Solubilization and characterization of the beta-adrenergic receptor binding sites of frog erythrocytes. Specific beta-adrenergic receptors present in membrane preparations of frog erythrocytes were identified by binding of (-)-[3H]dihydroalprenolol, a potent competitive beta-adrenergic antagonist. The (-)-[3H]dihydroalprenolol binding sites could be solubilized by treatment of a purified erythrocyte membrane fraction with the plant glycoside digitonin but not by treatment with a wide variety of other detergents. The binding sites appeared to be soluble by several independent experimental criteria including (a) failure to sediment of 105,000 X g for 2 hours; (b) passage through 0.22-mu Millipore filters; (c) chromatography on Sepharose 6B gels; and (d) electron microscopy. The soluble receptor sites retained all of the essential characteristics of the membrane-bound sites, namely rapid and reversible binding of beta-adrenergic agonists and antagonists; strict stereospecificity toward both beta-adrenergic agonists and antagonists; appropriate structure-activity relationships; saturability of the sites at low concentrations of ligand; no affinity for alpha-adrenergic drugs, nonphysiologically active catechol compounds, and catecholamine metabolites. Based on gel chromatography in the presence of detergent, the molecular weight of the soluble receptor is estimated to be no greater than 130,000 to 150,000. Equilibrium binding studies indicated a KD for the soluble receptor of 2 nM. Hill coefficients (nH) of 0.77 and curved Scatchard plots suggested the presence of negatively cooperative interactions among the solubilized receptors in agreement with previous findings with the membrane-bound sites. Kinetic studies indicated an association rate constant K1 = 3.8 X 10(6) M-1 min-1 and a reverse rate constant k2 = 2.3 X 10(-3) min-1 at 4 degrees. The kinetically derived KD (k2/k1) of 0.6 nM is in reasonable agreement with that determined by equilibrium studies. The soluble receptors were labile at temperature greater than 4 degrees but could be stabilized with high concentrations of EDTA. Guanidine hydrochloride and urea produced concentration-dependent losses of binding activity which were partially reversible upon dialysis. Trypsin and phospholipase A both degraded the soluble receptors but a variety of other proteases and phospholipases as well as DNase and RNase were without effect. Experiments with group-specific reagents indicated that free lysine, tryptophan, serine, and sulfhydryl groups may be important for receptor binding. These studies suggest that the receptor is probably a protein which requires lipids for functional integrity. Data obtained with the solubilized binding sites are consistent with the contention that these sites represent the physiologically relevant beta-adrenergic receptors which have been extracted from the membranes with full retention of their properties.


Reconstitution and characterization of the adenine nucleotide transporter derived from bovine heart mitochondria. 1. Adenine nucleotide exchange-transport was reconstituted in vesicles prepared from phospholipids and protein fractions derived from bovine heart submitochondrial particles. The transport, which was specific for ATP and ADP was measured either as ADP/ADP, ATP/ATP, or ADP/ATP exchange. The highest specific activity (370 nanomoles of ADP/ADP exchange/min/mg of protein at room temperature) was obtained with a protein fraction prepared by cholate extraction of partly resolved submitochondrial particles followed by ammonium sulfate fractionation. 2. At 200 muM external nucleotide, the exchange reactions were inhibited by low concentrations of bongkrekate, atractyloside, and palmitoyl-CoA, with Ki values of 1.8, 3.0, and 7.5 muM, respectively. The ADP/ADP nucleotide exchange was stimulated about 5-fold by 500 muM MgCl2 or MnCl2(km of 40 muM) and about 3-fold by 500 muM CaCl2(Km of 90 muM). It was optimal between pH 6.0 and 7.0 and decreased rapidly above pH 7.5. Arrhenius plots between 0 degrees and 40 degrees showed a break point at 15 degrees with soybean phospholipids and an activation energy of 29.5 kcal/mole from 0 degrees-15 degrees and 9.0 kcal/mole from 15 degrees-40 degrees. With mitochondrial phospholipids the break point was at 9 degrees and activation energies were 42.4 kcal/mole from 0 degrees-9 degrees and 7.6 kcal/mole from 9 degrees-40 degrees. 3. The phospholipid requirements for adenine nucleotide exchange were similar to those of oxidative phosphorylation. Optimal rates were observed with a phosphatidylethanolamine to phosphatidylcholine ratio of 4:1. Cardiolipin had a slight stimulatory effect. 4. The uptake of ADP into vesicles containing ATP was stimulated by KCl or by KPi as well as by hexafluoracetonylacetone, and uncoupler of oxidative phosphorylation. The uptake of ATP into vesicles containing ADP was inhibited by KCl or by KPi, but was also stimulated by hexafluoracetonylacetone. In both cases valinomycin reversed the effects of KCl, while mersalyl or N-ethylmaleimide prevented the effects of KPi. In contrast, none of these salts nor hexafluoracetonylactone affected the ADP/ADP or ATP/ATP exchange. These findings suggest that in the reconstituted system the ADP/ATP exchange is electrogenic.


Solubilization of thyroid peroxidase by nonionic detergents. We have examined the ability of nonionic detergents to solubilize thyroid peroxidase from a porcine thyroid particulate fraction, as measured by the release of peroxidase activity into the supernatant fraction after centrifugation at 105,000 X g for 1 hour and the retardation of the supernatant peroxidase of Sepharose 6B. The parameters of peroxidase solubilization by Triton X-100 have been investigated in detail. Under optimum conditions, 60 to 95% of the thryoid peroxidase and about 50% of the total protein is released into the 105,000 X g, 1-hour supernatant. Under the optimum conditions established with Triton X-100, a series of Brij detergents of different chemical structure were equally effective in releasing peroxidase and protein. The protein patterns of the supernatants obtained with these detergents were similar on sodium dodecyl sulfate-polyacrylamide electrophoresis gels, suggesting that the detergents studied release similar membrane proteins. The Triton X-100 and Brij 58 supernatants were chromatographed separately on Sepharose 6B equilibrated with 0.1% Triton X-100 or Brij 58, respectively. In both cases, 75 to 80% of the peroxidase activity was retarded, thereby indicating that the nonionic detergents effect solubilization of the peroxidase rather than dispersal of nonsedimentable membrane fragments. These studies report the first successful solubilization of thyroid peroxidase by nonionic detergents. Together with previous evidence from our laboratory, these experiments indicate that thyroid peroxidase is an integral membrane protein.


Carbon 13 magnetic resonance studies of DL-2-(alpha-hydroxyethyl) thiamin and related compounds. Relation of kinetic acidity to electronic factors in thiamin catalysis. Carbon 13 NMR spectra have been obtained for aqueous solutions of DL-2-(alpha-hydroxyethyl)thiamin, DL-2-(alpha-hydroxybenzyl)thiamin, DL-2-(alpha-hydroxybenzyl)oxythiamin, and related N-3 methyl and N-3 benzyl analogs. The unusually large downfield shift of the 13C resonance of C-2 of hydroxyethylthiamin suggests that this carbon bears a partial positive charge. This result stands in contrast to results of x-ray crystallographic studies of hydroxyethylthiamin, which place a partial negative charge on C-2 (Pletcher, J., and Sax, M. (1974) J. Am. Chem. Soc. 96, 155-165). A partial positive charge on C-2 helps to explain the facility of carbanion formation at the alpha carbon both enzymatically and in model systems. The rates of proton-deuteron exchange of (C-alpha)-H with solvent deuterium, and of release of aldehyde to regenerate thiamin have been measured for hydroxyethylthiamin and analogs. The differences in kinetic acidity of (C-alpha)-H and of rates of aldehyde release are rationalized in terms of differing electron-withdrawing abilities of the substituents attached to N-3, and appear not to be related to intramolecular basic catalysis of these processes by the C-4' amino group.


Oxygen and carbon monoxide kinetics of Glycera dibranchiata monomeric hemoglobin. Oxygen and carbon monoxide kinetics of Glycera dibranchiata monomeric hemoglobin have been studied using laser photolysis, air flash, and stopped flow techniques. The reactions of this hemoglobin with both ligands were found to be more rapid than the corresponding reactions involving myoglobin and were also biphasic in nature, the rate constants being approximately an order of magnitude different for the fast and slow phases in each case. No pH or hemoglobin concentration dependence of the pseudo-first order rate constants was apparent between pH 6 and 9 and in the concentration range of 1.25 to 40 muM heme. Both fast and slow pseudo-first order oxygen combination rate constants varied linearly with oxygen concentration between 16 and 1300 muM. A first order slow relaxation was also noted which was linearly dependent on heme concentration and inversely dependent on oxygen concentration. This reaction has been shown to be due to a replacement of oxygen by carbon monoxide. The presence of this reaction is a result of the high affinity of Glycera monomer for carbon monoxide as shown by the partition coefficient Mr = approximately 20,000 ana an equilibrium dissociation constant of the order L = 1.1 X 10(-9) M.


Analysis of phosphate metabolites, the intracellular pH, and the state of adenosine triphosphate in intact muscle by phosphorus nuclear magnetic resonance. 31P nuclear magnetic resonance spectra recorded from intact muophosphate, and the sugar phosphates. Quantitation of these metabolites by 31P nuclear magnetic resonance was in good agreement with values obtained by chemical analyses. The spectra obtained from various muscles showed considerable variation in their phosphorus profile. Thus, differences could be detected between (a) normal and diseased muscle; (b) vertebrates and invertebrates; (c) different species of the same animal. The time course of change in phosphate metabolites in frog muscle showed that ATP level remains unchanged until phosphocreatine is nearly depleted. Comparative studies revealed that under anaerobic conditions the Northern frog maintains its ATP content for 7 hours, while other types of amphibian, bird, and mammalian muscles begin to show an appreciable decay in ATP after 2 hours. Several lines of evidence indicated that ATP forms a complex with magnesium in the muscle water: (a) the phosphate resonances of ATP in the muscle were shifted downfield as compared to those in the alkaline earth metal-free perchloric acid extract of the muscle; (b) the coupling constants of ATP measured in various live muscles closely corresponded to those for MgATP in a solution resembling the composition of the muscle water; (c) in the muscle the gamma-phosphate group of ATP exhibited no shift change over a period of 10 hours under conditions where resonances of other phosphate compounds could be titrated. This behavior is similar to that of MgATP in model solutions in the physiological pH range, and it is different from that of CaATP. The chemical shifts of the phosphate metabolites were determined in several relevant solutions as a function of pH. Under all conditions only inorganic orthophosphate showed an invariant titration curve. From the chemical shift of inorganic phosphate observed during aging of intact muscle the intracellular pH of frog muscle was estimated to be 7.2.


