Regulation of steady state level of phosphoenzyme and ATP synthesis in sarcoplasmic reticulum vesicles during reversal of the Ca2+ pump. The role of the Ca2+ concentration gradient in ATP synthesis and membrane phosphorylation by Pi was investigated in sarcoplasmic reticulum vesicles isolated from rabbit skeletal muscle. The Pi concentration required to attain 50% of the maximal membrane phosphorylation varies significantly in the pH range of 5.5 to 4.5, the optimal being at pH 6.0. In the pH range of 6.0 to 7.0, this concentration of Pi was 4- to 10-fold higher in empty vesicles than in vesicles loaded with calcium phosphate, i.e. having transmembrane Ca2+ concentration gradient. ATP, ADP, and Ca2+ inhibit the membrane phosphorylation by Pi, the inhibition being greater at pH 7.0 than at pH 6.0. The pH profile for ATP synthesis shows a higher optimum than for membrane phosphorylation. The optimum pH for synthesis, but not for phosphorylation depends on whether the vesicles were previously loaded with calcium phosphate or with calcium oxalate. Addition of Ca2+ to the assay medium inhibits the extent of membrane phosphorylation and the rate of ATP synthesis to different extents. Evidence is presented that the rate of membrane phosphorylation by Pi is higher than the rate by which the phosphoprotein transfers its pohsphate to ADP for the ATP synthesis.


Acetyl coenzyme A carbosylase. Circular dichroism studies of Escherichia coli biotin carboxyl carrier protein. The biotin carboxyl carrier protein (BCCP) component of Escherichia coli acetyl coenzyme A carboxylase and three peptides derived from BCCP by proteolytic digestion have been examined by circular dichroism spectroscopy. BCCP, which has a peptide molecular weight of 22,500, has a spectrum typical of globular proteins with negative extrema at 222 nm and 208 nm. The two smallest peptides, BCCP(SC) and BCCP(9,100), with molecular weights of 8,900 and 9,100, respectively, exhibit unusual positive CD bands centered at 237 nm and 220 nm. BCCP(10,400), with a molecular weight of 10,400, has a CD spectrum intermediate between BCCP and that of the smallest peptides. Since d-biotin exhibits a positive CD band at 233 nm, it was suspected that the biotin prosthetic group might be the chromophore responsible for the 237 nm CD band seen in BCCP(SC) and BCCP(9,100). Enzymatic carboxylation of BCCP(SC) to form CO2-BCCP(SC) caused the CD spectrum to change with a shift of the 237 nm band to 232 nm. The positive CD band at 220 nm was unaffected by carboxylation of the biotin prosthetic group. These date suggest that the 237 nm signal may be due either to the biotin which acts as a chromophore directly or to a chromophore that is perturbed by the carboxylation of biotin. A spectropolarimetric titration was carried out to investigate the possible contribution of the single tyrosine residue of BCCP(SC) to the CD spectrum of this peptide. At pH values over 9 the CD spetrum changed with the disappearance of the 237 nm band, suggesting that tyrosine might contribute to this CD band. Denaturation of BCCP(SC) or BCCP(9,100) with 8 M urea of 6 M guanidine HCl abolished the positive CD bands and resulted in spectra typical of a random coil, whereas treatment of BCCP(SC) with 1% sodium dodecyl sulfate abolished the positive bands and left a spectrum exhibiting a shoulder at 222 nm and a negative band at 205 nm, suggestive of a high degree of ordered structure. It is concluded that the CD band at 237 nm in BCCP(SC) and BCCP(9,100) is prabably due to a noncovalent interaction of biotin with an amino acid residue(s) of the protein. It is suggested that the biotin prosthetic group is partially buried in the surface of the protein, rather than swinging free at the end of the lysine side chain through which it is covalently linked to the protein, to permit this interaction to occur.


Pulse radiolytic investigation of single heme group reduction in hum an methemoglobin. Reduction of one of the four heme groups of human aquomethemoglobin A has been investigated by the pulse radiolysis method. The reactivity of e-a-q, the hydrated electron, with methemoglobin was determined by observing this species directly. The separate reactions of the hydroxy yl radical and hydrogen atom, as well as of e-a-q, were studied by observing absorbance changes in the protein spectrum over the wavelength range 290 to 600nm, with appropriate scavengers in solution...


