings in this study have served to help select antibiotics most likely to be effective against this disease. The overall incidence of purulent meningitis was 0.68%. This figure decreased little throughout the period. As for the frequency of causative organisms, Neisseria meningitidis led the list, and Diplococcus pneumoniae ranked just behind. Haemophilus influenzae was rare. The frequency of N. meningitidis, however, decreased sharply in spite of the essentially unchanged overall incidence of this disease. The probable reason for the poor prognosis of this disease in spite of the remarkable strides in chemotherapy is the decreased frequency of N. meningitidis and the inversely increased organisms that are resistant to usual chemotherapy. The therapeutic effectiveness of cefazolin against this disease was studied in 15 children including eight newborns and four infants. The daily per kg bodyweight dose was 50 mg or less in four, 50 approximately 100 mg in five, and more than 100 mg in the remaining six. The route of administration was either intramuscular or intravenous. No deaths occurred. The rate of effectiveness was as high as 80%. Residual symptoms were recorded in six and, in as many as five of them, the cause was a-tributable to the delayed detection of the disease. Neither side effects nor aberrent laboratory findings attributable to large doses of cefazolin were recorded. Diffusibility of cefazolin into the CSF was studied in nine subjects. The CSF concentration of this antibiotic was shown to be somewhat lower than that of ampicillin or cephaloridine and to account on an average for 13% of the mean peak serum level. This relatively low diffusibility will be offset by its high serum concentration and safe large-dose therapy. These findings have clearly shown that the therapeutic effectiveness of cefazolin is as high as that of ampicillin, and that this excellent effectiveness holds true even when the causative organism happens to be Escherichia coli, Klebsiella, etc. that are resistant to ampicillin. The authors have furthermore scrutinized much literature on the frequency of the causative organisms, emergence of resistant strains, and the diffusibility of antibiotics into the CSF, and arrived at the conclusion that cefazolin is a promising antibiotic of choice for the treatment of purulent meningitis in newborn. The daily dose is preferably 150 mg/kg or more given in three divided intravenous doses. Meanwhile ampicillin proved to be useful as the antibiotic of choice for the treatment of purulent meningitis in infants and children.


A study on the rehabilitation of ischemic heart disease patients. The heart rate, beta-receptor blocking agents and strength-duration relationship of exercise.

Exercise tests with bicycle ergometer were performed in 9 normals and 54 patients with ischemic heart disease (IHD), in order to evaluate the heart rate (HR) as a parameter for determining the physical working capacity and controlling the daily activities, and to observe the circulatory and metabolic effects of beta-blockers and moreover to study the strength-duration relationship of exertion. The following results were obtained and discussed: 1. Reliable correlation between heart rate and O2 consumption during all phases of exercise, including the recovery period (R = 0.60-0.87, p less than 0.001) and parallelism of heart rate and pressure rate product allows the heart rate to be used as a parameter for myocardial O2 demand and ST-T changes. It is reasonable to evaluate the exercise effect by the net increment of heart rate during and after exercise, and to utilize the increment of heart rate in the recovery period for the evaluation of physical fitness and adaptability. 2. Similar to other studies made on beta-blockers, in this study too, the agents including Kö 1366 suppressed the exercise responses of heart rate (p less than 0.01), the product of heart rate and blood pressure (p less than 0.01), and O2 consumption (p less than 0.01). But a relatively slow recovery of O2 consumption from the peak response towards pre-exercise level and a higher O2 pulse in the early recovery period was observed after beta-blocker medication. 3. Capillary (ear lobe) pH decreased and recovered in correlation with the strength of exertion. This corresponded with the response of other factors such as ST-T, heart rate, pressure-rate product, and O2 consumption, showing that the exercise strength was one of the more dominant factors to determine allowable work load for ischemic heart disease patients. 4. Recovery of pH was delayed after medication with Kö 1366 (p less than 0.01).