5' leads to 3'-Exonucleases of bacteriophage T4. Two enzyme activities which release nucleotides preferentially from the 5' termini of DNA were found in T4-infected Escherichia coli. Since no corresponding activities were found in uninfected cells, the activities appeared to be induced by T4. Both activities are capable of excising pyrimidine dimers from ultraviolet-irradiated DNA which has been treated with T4 endonuclease V. One of the activities , referred to as T4 exonuclease B, was purified 400-fold from an extract of T4v 1- infected cells. The enzyme initiates hydrolysis of DNA specifically at the 5' termini to yield products which are mainly oligonucleotides of varying length. The hydrolysis reaction proceeds in a limited manner. The enzyme shows optimal activity at pH 7.0 and absolutely requires Mg2+. The molecular weight of the enzyme , as estimated by gel filtration, is approximately 35,000. Another activity, referred to as T4 exonuclease C, was purified 240-fold from the extract. This activity also excises pyrimidine dimers from ultraviolet-irradiated, incised DNA and releases nucleotides at 5' termini. It has a pH optimum at 7.5 and requires Mg2+. The molecular weight of the enzyme is approximately 20,000.


Nuclear magnetic resonance titration curves of histidine ring protons. A direct assignment of the resonances of the active site histidine residues of ribonuclease. One of the four titrating histidine ring C-2 proton resonances of bovine pancreatic ribonuclease has been assigned to histidine residue 12. This was accomplished by a direct comparison of the rate of tritium incorporation into position C-2 of histidine 12 of S-peptide (residues 1 to 20) derived from ribonuclease S, with the rates of deuterium exchange of the four histidine C-2 proton resonances of ribonuclease S under the same experimental conditions. The same assignment was obtained by a comparison of the NMR titration curves of ribonuclease S, the noncovalent complex of S-peptide and S-protein (residues 21 to 124) with the results for the recombined complex in which position C-2 of histidine 12 was fully deuterated. The second active site histidine resonance was assigned to histidine residue 119 by consideration of the NMR titration results fro carboxymethylated histidines and 1-carboxymethylhistidine 119 ribonuclease. This assignment is a reversal of that originally reported, and has important implications for the interpretation of NMR titration data of ribonuclease.


Nuclear magnetic resonance titration curves of histidine ring protons. Ribonuclease S-peptide and S-proteins. The histidine C-2 proton NMR titration curves of ribonuclease S-peptide (residues 1 to 20) and S-protein (residues 21 to 124) are reported. Although S-protein contains 3 histidine residues, four discrete resonances are observed to titrate. One of these arises from the equivalent histidine residues of unfolded S-protein. The variation in area of the four resonances indicate that there is a reversible pH-dependent equilibrium between the folded and unfolded forms of S-protein, with some unfolded material being present at most pH values. Two of the resonances of the folded S-protein can be assigned to 2 of the histidine residues, 48 and 105, from the close similarity of their titration curves to those in ribonuclease. These similarities indicate a homology of portions of the folded conformation of S-protein to that of ribonuclease in solution. These results indicate that the complete amino acid sequence is not required to produce a folded conformation similar to the native globular protein, and they appear to eliminate the possibility that proteins fold from their NH2 terminus during protein synthesis. The low pH inflection present in the titration curve assigned to histidine residue 48 in ribonuclease is absent from this curve in S-protein. This is consistent with our previous conclusion that this inflection arises from the interaction of histidine 48 with aspartic acid residue 14, which is also absent in S-protein. The third titrating resonance of native S-protein is assigned to the remaining histidine residue at position 119. The properties of this resonance are not identical with either of the titration curves of the active site histidine residues 12 and 119 of ribonuclease. The resonance assigned to histidine 119 is the only one significantly affected on the addition of sodium phosphate to S-protein, indicating that some degree of phosphate binding occurs. In both the absence and presence of phosphate this curve also lacks the low pH inflection observed in the histidine 119 NMR titration curve in ribonuclease. This difference presumably arise from a conformational between ribonuclease and the folded S-protein involving a carboxyl group.


Effects of formylation of vinyl side chains of heme on optical and ligand binding properties of horse heart ferric myoglobin. Effects of substitution of vinyl groups of hemin with formyl groups on the optical and ligand binding properties of horse heart ferric myoglobin were investigated. The peak positions as well as the line shapes of the absorption spectra of the ferric derivatives of three kinds of formylmyoglobin, 2-vinyl-4-formyl-, 2-formyl-4-vinyl-, and 2,4-diformylmyoglobins depend on the number and the position of the formyl groups. Absorption maxima in the Soret region of the acid forms of these ferric formylmyoglobins in 0.1 M potassium phosphate buffer, pH 6.0, at 20 degrees were 415.2, 422, and 429 nm, respectively. The acid forms of these formylmyoglobins exhibit absorption spectra of the mixture of high- and low spin states at ambient temperature. Since proto-, deutero- and mesomyoglobins have a high spin state under the same condition, the increase of the low spin iron in these formylmyoglobins may be due to the strong electron withdrawal by the formyl groups toward the periphery of the porphyrin ring. The affinities of these ferric formylmyoglobins and protomyoglobin for N3-, F-, OCN-, and SCN- increased in the order of proto-, monoformyl-monovinyl-, 2,4-diformyl-myoglobin, which corresponds to the increasing order of electron-withdrawing power of the porphyrin side chains. The pKa values of the acid-alkaline transition decreased in the same order. Although the ferric forms of the two isomeric monoformyl-monovinylmyoglobins exhibited different optical spectra, the dissociation constants of the complexes of these isomers for various ligands were similar to each other. The pKa values of the acid-alkaline transition were also similar. These results indicate that affinities of ferric myoglobin for ligands, in contrast to those of the ferrous form for oxygen and carbon monoxide (Sono, M., and Asakura, T. (1975) J. Biol. Chem. 250, 5527-5232 and Sono, M., Smith, P.D., McCray, J.A., and Asakura, T. (1976) J. Biol. Chem 251, 1418-1426), are not affected by the position of modifications at the two vinyl groups, but are determinedby the number of the formyl groups and that two vinyl groups at position 2 and 4 are equivalent in the binding of various ligands by ferric myoglobin. The electron density of the ferric iron appears to be similar for the two isomeric monoformyl-monovinylmyoglobins.


An essential residue at the active site of aspartate transcarbamylase. Reaction of phenylglyoxal with aspartate transcarbamylase and its isolated catalytic subunit results in complete loss of enzymatic activity. This modification reaction is markedly influenced by pH and is partially reversible upon dialysis. Carbamyl phosphate or carbamyl phosphate with succinate partially protect the catalytic subunit and the native enzyme from inactivation by phenylglyoxal. In the native enzyme complete protection from inactivation is afforded by N-(phosphonacetyl)-L-aspartate. The decrease in enzymatic activity correlates with the modification of 6 arginine residues on each aspartate transcarbamylase molecule, i.e. 1 arginine per catalytic site. The data suggest that the essential arginine is involved in the binding of carbamyl phosphate to the enzyme. Reaction of the single thiol on the catalytic chain with 2-chloromercuri-4-nitrophenol does not prevent subsequent reaction with phenylglyoxal. If N-(phosphonacetyl)-L-aspartate is used to protect the active site we find that phenylglyoxal also causes the loss of activation of ATP and inhibition by CTP. The rate of loss of heterotropic effects is exactly the same for both nucleotides indicating that the two opposite regulatory effects originate at the same location on the enzyme, or are transmitted by the same mechanism between the subunits, or both.


Physical and kinetic properties of homogenous bovine lens aldose reductase. Aldose reductase from calf lens was purified 15,000-fold. The homogeneity of the final preparation was demonstrated by molecular sieve chromatography, analytical ultracentrifugation, sodium dodecyl sulfate gel electrophoresis, Ouchterlony immunodiffusion, and polyacrylamide gel electrophoresis at three pH values. The monomeric nature of the enzyme is suggested by the molecular weight of 37,000 from both molecular sieve chromatography and sodium dodecyl sulfate-gel electrophoresis with beta-mercaptoethanol. This closely corresponds with a molecular weight of 40,400 estimated by using calculate physical constants in the Svedberg equation. The S20,w was 3.6 to 3.7 as determined from ultracentrifuge and sucrose density gradient data. The Stokes radius was found to be 2.5 +/- 0.2 nm and 2.75 +/- 0.15 nm by two different methods. The diffusion constant D20,w is (7.8 +/- 10(-7) +/- 0.45 X 10(-7) cm2/s). The molecule is nearly spherical as indicated by a frictional ratio f/fo = 1.14. The alpha-helical content was estimated from circular dichroism data to be 5% and did not change in the presence of added substrates, products, and some enzyme inhibitors. Homotropic cooperative effects were observed as shown by the concave downward curvature of the reciprocal plots.


L-Asparaginase of Klebsiella aerogenes. Activation of its synthesis by glutamine synthetase. An L-asparaginase has been purified some 250-fold from extracts of Klebsiella aerogenes to near homogeneity. The enzyme has a molecular weight of 141,000 as measured by gel filtration and appears to consist of four subunits of molecular weight 37,000. The enzyme has high affinity for L-asparagine, with a Km below 10(-5) M, and hydrolyzes glutamine at a 20-fold lower rate, with a Km of 10(-3) M. Interestingly, the enzyme exhibits marked gamma-glutamyltransferase activity but comparatively little beta-aspartyl-transferase activity. A mutant strain lacking this asparaginase has been isolated and grows at 1/2 to 1/3 the rate of the parent strain when asparagine is provided in the medium as the sole source of nitrogen. This strain grows as well as the wild type when the medium is supplemented with histidine or ammonia. Glutamine synthetase activates the formation of L-asparaginase. Mutants lacking glutamine synthetase fail to produce the asparaginase, and mutants with a high constitutive level of glutamine synthetase also contain the asparaginase at a high level. Thus, the formation of asparaginase is regulated in parallel with that of other enzymes capable of supplying the cell with ammonia or glutamate, such as histidase and proline oxidase. Formation of the asparaginase does not require induction by asparaginase and is not subject to catabolite repression.


Binding and degradation of insulin by human peripheral granulocytes. Demonstration of specific receptors with high affinity. The interaction of insulin with human circulating granulocytes was studied with the use of 125I-insulin. Human granulocytes, isolated from blood by the Böyum technique, showed high insulin-degrading activity in vitro which almost obscured the presence of specific, high affinity binding sites. Degradation, measured by trichloroacetic acid precipitation and by binding to well characterized insulin receptors on cultured human lymphocytes (IM-9 line), was due to extracellular as well as cell-bound enzymes. Degradation was enhanced by Ca2+ and thiols and inhibited by various protease inhibitors and sulfhydryl-blocking reagents. Phenylmethylsulfonyl fluoride (5 X 10(-4) M), a serine protease inhibitor, was the most potent and inhibited 125I-insulin degradation by 80 to 90%. Tert-butyl hydroperoxide (2 X 10(-3) M), a glutathione-oxidizing reagent, inhibited degradation by 35 to 50%, possibly due to an effect on a glutathione-insulin transhydrogenase. Neither of the inhibitors affected cell viability. In the presence of inhibitors of degradation, binding sites for insulin with high affinity were detected, which by multiple criteria were true insulin receptors. Binding to these sites was rapid, saturable, and reversible with about 1000 sites/cell. The Hill coefficient for binding was 0.7, and the Scatchard plot of B/F versus B was curvilinear, due to site-site interactions of the negative cooperative type; the latter were demonstrated directly by kinetic studies. As shown previously for all other insulin receptors, binding was highly pH-dependent, and insulin analogues had affinities for these sites that closely correlated with their biological potencies.