Enzymatic attack on side chains of synthetic polymers. Chymotrypsin-catalyzed hydrolysis of specific substrate groups attached to acrylamide or acrylic acid co-polymers. Three vinyl monomers, M-1, M-3, and M-5, in which L-phenylalanine p-nitroanilide was acylated with CH2==CHCONH(CH2)nCO--(n = 1, 3, 5) were synthesized. They were co-polymerized with a large excess of acrylamide (co-polymers PAm-1, PAm-3, and PAm-5) and with a large excess of acrylic acid (co-polymers PAc=1, PAc-3, and PCc-5). In addition, M-5 was co-polymerized with acrylamide containing 2.8 mol % of the hydrophobic monomer N-acrylyl-1-naphthylamine (co-polymer PAm-5N). The rates of the chymotrypsin-catalyzed hydrolysis of the nitroanilide groups of M-5 and the various co-polymers were determined over a range of pH. For some of the systems data were also obtained over a range of substrate concentrations to derive values for Vmax and Km. Results obtained with PAm-5 were found to be independent of the chain length of the co-polymer. At pH 7, 25 degrees and with 2.7 X 10(-6) M enzyme, Vmax values for M-5, PAm-k, PAm-5N, and PAc-5 were 5.5, 5.5, 10, and 3.6 X 10(-8) M/S, while Km values were 8.5, 16.5, 10, and 2.2 X 10(-5),respectively, With PAc-5, the pH activity profile was shifted to higher acidities as compared to the profiles obtained with M-5 and PAm-5. The susceptibility of the co-polymers to chymotrypsin attack decreases sharply with a decreasing spacing of the L-phenylalanine p-nitroanilide residue from the backbone of the polymer chains.


Purification and properties of a highly potent antitumor glutaminase-asparaginase from Pseudomonas 7Z. Crystalline glutaminase-asparaginase which is effective against solid as well as ascites tumors was prepared from soil isolate organism Pseudomonas 7A. This enzyme has a ration of Vmax for L-glutamine and L-asparagine of 2.0. The presence of glutamic acid in the growth medium is essential for optimal enzyme production and glucose inhibits the production of glutaminase-asparaginase. The purification procedure provides an overall yield of 40 to 45% from crude cell extract to homogeneous glutaminase-asparaginase and is adaptable to large scale production of the enzyme. The specific activity of homogeneous enzyme is 160 +/- 15 i.u./mg of protein and the E1% 280 is 9.8. No disulfide or sulfhydryl groups appear to be present on the enzyme. The isoelectric point of glutaminase-asparaginase by isoelectric focusing on ampholine polyacrylamide gel plates is 5.8. The Km values for L-glutamine and L-asparagine are 4.6 and 4.4 X 10(-6) M, respectively. The enzyme catalyzes the hydrolysis of the D isomers of glutamine and asparagine at 87 and 69% the rate of the respective L isomers. L-Glutamic acid gamma-monohydroxamate is hydrolyzed at approximately the same rate as L-glutamine. The enzyme is not inhibited by ethylenediaminetetraacetate (0.1 mM), L-glutamate (30 mM), or L-aspartate (30 mM). Ammonium sulfate (10 mM) inhibits the enzymatic activity. The plasma half-life of Pseudomonas 7A glutaminase-asparaginase if 13 hours in normal mice and 43 hours in mice infected with the lactate dehydrogenase-elevating virus.


Regulation of secretion from the adrenal medulla. Evidence for adenylate cyclase activity in secretory vesicle membranes. Adenylate cyclase activity has been found in purified secretory vesicle membranes from the adrenal medulla. Activity was detected both by formation of radioactive cAMP from [alpha-32P]ATP and by the competitive protein binding assay for cAMP. Activity was highest at pH 8.0 to 8.5, and was stimulated by sodium fluoride and GppNHp, a GTP analogue known to stimulate adenylate cyclase activity in plasma membrane preparations. The reaction rate was strongly dependent on the molar ratio of Mg2+:ATP in the system. This is the first demonstration of adenylate cyclase in a secretory vesicle membrane.


In vivo inhibition of superoxide dismutase in mice by diethyldithiocarbamate. Superoxide dismutase was assayed by a method which takes advantage of the inhibitory action of superoxide dismutase (or tissues which contain superoxide dismutase) on the rate of autooxidation of 6-hydroxydopamine. Incubation of pure superoxide dismutase of homogenates of brain or liver with 10(-3) M diethyldithiocarbamate for 1.5 hours resulted in total loss of superoxide dismutase activity. Inhibition of superoxide dismutase was not reversed by dialysis, but after dialysis, enzymatic activity was restored with CuSO4. When 1.5 g of diethyldithiocarbamate/kg were injected into mice, the superoxide dismutase activity at 3 hours was decreased by 86%, 71%, and 48%, respectively, in whole blood, liver, and brain. A dose of 0.5 g of diethyldithiocarbamate/kg lowered the superoxide dismutase activity by 42% in liver at 3 hours. A study of the time course for inhibiton of superoxide dismutase in liver after 1.5 g of diethyldithiocarbamate/kg, showed a maximum decrease (81%) within 1 hour, with a slow return to 64% of normal by 24 hours. Inhibition of superoxide dismutase in vivo and in vitro was confirmed with other assay systems based on the autooxidation of pyrogallol or epinephrine or on reduction of cytochrome c or intro blue tetrazolium. Treatment of animals with diethyldithiocarbamate may provide a useful experimental model to study the role of superoxide dismutase in various tissues.


Internal pH of isolated chromaffin vesicles. The passive permeability of isolated chromaffin vesicles to H+ and the internal pH of the vesicles under various conditions were measured. Potentiometric measurements of K+ and H+ fluxes in the presence of selected ionophores and uncouplers indicated that the membrane is highly impermeable to both protons and potassium. deltapH across the chromaffin granule membrane was measured by [14C]methylamine distribution. At pH 6.85, The deltapH WAS 1.16 WITH THE INTRAVESICULAR SPACE BEING FOUND ACIDIC. Varying the external pH produced an equivalent change in the deltapH, WITH EXTRAPOLATION TO ZERO DEltapH yielding a value of pH  5.5, WHICH IS TAKEN AS AN INDICATION OF THE PH of the intravesicular space. The pH gradient could be enhanced or collapsed by the addition of ionophores and uncouplers under varying ionic conditions. deltapH was constant for granules suspended in various ionic media, suggesting that the deltapH did not arise secondarily due to the establishment of a Donnan equilibrium. The significance of the proton impermeability and deltapH is discussed in terms of regulation of the uptake and storage of catecholamines in bovine chromaffin granules.


Triglyceride, diglyceride, monoglyceride, and cholesterol ester hydrolases in chicken adipose tissue activated by adenosine 3':5'-Monophosphate-dependent protein kinase. Chromatographic resolution and immunochemical differentiation from lipoprotein lipase. Hormone-sensitive lipase and cholesterol ester hydrolase of chicken adipose tissue were markedly activated by adenosine 3':5'-monophosphate (cAMP)-dependent protein kinase (on the average, 235 to 275%; occasionally as much as 1000%). Diglyceride and monoglyceride hydrolases were also activated, but to a lesser extent (60 to 87%). The activation of all four hydrolases was inhibited by protein kinase inhibitor and reversed by the addition of exogenous protein kinase. Following activation by cAMP-dependent protein kinase, all four hydrolases were deactivated in a Mg2+-dependent reaction and then reactivated to or near initial levels on incubation with cAMP and Mg2+-ATP. The reversible deactivation is assumed to reflect activity of one or more protein phosphatases. The maximum activation obtainable for the four hydrolases decreased when the tissue had been previously exposed to glucagon, indicating that the glucagon-induced activation was probably similar to or identical with the activation demonstrated in cell-free preparations. The pH optima for the four hydrolase activities were similar (7.13 to 7.38). Although the absolute activities and relative degrees of kinase activation differed according to the particular emulsified substrates used, the results do not rule out the possibility that all four hydrolase activities are referable to a single hormone-sensitive hydrolase. Hormone-sensitive acyl hydrolases were separated from lipoprotein lipase by heparin-Sepharose affinity chromatography. Lipoprotein lipase was active against triolein, diolein, and monoolein, but not cholesterol oleate. Incubation of lipoprotein lipase with exogenous protein kinase, cAMP, and Mg2+ATP had no effect on any of the three hydrolase activities. Lipoprotein lipase was further purified to homogeneity and used to prepare antiserum in rabbits. The immunoglobin G fraction from these antisera completely inhibited lipoprotein lipase eluted from heparin-Sepharose columns. However, the hormone-sensitive hydrolase activities (not retained on heparin-Sepharose affinity chromatography) were not inhibited by anti-lipoprotein lipase immunoglobin G, and anti-lopoprotein lipase immunoglobin G did not affect the activation process in crude fractions. Thus, hormone-sensitive lipase and lipoprotein lipase, functionally distinct enzymes, have been physically resolved and immunochemically distinguished. Apparently lipoprotein lipase activity is not regulated, at least directly, by cAMP-dependent protein kinase.