Triiodothyronine effects on some electrogenic properties of frog sartorius. At 21 degrees C in vitro, 0.2 and 2.0 muM of triiodothyronine (T3) produced an increase in resting membrane potential (RMP) of Rana pipens sartorius when the pH of the external solution was 7.4. The RMP was increased by 2.0 muM T3 in the presence of 10(-4) and 10(-3) M ouabain but not in 10(-3) M of 2,4 dinitrophenol. Small increases in RMP were observed with 2.0 muM T3 in solutions with low external Na. At pH 7.1 0.2 muM T3 produced a small transient increase in RMP. Membrane resistance (Rm) was found to decline gradually during exposure to 0.2 muM at a pH of 7.4. Treatment with 2.0 muM T3 at pH 7.4 was accompanied by a transient reduction in Rm. Similar transient changes in Rm were produced by 0.2 and 2.0 muM T3 at pH of 7.1 T3 reduced membrane resistance in isotonic K2SO4 and tris-buffered Mn (20 mM) solutions indicating that T3 increases potassium permeability. Direct action potentials were studied at pH 7.1. Overshoot, amplitude and rate of rise of the action potential underwent a gradual decrease in the presence of 0.2 muM T3 while thresholds remained unchanged. Thresholds were increased during exposure to 2.0 muM T3 whereas overshoot, amplitude and rate of rise underwent transient decreases followed by a return toward control levels.


[Acute amebic abscess of the liver followed by colonic ulcers. Report  of 2 cases]. Two unusual cases of ruptured acute amebic liver abcess are presented one with inferior vena caval compression. Surgical drainage was followed by the development of colonic complications (haemorrhage and perforation) although the patients were under specific therapy. After a new surgical operation both cases were cured. The relation with amebiasis is discussed. The possibility of such an association must always be kept in mind. The value of immunofluorescence test in the diagnosis is emphasised.


High-performance ion-pair partition chromatography of sulfa drugs. Study and optimization of chemical parameters. High-performance ion-pair partition chromatography is shown to be a versatile, efficient method for separating sulfonamides. For a group of fourteen sulfa drugs varying widely in pKA and hydrophobicity, the effect of mobile phase composition, counterion composition, pH, and ionic strength on their ion-pair partition chromatographic separation using tetrabutylammonium as the counterion and n-butanol-n-heptane as the mobile phase is shown. Wide variation in k' and a is possible by changing these parameters. Silica columns coated with buffered aqueous solutions of tetrabutylammonium sulfate resulted in efficiencies of 4000-6000 theoretical plates per 25 cm. These columns are stable for long periods of time, and can be stripped and re-used in the adsorption mode with little or no loss in efficiency. Several chromatograms are presented in order to illustrate the performance of ion-pair partition chromatography.


Gas chromatographic determination of carboxylic acid chlorides and residual carboxylic acid precursors used in the production of some penicillins. An improved gas chromatographic method is described for the simultaneous determination of carboxylic acid chlorides and related carboxylic acids used in the production of some commercial semisynthetic penicillins. The acid chloride reacts with diethylamine to form the corresponding diethylamide. Carboxylic acid impurities are converted to trimethylsilyl esters. The two derivatives are separated and quantitated in the same chromatographic run. This method, an extension of the earlier procedure of Hishta and Bomstein (1), has been applied to the acid chlorides used to make oxacillin, cloxacillin, dicloxacillin, and methicillin (Figure 1); it shows promise of application to other acid chlorides. The determination is more selective than the usual titration methods, which do not differentiate among acids with similar pK's. Relative standard deviations of the acid chloride determination are 1.0-2.5%. Residual carboxylic acid can be repetitively determined within a range of 0.6% absolute.


Demonstration and some properties of cytosol-binding proteins for thyroxine and triiodothyronine in human liver. Cytosol-binding proteins for L-thyroxine (T4) and triiodo-L-thyronine (T3) were studied in human liver specimens obtained at autopsy from 5 male and 2 female subjects. The liver cytosol containing 131I-T4 or T3, together with or without added stable hormones, was fractionated by Pevikon thin-layer electrophoresis at pH 8.6, 8.0, and7.4. It was demonstrated in all the specimens that besides a small amount of serum T4-binding globulin, there existed three T4-binding proteins, termed hT4-1, hT4-2 and hT4-3, with the electrophoretic mobilities of alpha2- and beta-globulins, and two T3-binding proteins, termed hT3-1 and hT3-2, with the mobilities of gamma-globulin. Binding of hormones by the cytosol proteins was pH-dependent, and a preliminary dialysis had no effect on the hormone binding. The major band of T4, hT4-2, bound more than half the tracer T4, and possessed the maximal binding capacity of 110 mug/100 ml of 33% cytosol at pH 7.4. However, it showed no apparent affinity for T3, because the bound T4 could not be displaced with a T3 load of 600 mug/100 ml. The major band of T3, hT3-2, bound more than 70% of the tracer T3, and appeared to have a large capacity for the hormone although secondary binding sites on the same molecule might be responsible for the large capacity. The binding sites appeared almost specific for T3, because only a small, insignificant displacement was noted with a T4 load of 600 mug/100 ml. The results provide evidence for distinct binding proteins for T4 and T3 in the human liver cytosol, though their physiological roles remain to be elucidated.


Glucagon-sensitive adenylate cyclase in human renal medulla. In cell-free preparations (washed 600 x g pellets) of human renal medulla, glucagon produced a dose-dependent stimulation of adenylate cyclase. The stimulation of renal medullary adenylate cyclase by saturating concentrations of glucagon was additive to the saturating doses of vasopressin. Furthermore, L-isoproterenol stimulated renal medullary adenylate cyclase in a dose-dependent manner, and this stimulation was blocked by DL-propranolol. Stimulation of the renal medullary adenylate cyclase by maximal doses of glucagon and L-isoproterenol was additive. DL-Propranolol did not inhibit stimulation of glucagon. Thus, the results indicate the existence of a specific adenylate cyclase that is responsive to glucagon--distinct from the isoproterenol-sensitive adenylate cyclase and the previously described vasopressin-sensitive adenylate cyclase in human renal medulla. We suggest that the renal tubular effect of glucagon may be mediated by glucagon-dependent cyclic-AMP production in renal tissue.


Simultaneous comparison of delta 5-3beta-hydroxysteroid levels in the fetoplacental circulation of normal pregnancy in labor and not in labor. Concentrations of pregnenolone (delta5P), dehydroepiandrosterone (DHEA), 16alpha-hydroxydehydroepiandrosterone (16alpha-OH DHEA), pregnenolone sulfate (delta5P-S), and dehydroepiandrosterone sulfate (DHEA-S) were measured simultaneously by radioimmunoassay in individual, paired umbilical artery (UA) and vein (UV) sera from 18 normal term pregnancies, 6 in labor, 12 not in labor. Mean UA and UV levels +/- SEM (ng/ml) were for delta5P: 30.39 +/- 1.69, 35.55 +/- 3.06; DHEA: 12.31 +/- 2.34, 3.66 +/- 0.38; 16alpha-OH DHEA: 7.48 +/- 0.63, 10.59 +/- 0.78; delta5P-S: 1,652 +/- 154, 1,486 +/- 130; DHEA-S: 2,122 +/- 134, +/- 134, 1,906 +/- 134. Umbilical artery delta5P-S, DHEA-S, and DHEA levels were significantly higher than UV levels, whereas the reverse was true for delta5P and 16alpha-OH DHEA. The inverse arterio-venous (A-V) gradient for 16alpha-OH DHEA was contrary to previous published reports using pooled samples. Comparison by linear regression of paired UA and UV steroid concentrations of delta5P, delta5P-S, DHEA, and DHEA-S revealed a significant correlation (P less than 0.01) for each steroid. Labor was associated with a significant increase in UA levels of DHEA-S and a smaller, but not quite significant, increase in UA levels of delta5P-S, while similar changes for unconjugated delta5-3beta-hydroxysteroids were not observed. Mean A-V gradients between the group of patients in labor and those not in labor were not significantly different. These data demonstrate that: 1) a significant difference between UA and UV concentrations exists for delta5P, DHEA, 16alpha-OH DHEA, delta5P-S, and DHEA-S; 2) there is a significant correlation between UA and UV concentrations for delta5P, DHEA, delta5P-S, and DHEA-S, implying that each fetoplacental unit maintains an equilibrium relative to these steroid concentrations in the umbilical circulation; 3) labor is associated with a significant increase in UA levels of DHEA-S and probably of delta5P-S.


Isolation of human eosinophil phospholipase D. Phospholipase D preferentially contained in human eosinophil polymorphonuclear leukocytes as compared to other leukocytes was isolated by sequential asion and cation exchange chromatography and gel filtration. The purified eosinophil enzyme specifically liberated choline from I-alpha-phosphatidyl choline with a pH optimum of 4.5-6.0 and exhibited a pI of 5.8-6.2 on polyacrylamide-gel isoelectric focusing, which are properties shared by phospholipase D from plant sources; however, its apparent mol wt of 60,000 is approximately one-half that of the plant enzymes. Eosinophil and cabbage phospholipase D inactivated a partially purified rat platelet-activating factor (PAF) in a time- and dose-dependent reaction. The cleavage of this PAF activity was attributed to the inherent phospholipase D activity of the eosinophil enzyme since the two activities chromatographed together at each purification step, and there was apparent reciprocal inhibition of choline-generating activity by PAF and of PAF-inactivating activity by phosphatidyl choline. Thus, possible regulatory functions of the eosinophil in immediate hypersensitivity reactions include inactivation of a PAF by phospholipase D as well as degradation of slow-reacting substance of anaphylaxis by arylsulfatase B.