Binding studies of polypeptide hormones to bovine neurophysins. Experimental binding isotherms of [9-glycinamide-1-(14)C]oxytocin and [9-glycinamide-1-(14)C]arginine vasopressin to purified neurophysins I and II at pH = 4.4, 5.4, 6.5, 7.4, and 8.5 and 6 degrees, 22 degrees, and 37 degrees in aqueous buffers are reported. For purposes of comparison, binding isotherms for [4-glycine-1-(14)C]oxytocin to neurophysin II and I in aqueous buffer, and [9-glycinamide-1-(14)C]oxytocin to neurophysin II in dimethylsulfoxide under selected conditions are also reported. A brief discussion of the interpretation of binding isotherms is entered into and apparent binding constants are derived. The results indicate that the interpretations presented in the literature up to now are much too simple. There are, in contrast, multiple binding sites of oxytocin and vasopressin to the neurophysins and large temperature dependences of the number of sites and their binding constants. We find, in fact, that at 37 degrees the binding of neurohypophysial hormones to the supposed storage proteins is rather weak even at the pH of maximum binding.


Inhibition of bovine hepatic fructose-1,6-diphosphatase by substrate analogs. Purified bovine hepatic fructose-1,6-diphosphatase, which exhibits maximal activity at neutral pH, is competitively inhibited by several analogs of its substrate, fructose 1,6-diphosphate. These include glucose 1,6-diphosphate (Ki = 9.4 X 10(-5) M), hexitol 1,6-diphosphate (Ki = 2.3 X 10(-4) M), and 2,5-anhydro-D-mannitol 1,6-diphosphate (Ki = 3.3 X 10(-8) M), and 2,5-anhydro-D-glucitol 1,6-diphosphate (Ki = 5.5 X 10(-7) M). The Ki values for both 2,5-anhydro-D-mannitol 1,6-diphosphate and 2,5-anhydro-D-glucitol 1,6-diphosphate are lower than the Km of 1.4 X 10(-6) M for fructose 1,6-diphosphate. Since 2,5-anhydro-D-mannitol 1,6-diphosphate is an analog of the beta anomer of fructose 1,6-diphosphate and 2,5-anhydro-D-glucitol 1,6-diphosphate is an analog of the alpha anomer, the lower Ki for the mannitol analog may indicate that the beta anomer of fructose 1,6-diphosphate, which predominates in solution, is the true substrate. The substrate analog 1,5-pentanediol diphosphate inhibits slightly (K0.5 = 5 X 10(-3) M), but 1,4-cyclohexyldiol diphosphate does not. The Ki for product inhibition by sodium phosphate is 9.4 X 10(-3) M. 2,5-Anhydro-D-mannitol 1,6-diphosphate and alpha-D-glucose 1,6-diphosphate are substrates at pH 9.0, but not at pH 6.5.