Role of expectancy set in the systematic desensitization of speech anxiety: an extension of prior research. The influence of expectancy set with regard to therapy outcome on the effectiveness of systematic desensitization (SD) for reducing public speaking anxiety was investigated. The 7 Ss given a high expectancy set for favorable therapy outcome were informed about psychological research that indicates that SD is effective to reduce public speaking fears. SD was administered with the standard instructions to the 11 Ss given a neutral expectancy set. This expectancy manipulation did not require deception and perhaps could be used with actual SD therapy clients. As in previous research by Woy and Efran, the expectancy set manipulation significantly modified Ss' self-report of subjective perceptions of anxiety from pretreatment to posttreatment speeches, but did not affect overt behavioral or physiological indices of anxiety. Since subjective perceptions of anxiety responses are psychologically significant behaviors, these data suggest the importance of conveying a high expectation of improvement to SD and perhaps also to other types of therapy clients. SD sessions administered to small groups of clients on consecutive days, as in this study, appeared to be as effective to reduce speech anxiety as SD sessions administered to each client individually at 1-week intervals, as in the Woy and Efran study.


Graduated approach modeling in an aversive task. Sixty female Ss were assigned randomly to six conditions that involved self-administration of electrical shock. These were: (1) no model-one trial S response; (2) no model-graduated trial S response; (3) one trial model-one trial S response; (4) one trial model-graduated S response; (5) graduated model-one trial response; and (6) graduated model-graduated response. Results indicated that graduated exposure of S to an aversive stimuli was more effective than a single such exposure and that type of exposure provided by a model had little effect.


Cephradine excretion in humans with pH-altered urine. In a random crossover study, ten healthy men were pretreated with ammonium chloride or sodium bicarbonate to alter urinary pH, and then were given a single 1-Gm dose of cephradine, either orally or intravenously. No significant changes were found in the terminal phase rate constant nor in the time or magnitude of peak concentration. Total recovery was greater than 90 per cent whenever degradation of cephradine did not occur. An unexplained but significant reduction in area under curve was present in data for prealkalinized subjects and for all subjects who took cephradine orally. Trapping of cephradine was not significant in the excretory pathway.


Partial agonist properties and toxicity of oral oxilorphan. Alternative pharmacologic adjuncts are needed for the management of opiate abuse. Oxilorphan, a narcotic antagonist, was studied at 5 different dose levels (1, 2, 4, 6, and 8 mg) in 30 normal subjects to determine the relation of single oral doses and toxicity. The drug causes pupillary constriction and mild central nervous system side effects (nausea, dizziness) at all doses. Mean urine volume increased (P less than 0.05) during the 12 hours after 1 and 2 mg. Oxilorphan has partial agonist properties similar to dl-cyclazocine.


Characteristics of the release of adenosine 3':5'-monophosphate from micropipets by microiontophoresis. The transfer number for radio-labelled cyclic AMP released from microiontophoretic pipets into brain pieces was determined for a large number of samples by radioassay. Release of cyclic AMP was linearly related to both iontophoretic current intensity and time as predicted by Faraday's Law. The results revealed that cyclic AMP has a rather low transfer number. In addition, an unusually large amount of variation of release, both within and among pipets was found under a variety of times and currents. The cause of the variation is not known but could be due to the unusual structure of the cyclic AMP molecule and the fact that it must be iontophoresed as a negative ion. These characteristics of cyclic AMP release may contribute to the difficulty in obtaining positive responses from appropriate neuronal target cells in vivo.


Effects of egg factors on cyclic nucleotide metabolism in sea urchin sperm. Cyclic AMP in Strongylocentrotus purpuratus sperm was elevated approximately 2-fold by theophylline or 1-methyl-3-isobutylxanthine. Factors released from sea urchin eggs (FRE) elevated sperm cyclic AMP by about 7-fold within 1 min, and the combination of FRE with theophylline increased sperm cyclic AMP up to 100-fold within 1 min. Cyclic GMP in sea urchin sperm was slightly elevated by theophylline, but was lowered by FRE. Cyclic GMP in sperm treated with FRE plus theophylline was not higher than in sperm treated with theophylline alone. The ability of FRE-containing sea water to increase sperm cyclic AMP in the presence of theophylline was altered only slightly if at all by boiling, but it was decreased by about 50% by dialysis and destroyed by ashing. Filtration of FRE on Sephadex G-50 columns yielded two peaks of cyclic AMP-elevating activity. One peak (peak I) was eluted at the column void volume, and the other (peak II) was retained by the column. The cyclic GMP-lowering activity was located in fractions approximately corresponding to peak I of cyclic AMP-elevating activity. Dialysis of FRE-containing sea water before its application to the G-50 column virtually eliminated peak II of the cyclic AMP-elevating activity. When the cyclic AMP-elevating activity in peak I was filtered on Bio Gel A-5m columns, it also migrated at or near the column void volume. Fractions corresponding to peak I contained material that inhibited both guanylate and adenylate cyclase activities in broken cell preparations of sperm and guanylate cyclase from rat lung. The inhibitory material was stable to boiling, non-dialyzable, and destroyed by ashing. Under a variety of conditions, FRE-containing sea water or cyclic AMP-elevating peaks I or II did not stimulate sperm adenylate cyclase activity in broken cell preparations.


Dephosphorylation of bovine casein by milk alkaline phosphatase. The pH of optimum activity of alkaline phosphatase from cow's milk depended on the substrate, being 10-1 for rho-nitrophenylphosphate, 8-6 for phosphoserine, 8-0 for phosvitin and 6-8 for casein. Individual casein components were dephosphorylated more rapidly than mixtures of alphas- and beta-caseins or of alphas-, beta-and kappa-caseins and micellar casein. Mixtures of 2 components involving kappa-casein were more readily dephosphorylated than alphas- and beta-casein mixtures. At pH 6-8, lactose, whey proteins and phosphate ions had an inhibitory effect. beta-Lactoglobulin had an inhibitory effect only when the pH of the reaction was lower than the optimum pH value of the enzyme. Mg2+ and Zn2+ were not inhibitory. The optimum conditions for dephosphorylation of casein are described.


Effect of flurazepam on common clinical laboratory tests. Twenty-two clinical laboratory tests performed on blood samples from 16 normal subjects following one week of either flurazepam or placebo administered in a double-blind method showed no apparent chemical interference by flurazepam on any of the testing procedures.


A kinetic concepto of lipid transport in ruminants. Summarization of the literature shows a strong correlation between dietary fatty acid intake and total lipid concentration in plasma in lactating cows whereas total milk fat secreted is related to neither of these. In the process of plasma triglyceride removal, chylomicra and very low density lipoproteins are converted to low density lipoproteins. Limited kinetic data indicate that the fractional removal rates for chulomicra and very low density lipoproteins are rapid in lactating cows whereas fractional removal of low density lipoproteins is slower, resulting in accumulation of the latter in plasma. Under such conditions, low density lipoprotein concentrations of plasma would not be expected to reflect quantitatively the transfer of plasma triglyceride fatty acids to milk fat. Quantitative analysis or triglyceride fatty acid turnover in density less than 1.006 lipoproteins should delineate the role of plasma lipid transport in milk fat synthesis. High fat diets protected from rumen biohydrogenation have proven to be a useful approach in studying ruminant fat metabolism and may be used more extensively to elucidate the role of cholesterol in plasma lipid transport and the metabolism of essential fatty acids in ruminants.


Bovine pancreatic lipase.I.Isolation, homogeneity, and characterization. Bovine pancreatic lipase was isolated in pure form by lyophilization of fresh bovine pancreas, extraction of the enzyme with sucrose solution, fractional precipitation with ammonium sulfate and acetone, followed by chromatography on Sephadex G-100. The specific activity of the purest lipase fraction was 1750 micromoles fatty acid, liberated in 30 min per milligram of protein, indicating a purification of approximately 473-fold, with an overall yield of about 42%. Homogeneity of the enzyme was confirmed by rechromatography on Sephadex G-100 as well as with the gel electrophoretic and ultracentrifugal techniques. The purified enzyme gave a typical protein ultraviolet absorption spectrum with maximum absorption at 276 nm and minimum at 252 nm. The purified enzyme exhibited a single pH optimum of 8.8 and an isoelectric point near pH 5.5. Its optimum temperature was 37 C, and its optimum substrate concentration was 10%. These properties resembled those of milk lipase.


Distribution and removal of cadmium from milk. Distribution of added cadmium in milk systems and the feasibility of removing cadmium were investigated. In milk containing 1 ppm cadmium, 96% of the added cadmium chloride was dispersed in the skim milk fraction, and 3% was associated with the cream fraction. Cadmium was not bound strongly to any protein fraction. The association of cadmium with acid casein, whey proteins, and the fat globule membrane was 18,6, and .5% of total added cadmium. With a 5-min exposure thiosuccinylated amino-ethyl cellulose, thionitrocarboxyphenylated aminoethyl cellulose, and reduced human hair removed 72, 70, and 30%, respectively, of added cadmium in skim milk previously equilibrated for 2 h at 37 C. Increasing equilibration time beyond 24 h had no effect on removal efficiency whereas increasing pH decreased cadmium removal markedly.


Influence of viable yogurt microflora on digestion of lactose by the rat. Laboratory rats were fed experimental diets including yogurt, pasteurized yogurt, and simulated yogurt with sucrose or lactose for 7 days followed by a single experimental meal of yogurt, pasteurized yogurt, or simulated yogurt. Assays of blood galactose demonstrated that animals fed natural yogurt containing the viable culture microflora were able to absorb galactose more efficiently. Intestinal lactase activity of yogurt-fed animals was greater than in animals fed other experimental diets including pasteurized yogurt. Gastrointestinal survival of culture organisms was demonstrated in vivo up to 3 h after feeding, and thus, the viable cells resulted in more efficient hydrolysis which favored lactose digestion in natural yogurt.


Nonimmunologic aspects of caries resistance. A variety of components provide salivary secretions with an array of potentially effective means of combating cariogenic challenges. These defense factors range from a laissez-faire mechanical cleansing to exquisitely controlled production of highly specific antibodies. In between the two extremes are antibacterial systems whose operating characteristics are only beginning to be understood. These systems are well worth our attention. They may be the key to our understanding of variations in individual susceptibility, and could provide valuable leads for development of anticaries agents.


Adherence of serotype e Streptococcus mutans and the inhibitory effect of Lancefield group E and S mutans type e antiserum. S mutans strain MT703 from an active carious lesion in the tooth of a child had type e specificity and showed a cross-reaction with the Lancefield group E cell wall streptococcal polysaccharide antigen. Heat-killed cells MT703 adhered to a glass surface in the presence of CGT MT703 and sucrose. Pretreatment of the cells with anti-MT703 whole cell serums inhibited adherecne. The removal of glycerol teichoic acid antibody and group E antibody from the MT703 serum did not result in a loss of inhibitory activity. Antiserum with or without adsorption significantly inhibited glucan synthesis by CGT from sucrose. Antibodies specific for the polyglycerol phosphate of teichoic acid did not inhibit adherence. Anti-group E serum and serums specific for other types of S mutans, did not show adherence inhibitory activity except for an occasional type c specific antiserum. Antibody specific for the type e antigen produced significant inhibition of the binding of CGT to the MT703 cell wall, and adherence of these cells did not occur. Antibody to CGT inhibited glucan synthesis. Treatment of the cells with dextranase, dextran antibody, or trypsin caused a significant reduction in adherence. The results suggest that the type antigen and dextran on the surface of the S mutans type e cell are functional in adherence, and that these polymers are associated with cell wall protein.


The effect of beta adrenergic blockade on bronchial sensitivity to acetyl-beta-methacholine in normal and allergic rhinitis subjects. The effect of propranolol inhalation on sensitivity to methacholine inhalation was studied in normal and allergic rhinitis subjects to determine whether beta adrenergic blockade alters sensitivity to mediators in nonasthmatic atopic individuals. A partial beta adrenergic blockade is suggested as being instrumental in asthma. Hay fever patients studied showed similar effects and also developed asthma for the first time.


Effect of beta adrenergic stimulation and blockade on cutaneous reactivity to histamine. It has been previously demonstrated that iontophoresis of beta adrenergic agents will alter the size of immediate hypersensitivity skin tests. It was unclear whether this alteration was due to an effect on the dermal mast cell (inhibition of histamine release) or on the cutaneous vasculature (inhibition of capillary permeability). For this reason isoproterenol, propranolol, diphenhydramine as a positive control, and saline as a negative control were iontophoresed onto the forearm of 10 atopic and 10 nonatopic adult subjects. In order to bypass histamine release from mast cells the patients were then challenged directly with histamine by the "prick" technique. The size of the resultant wheals was noted. The data obtained allowed the following conclusions: (1) The atopic group responded to histamine with greater wheal size than the nonatopic group. (2) Iontophoresis of diphyenhydramine effectively reduced the magnitude of the histamine wheal in both groups. (3) Isoproterenol decreased the wheal size in both groups. (4) Propranolol increased the wheal size in only the nonatopic group. (5) The successful modulation of the histamine-induced wheal and flare indicated that these drugs, regardless of their effect on the dermal mast cell, exert a measurable effect on the target organ (vasculature).


Further observations on the potentiation of the antibacterial effect of methenamine by acetohydroxamic acid. The use of methenamine in the treatment of urinary tract infections due to Proteus species is limited by urine alkalinity. Acetohydroxamic acid, an inhibitor of urease, maintains acidity despite growth of Proteus in urine. Easily achievable concentrations of acetohydroxamic acid in vitro systems that simulated the dynamics of the urinary tract potentiated the antibacterial effect of methenamine against Proteus species. The combined use of a urease inhibitor and methenamine may be effective in the treatment of urinary infection caused by these organisms.


Hematopoietic thymocyte precursors. I. Assay and kinetics of the appearance of progeny. A quantitative assay for the hematopoietic precursor of thymocytes has been developed. Using this assay the kinetics of appearance of the progeny of transfused bone marrow and spleen cells in the thymus of irradiated (760 R) mice has been studied. Precursor cells are seven to eightfold more common in bone marrow than in spleen and are absent from peripheral lymph nodes. They decline in number as the animals age. When hematopoietic cells are injected immediately after lethal irradiation only a small number of cells actually enter the gland. Their progeny are not detectable in the thymus for 8-12 days. The time of their detection depends both upon the size of the residual endogenous thymocyte population and the number of progenitor cells injected. Evidence has been presented that excludes thymic injury as the basis for the delay in the appearance of donor type cells and indicates that neither the production of a "homing" signal in the irradiated animal nor the development of precursor cells are limiting factors in the rate of thymic repopulation. These studies indicate that only an exceedingly small number (less than 100) of prothymocytes are required to repopulate the thymus of an irradiated mouse. This restricted number of progenitors must produce the entire repertory of T-cell immunologic responsiveness seen in the first weeks after repopulation.


Frog lysozyme. I. Its identification, occurrence as isozymes, and quantitative distribution in tissues of the leopard frog, Rana pipiens. In the course of examining the etiology of the Lucké renal adenocarcinoma of the frog, Rana pipiens, it was found that organs of the normal adult contain bacteriolytic enzymes. These enzymes all satisfied the six criteria for the identification of lysozymes and at least eight forms were separable by polyacrylamide gel electrophoresis. Their qualitative and quantitative distribution was organ-specific. All eight isozymes were found in normal kidney, while liver and spleen contained seven forms; skin, six; ovarian egg, five; and serum, two. In quantitative assays using a radial diffusion test, spleen had the greatest lysozyme concentration, followed in descending order by kidney, liver, skin, and ovary. Serum contained very low amounts. In terms of enzyme activity per animal, ovary was the highest ranking organ. As such a large number of lysozyme isozymes has not been reported in any other organism, their origins and functions are considered in the context of their presence in an ectotherm.


Kinetic and pharmacological properties of the sodium channel of frog skeletal muscle. Na channels of frog skeletal muscle are studied under voltage clamp and their properties compared with those of frog myelinated nerve. A standard mathematical model is fitted to the sodium currents measured in nerve and in muscle to obtain a quantitative description of the gating kinetics. At 5 degrees C the kinetics in frog nerve and skeletal muscle are similar except that activation proceeds five times faster in nerve. Block of Na channels by saxitoxin is measured in nerve and in muscle. The apparent dissociation constants for the inhibitory complex are about 1 nM and not significantly different in nerve and muscle. Block of Na channels by external protons in muscle is found to have an apparent pKalpha of 5.33 and a voltage dependence corresponding to action of 27% of the membrane potential drop. Both values are like those for nerve. Shift of the peak sodium permeability-membrane potential curve with changes of external pH and Ca++ are found to be the same in nerve and muscle. It is concluded that Na channels of nerve and muscle are nearly the same.


Cation transport in Escherichia coli. VIII. Potassium transport mutants. Analysis of K transport mutants indicates the existence of four separate K uptake systems in Escherichia coli K-12. A high affinity system called Kdp has a Km of 2 muM, and Vmax at 37 degrees C of 150 mumol/g min. This system is repressed by growth in high concentrations of K. Two constitutive systems, TrkA and TrkD, have Km's of 1.5 and 0.5 mM and Vmax's of 550 and 40 at 37 and 30 degrees C, respectively. Mutants lacking all three of these saturable systems take up K slowly by a process, called TrkF, whose rate of transport is linearly dependent on K concentration up to 105 mM. On the whole, each of these systems appears to function as an independent path for K uptake since the kinetics of uptake when two are present is the sum of each operating alone. This is not true for strains having both the TrkD and Kdp systems, where presence of the latter results in K uptake which saturates at a K concentration well below 0.1 mM. This result indicates some interaction between these systems so that uptake now has the affinity characteristic of the Kdp system. All transport systems are able to extrude Na during K uptake. The measurements of cell Na suggest that growing cells of E. coli have very low concentrations of Na, considerably lower than indicated by earlier studies.


Some properties of the pyruvate carboxylase from Pseudomonas fluorescens. The pyruvate carboxylase of Pseudonomas fluorescens was purified 160-fold  from cells grown on glucose at 20 degrees C. The activity of this purified enzyme was not affected by acetyl-coenzyme A or L-aspartate, but was strongly inhibited by ADP, which was competitive towards ATP. Pyruvate gave a broken double reciprocal plot, from which two apparent Km values could be determined, namely 0-08 and 0-21 mM, from the lower and the higher concentration ranges, respectively. The apparent Km for HCO3 at pH 6-9, in the presence of the manganese ATP ion (MnATP2-), was 3-1 mM. The enzyme reaction had an optimum pH value of 7-1 or 9-0 depending on the use of MnATP2- or MgATP2-, respectively, as substrate. Free Mg2+ was an activator at pH values below 9-0. The enzyme was strongly activated by monovalent cations; NH4+ and K+ were the better activators, with apparent Ka values of 0-7 and 1-6 mM, respectively. Partially purified enzymes from cells grown on glucose at 1 or 20 degrees C had the same properties, including the thermal stability. In both cases 50% of the enzyme activity was lost after pre-incubation for 10 min at 46 degrees C. The molecular weight was estimated to be about 300000 daltons by gel filtration on Sephadex G-200. The regulatory properties and molecular weight are thus similar to those determined for the pyruvate carboxylases from Pseudomonas citronellolis and Azotobacter vinelandii.


Cyanide intoxication in the rat: physiological and neuropathological aspects. Sodium cyanide was given to rats by intravenous infusion at a rate that would avert apnoea (the first sign of overdosage) in the majority. There was full physiological monitoring in a group under anaesthesia and more limited monitoring in an unanaesthetized group. White matter was damaged in six animals and grey matter additionally in only one. It was concluded that cyanide can damage neurones only through the medium of secondary effects on circulation and respiration.


Summer sequential graduate programs in nursing. In 1970 Texas Woman's University, College of Nursing instituted summer sequential programs in nursing whereby a student could study for a master's degree by attending the University for three summers. A different clinical focus was added each year from 1970 to 1973. Three sequences have been completed, i.e., clinical focuses of psychiatric-mental health nursing, medical-surgical nursing, and maternal-child nursing. Community health nursing is still in progress. Table II is a tabulation of the number and percentage of students who have entered, have withdrawn, are incomplete, or have graduated. Faculty and students think that the program is successful. Enrollment for the first summer of a three-summer sequence beginning the summer of 1974 was twenty-five students in medical-surgical nursing and fifteen students in psychiatric-mental health nursing.


The multiple assignment: an effective alternative for laboratory experiences. The purpose of the study was to test out the efficacy of both the Traditional and the Multiple Assignment approaches to the laboratory experiences of students of nursing. The subjects were 22 students enrolled in the first quarter of an Associate Degree Nursing Program and placed in either the multiple or the traditional laboratory assignment. Results showed the students in the peer experience to be superior in overall performance on tests of Nursing Knowledge, Concept Usage and Self-Esteem. It was concluded that the Multiple Assignment approach to laboratory experiences is a viable and useful method for the education of student nurses.


Stability of some pyridoxal phosphate-dependent enzymes in vitamin B-6 deficient rats. The effects of lowering the liver pyridoxal phosphate (PLP) concentration by vitamin B-6 deficiency on the stability of several rat liver enzymes were examined. Three PLP-dependent enzymes (serine dehydratase, ornithine-delta-aminotransferase, and tyrosine aminotransferase) and two non-PLP-dependent enzymes (glucose-6-phosphate dehydrogenase and phosphoenolpyruvate carboxykinase) were induced in vitamin B-6 deficient and control rats by feeding them high-protein diets or by injecting them with glucagon or dexamethasone. The decline of each activity was followed after withdrawal of the inducer. Serine dehydratase activity declined more rapidly in vitamin B-6 deficient than in control liver; however, ornithine aminotransferase and tyrosine aminotransferase activities were equally stable in deficient and control liver. Ornithine aminotransferase was predominantly in holoenzyme form in both control and deficient rats, whereas tyrosine aminotransferase was predominantly in apoenzyme form in both groups. The proportion of serine dehydratase in apoenzyme was less stable than the holoenzyme. Activity changes of glucose-6-phosphate dehydrogenase and phosphoenolpyruvate carboxykinase in control and vitamin B-6 deficient rats were similar. The results suggest that differences in the stability of PLP-dependent enzymes in vitamin B-6 deficient rats depend upon differences in the proportions of these enzymes existing as holo- and apoenzyme.


Adsorption sites of kaolin.

Adsorption sites of kaolin. The electrophoretic mobility and adsorptive properties of kaolin and kaolin pretreated with anionic and cationic materials were examined over a range of pH values. Pretreatment with anionic compounds had little effect on mobility, while adsorption of cationic species markedly reduced mobility. These results are explained by reference to the structure of kaolin, and the interdependence of adsorption properties and electrophoretic mobility are discussed.


Electrode sensitive to sulfa drugs.

Electrode sensitive to sulfa drugs. An electrode sensitive to sulfa drugs was constructed by using the iron(II)-bathophenanthroline chelate embedded in a liquid membrane. Rapid and Nernstian responses were exhibited against solutions of sulfamerazine and sulfisomidine ranging between 10(-3) and 10(-1) M in concentration. High selectivity was observed in the presence of urea, glycine, aminopyrine, or p-amino-benzoic acid. These chemicals are known to interfere in the usual colorimetric analysis of sulfa drugs.


Liquid-solid chromatographic determination of 6-demethylgriseofulvin in urine. A specific and quantitative liquid-solid chromatographic method for the determination of 6-demethylgriseofulvin in human urine is reported. The method consists of extraction into an organic solvent, addition of internal standard, and analysis by liquid-solid chromatography using a UV detector. Griseofulvin, if present, can be determined simultaneously. The sensitivity of the method is 6 mug/ml of urine. Total 6-demethylgrisefulvin is determined after hydrolysis of the glucuronide conjugate with glucuronidase-sulfatase enzyme solution. The method is well suited for the analysis of a large number of samples.


Mass fragmentography of morphine: relationship between brain levels and analgesic activity. Morphine levels in rat brain were measured by the multiple ion detection method (mass fragmentography), using a computer-controlled gas chromatograph-mass spectrometer, and were correlated with the analgesic activity of the narcotic at intervals up to 6 hours after the injection. Morphine levels in brain reached a peak of 346 ng/g of tissue wet weight 30 minutes after the subcutaneous injection of 10 mg/kg of morphine sulfate and then declined rapidly over the next 3 hours. Between 3 and 6 hours after the injection of morphine, the brain concentration decreased slightly but was still readily detectable at 6 hours after injection. An excellent correlation (r = 0.923) was found between the concentration of morphine in brain and analgesic activity, as measured by the hot plate method. The multiple ion detection method for the measurement of morphine appears to meet all of the criteria necessary for any drug assay: sensitivity, specificity and ease of analysis.


Vasoconstrictor actions of delta8- and delta9-tetrahydrocannabinol in the rat. Cardiovascular effects of delta8- and delta9-tetrahydrocannabinol (THC) were studied after systemic intravenous administration and intra-arterial administration into a perfused vascular bed in the urethane-anesthetized rat. Intravenous administration of delta8- and delta9-THC produced dose-related transient increases in blood pressure followed by more prolonged hypotensive responses and bradycardia. Intra-arterial administration of delta8- and delta9-THC into the perfused hindquarters of the rat produced an increase in perfusion pressure indicative of vasoconstriction. The vasoconstrictor response to the cannabinoids corresponded temporally to a similar response produced by i.a. norepinephrine and was in contrast to the more prolonged vasoconstrictor responses produced by vasopressin. Phentolamine, in a dose which reduced the vasoconstrictor effect of norepinephrine by 90%, significantly reduced the response to i.a. delta9-THC while having no effect on the actions of i.a. vasopressin. It was demonstrated that reserpine pretreatment significantly reduced vasoconstrictor actions of i.a. tyramine and delta9-THC but did not alter the responses to norepinephrine. These data suggest that delta8- and delta9-THC have peripheral vasoconstrictor activity in the rat which may be mediated, in part, through a tyramine-like action on adrenergic nerve terminals.


Possible involvement of a transmitter different from norepinephrine in the residual responses to nerve stimulation of the cat nictitating membrane after pretreatment with reserpine. Pretreatment with reserpine (0.3 or 3 mg/kg, 24 hours before the experiment) reduced the norepinephrine (NE) levels in the medial muscle of the cat nictitating membrane to approximately 2% of the control values. Under these experimental conditions, the responses to postganglionic nerve stimulation were not abolished, reaching up to 50% of the maximum development of tension to exogenous sympathomimetic amines both in vivo and in vitro. In contrast to the responses to nerve stimulation obtained in normal nictitating membranes, the residual responses to nerve stimulation obtained after pretreatment with reserpine were not blocked by phentolamine (3.1 and 31 muM) or by 0.29 muM phenoxybenzamine. The effectiveness of phentolamine and phenoxybenzamine in blocking responses to exogenous NE was the same when the normal nictitating membrane was compared to the smooth muscle obtained from cats pretreated with reserpine. The residual responses to nerve stimulation were reduced when the calcium concentration in the medium was decreased to 0.65 mM. These residual responses were abolished in the presence of tetrodotoxin. Scopolamine, 0.078 muM, did not reduce the residual responses to nerve stimulation while it antagonized the responses to exogenous acetylcholine, indicating that a cholinergic mechanism is not involved in this phenomenon. Adenosine triphosphate (ATP) and adenosine diphosphosphate (ADP) behaved as agonists on the smooth muscle of the normal and of the reserpine-pretreated nictitating membrane and the responses to ATP were not blocked by phentolamine. It is concluded that the residual responses to nerve stimulation obtained after pretreatment with reserpine could be due to the release of a transmitter different from NE. The possibility that ATP or ADP might be involved in these residual responses to nerve stimulation is discussed.


Pineal beta adrenergic receptor: correlation of binding of 3H-l-alprenolol with stimulation of adenylate cyclase. 3H-l-Alprenolol, a potent competitive beta adrenergic antagonist, binds to sites in rat pineal gland membranes. The properties of these binding sites were compared to those of the receptors which mediate the beta adrenergic activation of pineal adenylate cyclase. Both sites are highly stereospecific. The l-stereoisomers of alprenolol and propranolol were at least two orders of magnitude more potent than the d-stereoisomers in inhibiting isoproterenol-stimulated adenylate cyclase or 3H-l-alprenolol binding. The dissociation constants (Kd) of the l-stereoisomers of both alprenolol and propranolol were 10 to 22 nM as determined by competition for binding sites or by inhibition of isoproternol-stimulated adenylate cyclase. Beta adrenergic agonists which stimulated adenylate cyclase also competitively inhibited the binding of 3H-l-alprenolol. They showed the same order of potency (isoproterenol greater than norepinephrine greater than or equal to epinephrine) and the same individual affinities in the two systems. Alpha adrenergic blockers were ineffective in inhibiting either adenylate cyclase stimulation or 3H-l-alprenolol binding. Isoproternol stimulation of adenylate cyclase acrivity, and 3H-l-alprenolol binding, were rapid and rapidly reversible. The 3H-l-alprenolol binding sites were saturable and bound 0.6 pmol of ligand per mg of added protein. The data suggest that the binding of 3H-l-alprenolol occurs at sites indistinguishable from the pineal beta adrenergic receptor.


A pharmacological analysis of neurally induced inhibition of carotid body chemoreceptor activity in cats. Experiments were performed to determine the mechanism by which centrifugal impulses in the carotid sinus nerve (CSN) reduce the frequency of impulse traffic in afferent chemoreceptor fibers from the carotid body in cats. Recordings of chemoreceptor activity were made from single- or few-fiber preparations dissected off the CSN, while the remainder of the CSN was stimulated electrically to produce neurally induced inhibition of chemoreceptor activity. Various drugs were injected either intravenously or directly into the arterial blood supply to the carotid body. We found that catecholamines (dopamine, norepinephrine and epinephrine) inhibited spontaneous chemoreceptor activity, and that alpha adrenergic antagonists abolished both this inhibition and that produced by electrical stimulation of the CSN in the same preparation. Atropine, but not nicotinic antagonists of acetylcholine, consistently blocked neurally induced inhibition but not that produced by catecholamines. Muscarinic agonists had no effect on spontaneous chemoreceptor activity. We conclude that centrifugal activity in the CSN causes release of endogenous catecholamines in the carotid body, and that these catecholamines mediate neurally induced inhibition of chemoreceptor activity is due to the vasomotor effects of acetylcholine.


On the ability of narcotic antagonists to produce the narcotic cue. The ability of narcotic antagonists to produce the narcotic cue was investigated in rats trained to discriminate fentanyl (0.04 mg/kg) from solvent. The partial antagonists pentazocine, cyclazocine and nalorphine were found to possess narcotic cuing activity whereas naloxone lacked any such action at doses up to 160 mg/kg. The relationship between the present findings and the ability of these drugs to produce opiate-like subjective effects in humans is discussed. The conclusion was reached that the experimental procedure used may contribute significantly to the preclinical evaluation of drug abuse liability.


Effect of para-aminohippurate on renal glutamine metabolism in the rat. After para-aminohippurate (PAH) infusion into rats, urine pH decreased and urine ammonium excretion increased. Because augmented urine flow and decreased urine pH could not explain entirely the enhanced ammonium excretion, an increased ammonia production was postulated as a contributing influence. This was supported by the in vitro findings that PAH could increase slice ammoniagenesis from glutamine. The ability of PAH to stimulate ammoniagenesis in vitro was attributed to enhanced phosphate-dependent glutaminase activity. We conclude that PAH infusions at certain concentrations in vivo can alter ammonium excretion through increased renal ammonia production. The latter may be secondary to enhanced phosphate-dependent glutaminase activity.


Inhibition by sulfobromophthalein of mitochondrial translocation of anions and adenine nucleotides: effects upon liver adenosine triphosphate and possible correlation with inhibition of bile flow in the rat. Unconjugated sulfobromophthalein (BSP) inhibits state III respiration of rat liver mitochondria. It competitively inhibits the translocation into mitochondria of citrate, malate, phosphate and adenosine diphosphate, as studied by the inhibitor stop method. A double-beam spectrophotometric study strongly suggests that glutamate translocation is similarly inhibited. After perfusion of 65 mumol/hr/100 g for 90 minutes, bile flow is inhibited by 82% and liver adenosine triphosphate (ATP) falls by 60%. The amount of mitochondrial BSP can be computed form the amount of [35S] BSP still bound to mitochondria that are prepared at the end of such experiments; the amount of BSP lost during the isolation procedure is estimated from parallel experiments following binding of BSP in vitro. Comparison of the kinetic constants of mitochondrial transport and of their inhibition by BSP on the one hand and of liver concentration of substrates and BSP on the other gives rise to the conclusion that a strong inhibition of transports, mainly of phosphate, occurs in vivo and is responsible for the concomitant decrease in bile flow.


Dissociation constants and relative efficacies of agonists acting on alpha adrenergic receptors in rabbit aorta. The dissociation constants (KA values) of l-norepinephrine (l-NE) and seven other agonists acting on alpha adrenergic receptors in rabbit aorta strips were determined by analysis of concentration-response data before and after fractional inactivation of receptors with Dibenamine. In experiments to determine KA values, propranolol was added to block activation of beta receptors and cocaine to block the neuronal uptake mechanism. The KA of l-NE and the KA of a second agonist, when determined on paired strips from the same aorta, were used to calculate the relative affinity and the relative efficacy (er) of the second agonist as compared to l-NE. The validity of the method used for determining KA and er values was supported by the following findings. 1) The dissociation constant (KB) of the competitive antagonist, phentolamine, determined with each of the agonists, was the same as that determined with l-NE. 2) The KA determined for l-NE was independent of the fraction of active receptors remaining (q) after pretreatment with different concentrations of Dibenamine. 3) The KB of phentolamine determined with l-NE as the agonist was the same before and after fractional inactivation of receptors. 4) After inactivation in paired strips by equal exposures to Dibenamine, the q value determined with each agonist was the same as that determined with l-NE. The mean KA value for l-NE was 3.39 +/- 0.15 X 10(-7) M. The mean relative affinities of the agonists for the alpha receptor were: l-NE, 1;L-EPINEPHRINE, 1.25; L-PHENYLEPHRINE, 0.200; L-norphenylephrine, 0.217; epinine, 0.136; dopamine, 0.0055; l-alpha-methylnorepinephrine, 0.095; dl-alpha-ethylnorepinephrine, 0.0048. The mean er of each agonist was not significantly different from that of l-NE, except for l-norphenylephrine with an e of 0.71, and dl-alpha-ethylnorepinephrine with an er of 0.41. The results are discussed from the standpoint of structure-activity relationships.


The effect of carbon dioxide on the intracellular pH and buffering power of snail neurones. 1. Intracellular pH (pHi) was measured using pH-sensitive glass micro-electrodes. The effects on pHi of CO2 applied externally and HCO3-, H+ and NH4+ injected iontophoretically, were investigated. 2. The transport numbers for iontophoretic injection into aqueous micro-droples were found by potentiometric titration to be 0-3 for HCO3- and 0-94 for H+. 3. Exposure to Ringer, pH 7-5, equilibrated with 2-2% CO2 caused a rapid, but only transient, fall in pHi. Within 1 or 2 min pHi began to return exponentially to normal, with a time constant of about 5 min. 4. When external CO2 was removed, pHi rapidly increased, and then slowly returned to normal. The pHi changes with CO2 application or removal gave a calculated intracellular buffer value of about 30 m-equiv H+/pH unit per litre. 5. Injection of HCO3- caused a rise in pHi very similar to that seen on removal of external CO2. 6. The pHi responses to CO2 application, CO2 removal and HCO3- injection were slowed by the carbonic anhydrase inhibitor acetazolamide. 7. H+ injection caused a transient fall in pHi. In CO2 Ringer pHi fell less and recovered faster than in CO2-free Ringer. Calculation of the internal buffer value from the pHi responses to H+ and HCO3- injection gave very similar values. 8. The internal buffer value (measured by H+ injection) was greatly increased by exposure to CO2 Ringer. Acetazolamide reduced this effect of CO2, suggesting that the function of intracellular carbonic anhydrase may be to maximize the internal buffering power in CO2. 9. It was concluded that the internal HCO3- was determined primarily by the CO2 level and pHi, that internal HCO3- made a large contribution to the buffering power, and that after internal acidfication pHi was restored to normal by active transport of H+, OH- or HCO3- across the cell membrane. The active transport was much faster in CO2 than in CO2-free Ringer.


[Blood acid-base changes produced by variations of water oxygenation in the crab Carcinus maenas (author's transl)]. 10 Blood acid-base changes were studied at 17 degrees C in immersed crabs (Carcinus maenas) exposed to hypoxic and hyperoxic conditions, by measuring the pH and the CO2 partial pressure, PbCO2, and by calculating the bicarbonate concentration. 20 Hyperoxia first induces a marked respiratory acidosis with a rise of PbCO2. This acidosis is compensated thereafter by a non-ventilatory increase of the blood buffer base concentration. These results are discussed in relation to the general problems concerning the control of the blood acid-base balance in aquatic animals.


The diagnosis and treatment of acid-base deranged dogs infected with Babesia canis. A study was made of the acid-base status of Babesia canis infected dogs judged unlikely to recover after specific babesicidal drug therapy despite the use of blood transfusion and other conventional supportive measures. Such cases were invariably acidotic and responded well and often dramatically to supportive intravenous sodium bicarbonate administration. Elevated blood urea nitrogen, also responded gratifyingly to this procedure. The rationale is discussed in some detail.


Regulation of cell volume and ion concentrations in a Halobacterium. Changes in cell volume and ion content of a Halobacterium species are described in terms of the NaCl concentration (0.5--3.5M) and pH(4-8) of the suspending medium. Cell volume, per unit content of protein of bacteria in stationary phase cultures, rose as the [NaCl] of the growth medium was increased. Logarithmic-phase bacteria shrank as the pH fell from 7 to 5.5. These changes are characteristic of bacteria with a moderate or rapid rate of O2 consumption. Starving (i.e. nonmetabolizing) bacteria, on the other hand, did not change in size within the above ranges of [NaCl] and pH. At lower values, however, such bacteria swelled and eventually lysed. Effects of low pH on cell ions are compared in metabolizing and starving bacteria, and it is shown that changes in the state of the cell K are correlated with movements of cell Na. It appears that the cell K is used to maintain cell [Na] below the NaCl concentration of the medium. The results are explained in terms of a model involving interactions between polyelectrolytes, salts and water in the concentrated cytoplasm of these halophilic organisms.


Behavioral and psychodynamic dimensions of the new sex therapy. The new sexual therapy, a brief outpatient treatment of sexual dysfunction consisting of structured sexual exercises and conjoint therapeutic sessions, is a systematic intergration of behavioral and psychodynamic elements. Formally consisting of many defining characteristics of a behavior therapy including the direct treatment of a specified problem and the application of behavioral principles, the new sex therapy also relies on psychodynamic understanding. The result is synergistic. Psychodynamic understanding underlies appropriate behavioral intervention, and psychodynamic exploration of resistance enhances the effectiveness of behavioral methods. Behavioral techniques facilitate the most rapid implementation of therapeutic insight. The integration of approaches in the new sex therapy has general significance for psychotherapeutic theory and practice.


The use of microchemical techniques for the identification of new transmitter molecules in neurons. The author reviews available microtechniques and results on cellular analysis of single neurons emphasizing the identification of putative neurotransmitter molecules. Highly sensitive microquantitative methods for identification and assay of known and putative neurotransmitters have been developed within the last 15 years. Several of the methods reported in the article fulfill the requirements of both specificity and sensitivity for exploring and measuring the level of transmitter molecules in single neurons. Some of the available techniques, mostly those which associate GC or TLC with MS, have been able to unambiguously demonstrate the presence of putative neurotransmitter molecules, or at least of substances which are strongly suspected of fulfilling a functional role in the nervous system.


Studies of introital colonization in women with recurrent urinary infections. V. The inhibitory activity of normal vaginal fluid on Proteus mirabilis and Pseudomonas aeruginosa. Normal vaginal fluid from premenopausal volunteers was inoculated with 10 strains of Proteus mirabilis and 14 strains of Pseudomonas aeruginosa at pH's of 4.3, 4.6 and 4.9. All bacteria were killed at pH 4.3. Nine of 10 strains of Proteus mirabilis and 12 of 14 Pseudomonas aeruginosa were killed at pH 4.6. Only 4 of 14 strains of Pseudomonas aeruginosa were killed at pH 4.9, while 8 of 10 strains of Proteus mirabilis were killed at the same pH. We conclude that in comparison to the common 0 group strains of Escherichia coli, vaginal fluid is more bactericidal to Proteus mirabilis and Pseudomonas aeruginosa and that these observations may help explain the relative infrequency of bacteriuria owing to the organisms.


Successful autotransplantation of an intra-abdominal testis to the scrotum by microvascular technique. An intra-abdominal testis in a child with prune belly syndrome was successfully transplanted to the scrotum by a microvascular technique. Immediate results were good with no palpable atrophy of the testis. Long-term results (fertility) will not be known for many years. The technique of microvascular anastomosis and its application to orchiopexy are described.


Two rare cases of ectopic testis.

Two rare cases of ectopic testis. A case of pubopenile testis and a case of perineal ectopic testis are presented. The mechanism of descensus is largely positive, possibly facilitated by raised intra-abdominal pressure. The testis is usually guided by the gubernaculum and ectopia results from gubernacular failure. The ectopic testis is relatively rare but is easily recognized and treated by orchiopexy.


Familial persistent Müllerian duct syndrome. Two phenotypically normal pre-adolescent brothers with bilateral undescended testes were found to have bilateral fallopian tubes, a uterus and a vagina that drained into the prostatic utricle. We have documented this condition radiographically for the first time, gained some insight into the pattern of inheritance and made recommendations for surgical management.


Studies on the mode of antagonism between adrenergic beta-mimetics and beta-blocking agents (II). Analysis by the uptake saturation model. Curves of experimentally plotted log (dose ratio-1) vs.-log [B] for the antagonism between adrenergic beta-mimetics, isoproterenol (ISO) and trimetoquinol (TMQ), and various beta-antagonists in relaxation of guinea-pig trachea could not be reasonably fitted to Schild's equation which has been commonly used in the analysis of drug-antagonism. Taking into consideration the saturable uptake process of the drug used herein, the equation presented in this paper fitted fairly well to the experimental curves and explains the following results: 1, TMQ was more strongly antagonized than ISO by all the blocking agents tested, that is, the apparent modes of antagonism were different between ISO and TMQ although they are considered to interact with the same receptor site. 2, The slope of the curve for a given antagonist markedly differed between ISO and TMQ. It is hypothesized that ISO is more easily taken up than TMQ. This was experimentally confirmed: i.e., ISO was potentiated about 8 fold by inhibiting the uptake process with dibenamine while TMQ was not. By pretreatment with dibenamine, the log (dose ratio-1) vs.-log [B] curve for the ISO-propranolol antagonism was shifted upward and superimposed with the theoretical curve of antagonism in which uptake of the agonist was neglected.


pH of sweat of patients with cystic fibrosis. pH of the sweat from patients with cystic fibrosis and in controls was measured as a function of the sweat-rate using a fluorescence-pH-indicator (umbelliferone). In both populations sweat is acid at low sweat-rates and alkaline at high ones. The results do not favour an abnormality of the ductal H+-secretion as the pathomechanism of cystic fibrosis.


Morphologic and biochemical changes in autolysing dog heart muscle. The progressive changes in pH, lactate content, and light and electron microscopic appearances were studied in dog myocardium undergoing a 6-hour period of autolysis in vitro at 37 degrees C. and at room temperature. At 37 degrees C. there was a rapid cumulative fall in pH during the 1st hour after excision of the heart and a corresponding increase in lactate content, but little additional change in either subsequently. The nature and sequence of the morphologic alterations at this temperature were generally similar to those which occur in ischemic myocardium in vivo. At room temperature, a much slower cumulative decrease in pH and increase in lactate content took place throughout the whole period of investigation and was paralleled by a slower rate of development of morphologic change.


[Conditioning of water regenerated from moisture-containing wastes]. Conditioning of reclaimed water with minerals and trace elements by means of filters made of silver-plated natural minerals containing calcium, magnesium, fluoride and iodine ions has been investigated. On the basis of the investigations a complex filter made of silver plated minerals--dolomite and fluorite--has been developed. This filter allows simultaneous mineralization and decontamination of reclaimed water and preparation of biologically complete potable water.


[Problem of "biological compatibility" of crew members during prolonged space flight and possible directions for its solution]. The concept of biological compatibility of men can be defined as a phenomenon which includes an effect (that might be favorable or unfavorable) of one individuum on the other via trace amounts of chemicals released into their environment and micro-organisms. The paper presents the main results of studies of biological compatibility of crewmembers of space vehicles and outlines possible directions of research to be carried out in this area.


Proteins of the hepatoma tissue culture cell plasma membrane. The specificity of lactoperoxidase-catalyzed iodination for the proteins of the hepatoma tissue culture cell plasma membrane was examined by histochemical, biochemical, and cell fractionation techniques. Light microscope autoradiography of sectioned cells shows the incorporated label to be localized primarily at the periphery of the cell. Most of this label can be released from the cell by trypsin but not by collagenase or hyaluronidase. The label is recovered from the cells as either monoiodotyrosine or diiodotyrosine after hydrolysis of cell extracts with a mixture of proteolytic enzymes. The label co-purifies during cell fractionation with an authentic liver cell plasma membrane marker enzyme, 5'-nucleotidase. Thus, the incorporated iodide is itself a valid marker for those membrane polypeptides having tyrosine residues accessible to the lactoperoxidase. The polypeptide complexity of the purified plasma membrane was examined by high resolution dodecyl sulfate-polyacrylamide gel electrophoresis. At least 50 polypeptides in the membrane are accessible to iodination. These polypeptides probably represent the bulk of the protein mass of the membrane and iodinating them does not affect cell viability, growth rate, or cell function. Labeling experiments with fucose and glucosamine show that at least nine of the iodinated peptides may be glycoproteins.


Lesion-induced synaptogenesis in brain: a study of dynamic changes in neuronal membrane specializations. When incoming fibers to a given brain region are damaged and degenerate, the remaining undamaged fibers can, in some cases, form new synapses, and restore physiologically functional circuitry. Synaptic membrane events underlie this reconstruction: the connection between membranes is broken and reformed.


Preliminary characterization of the acetylcholine receptor in human erythrocytes. The response of human erythrocytes to cholinergic ligands was studied with an electron spin resonance assay. The membrane response to carbamyl choline was found to be antagonized by atropine and, in the absence of calcium, by tetrodotoxin. Experiments with resealed ghosts showed that the membrane response to carbamyl choline required ATP and calcium. Reductive alkylation of intact cells eliminated the cholinergic response, but the presence of saturating amounts of carbamyl choline protected the putative receptor against inactivation. Affinity labeling was used to demonstrate an apparent molecular weight of 41,000 for the carbamyl choline-binding species. A lipid vesicle extraction technique was used to induce a specific cation permeability defect in intact cells. Preliminary investigation of this phenomenon is described.


Effect of alcohol on elicited male sexual response. Sixteen young men drunk three doses of alcohol and their sexual arousal was measured by the changes in penile diameter. The lowest alcohol dose (BAC of 0.025%) was associated with maximum penile diameter increase; marked suppression of response was found at BACs of 0.05% and above.


"Feeling no pain" differential responses to pain by alcoholics and nonalcoholics before and after drinking. Whisky reduced the level of pain reported by alcoholics but had no effect on that reported by nonalcoholics. The results appear to be based on the joint effects of the alcoholic's expectation that alcohol has an analgesic effect and the physiological cues accompanying alcohol consumption.


Decrease of iconic memory after alcohol. Alcohol did not alter the rate of information loss from iconic memory. However, there was a dose-related decrease in the total amount of information reported which was independent of the rate of information loss.


The relationship between alcohol dosage and performance decrement in humans. The effects of alcohol on human perceptual, cognitive and motor performance was assessed in a battery of tests, and the dose-response relationships for alcohol, important for the study of drug-alcohol interactions, established.


Personality impairment in alcoholism. Its relation to regional cerebral blood flow and psychometric performance. The psychiatric symptoms constituting three main syndromes of personality impairment ("defense," "low vitality" and "emotional deficiency") were homogeneous in alcoholic patients. Only the "low vitality" syndrome was associated with impaired psychometric performance and reduced cerebral blood flow.


Group assertiveness training for alcoholics. Six men alcoholics showed increased assertiveness and improvement in social and occupational status after attending a group assertiveness training program.


Viability of cells in ethanol. Role of alcohol dehydrogenase. Rodent cells were found to contain a high level of alcohol dehydrogenase activity which was not inducible. Other hepatoma and nonhepatoma cell lines were tested and found to contain lower but measurable levels of alcohol dehydrogenase.


Alcohol-induced conditioned taste aversion in rats. Effect of concentration and prior exposure to alcohol. The effects of prior opportunities to drink alcohol solutions on the subsequent production of conditioned taste aversion by the oral ingestion of alcohol were evaluated. The consumption of 5 and 7% alcohol solutions produced conditioned aversion; the consumption of 3% alcohol solution did not result in aversion. Prior exposure to alcohol did not alter the extent of the aversion.


Plasma immunoreactive insulin and somatotropin in delirium tremens and alcoholic hallucinosis. The glucose tolerance curve in alcoholics in delirium tremens was similar to that seen in hepatogenic diabetes. The secretion of immunoreactive insulin and somatotropin after glucose was similar in patients with delirium tremens and alcoholic hallucinosis.


Alcohol toxicity, blood alcohol concentration and body water in young and adult rats. Young rats, with more body water, had lower blood alcohol concentrations than did older ones at several times after various doses. The young also recovered sooner from intoxication. The age difference in recovery increased with increasing dose.


Estimated U.S. alcoholic beverage consumption, 1790-1860. From 1790 to 1830 American consumption of alcoholic beverages generally rose, with an increased use of spirits, a high but declining consumption of hard cider, and a negligible intake of wine and beer. Intake of absolute alcohol peaked in 1830, at a rate twice that estimated for 1970. From 1830 to 1860 consumption fell sharply, but since 1860 consumption has fluctuated much less.


Philosophies for educating about alcohol and other mood-modifying substance. Personal or social controls. Groups of students, teachers, adult advisers to youth, civil-service supervisors and occupational-program consultants were in basic agreement on philosophies for educating about alcohol, marihuana, lysergide (LSD) and heroin.


Drinking during pregnancy.

Drinking during pregnancy. Pregnant women reported a decrease in the use of alcoholic beverages during pregnancy, often citing adverse physiological effects as a reason for the decline.


The APUD cell concept.

The APUD cell concept. Embryonic neural crest cells have been traced to the primitive entoderm where they differentiate into a family of hormone-producing cells, APUD cells. The APUD cell concept explains many otherwise seemingly dissociated clinical circumstances involving endocrine glands and hormone production by tumors.


Effect of narcotic analgesics on the striatal homovanillic acid content in mice; relation to antinociceptive effect. The effects of various narcotic analgesics on striatal homovanillic acid (HVA) content, hot plate time and rectal temperature in mice were compared in relation to dose and time. The hypothermia induced by narcotic analgesics did not correlate with the striat"al HVA increase. Pentazocine, cyclazocine and thebaine had no effect on the hot plate time. The maximum prolongation of hot plate time induced by morphine, methadone or piminodine occurred before the highest HVA increase. The highest increase induced by narcotic analgesics in striatal HVA content was twice the original concentration. This occurred 2 hr after 40 mg/kg of morphine; 2 hr after 20 mg/kg of methadone; 1/2 hr after 20 mg/kg of piminodine; and 1 hr after 60 mg/kg of pentazocine. Cyclacozine (10 and 20 mg/kg) and thebaine (10 mg/kg) did not alter the HVA content. With the exception of pentazocine, those doses of narcotic analgesics that caused equal increases in striatal HVA content were also equianalgesic. These results suggest that there are similarities in the structural requirements for antinociceptive and striatal HVA-increasing effects of narcotic analgesics. The neuroleptic compound haloperidol (0.5 mg/kg) caused a fourfold increase in striatal HVA content making it twice as efficient as narcotic analgesics. This finding suggests that narcotic analgesics do not act on the same sites as neuroleptics when causing an increase in striatal HVA content.


The role of new health practitioners in a prepaid group practice: provider differences in process and outcomes of medical care. Practice patterns and patient-reported outcomes of care are compared in detail for ten physicians and 12 new health practitioners delivering ambulatory care in two departments of a prepaid group practice, the Columbia Medical Plan (CMP). All providers completed questionnaires for a 50 per cent random sample of patients seen during a two-week period. Patients completed questionnaires prior to receiving care and were interviewed one week and one month after their clinic visits. New health practitioners deliver approximately 75 per cent of well-person care, 56 per cent of problem-oriented care in adult medicine, and 29 per cent of problem care in pediatrics. They have become increasingly involved over time in the treatment of acute conditions and injuries while physicians have retained their predominant role in treating patients with chronic conditions. Thirty-two per cent of visits with new healh providers involved a physician in one or more of the following: decision-making, direct supervision, consultation, or seeing the patient as a second provider of care. Degree of autonomy varied by type of task performed, category of problem treated, and specialty. The following outcomes of care were examined by type of provider: patient-reported change in problem status,including frequency and intensity of pain or discomfort, level of anxiety, and degree of activity limitation; the degree to which physician-specified criteria for the most commonly occurring conditions were met with respect to change in problem status; and patient satisfaction with a number of dimensions of the clinic visit. The analysis suggests that the new health practitioners at the CMP are providing care, within their areas of responsibility, of comparable quality to that delivered by physicians.