Genetic variants of human erythrocyte glucose-6-phosphate dehydrogenase. Kinetic and thermodynamic parameters of variants A, B, and A- in relation to quaternary structure. The values of Vmax and Km for the three genetic variants A, B, and A- of erythrocyte glucose-6-phosphate dehydrogenase have been determined at 10 different pH values in the range from 5.5 to 9.5, and at four different temperatures in the range from 18.5-40.0 degrees. The log Vmax versus pH curve for each of the enzymes shows a monotonic increase between pH 5.5 and 7, and a plateau from pH 7.5 upwards. These curves, and their temperature dependence, are compatible with the presence of a single ionizable group which, in its conjugate acid form, renders the enzyme-substrate complex inactive. The pK of this group is 6.94 at 18.5 degrees, and its enthalpy of ionization is 7.0 kcal mol-1. The log Km versus pH curves show a broad plateau between pH 6.2 and 8.2, interrupted by a sharp minimum at pH 7.2 for variant B, while variants A and A- show sharp maxima at pH 7.2 and 7.45, respectively. It is proposed that this unusual behavior depends on the dissociation of the tetrameric enzyme to dimers in this pH region. Specifically, it is shown that  a sharp maximum or minimum of Km can arise if cooperative uptake or release of protons is linked to dimer formation, and if the degree of cooperativity is different for the free enzyme compared to the enzyme-substrate complex. The pH dependence of the equilibrium between the tetrameric and the dimeric form of the enzyme has been determined by gel filtration for the same three genetic variants B, A, and A-. In agreement with previous ultracentrifugal data, the enzyme is a tetramer in acid solution and a dimer in alkaline solution. The pH at which half of the enzyme is in dimeric form, under our experimental conditions, is 7.15 +/- 0.05 for variants A and B, and 7.35 +/- 0.05 for variant A-. These pH values correspond closely, for all three variants, to the sharp extrema in the pH dependence of their Km values for glucose 6-phosphate. From the measured dissociation equilibria, it can be inferred that the tetramer-dimer transition entails cooperative release of protons. The degree of cooperativity estimated from these data agrees closely with the independent estimate based on the pH dependence of Km.


Studies on tyrosine residues in porcine muscle adenylate kinase. Circular dichroism spectra and chemical modification with tetranitromethane. Substrate-induced conformational change of porcine muscle adenylate kinase (EC 2.7.4.3) is evidenced by a change in circular dichroism spectra in the near ultraviolet. In the absence of tryptophan in porcine muscle adenylate kinase, the spectral change may be assigned to a perturbation of tyrosine chromophore(s). The spectral change was specific for adenine nucleotide binding and was greater with ATP than with AMP. In the x-ray model, Tyr153 and Tyr154 are located at a hinge region of two domains which form a deep active site cleft and are therefore susceptible to conformational change on substrate binding. Adenylate kinase was treated with equimolar tetranitromethane. The yellow-colored product, separated from unmodified enzyme by substrate gradient elution on a phosphocellulose column, had about 1 mol of nitrotyrosine per mol of the enzyme by amino acid analysis and showed a slightly higher Km value than native enzyme for ADP (Km = 0.50 mM compared with 0.25 mM for native adenylate kinase). Spectrophotometric titration of nitroadenylate kinase gave pKa 8.4 for the dissociation constant of the nitrotyrosyl hydroxyl group. On binding ATP the pKa value increased to 9.0 while AMP binding caused very little change. By peptide mapping of the carboxypeptidase digestion product, 0.70 mol of nitro group per mol of adenylate kinase was detected on Tyr153 and a small amount of nitro group was also found on Tyr95. From these results it is proposed that Tyr153 is directly or indirectly involved in the binding of ATP.


Antifreeze glycoproteins from Antarctic fish. Inactivation by borate. Antifreeze glycoprotein, which has previously been shown to be inactive in the presence of borate, migrates electrophoretically as the borate complex, presumably through formation of borate complexes with hydroxyl groups on the sugar side chains. Antifreeze glycoprotein (5 mg/ml) has been found to be completely active in the presence of 0.1 M borate at pH 7, but inactive at pH 9. A titration curve of pH versus the antifreeze activity of glycoprotein (5 mg/ml) in 0.1 M borate showed a progressive decrease in antifreeze activity as the pH was increased. Concomitant with decreases in activity were increases in binding of borate. At pH 9.0, nearly 2 mol of borate were complexed per glycotripeptide. Ultracentrifuge analyses showed similar molecular weights and laser quasi-elastic light scattering showed similar diffusions at pH 7.0 and 9.0 in borate and in the absence of borate. The binding of borate, rather than a change in conformation, is thus directly related to the loss of antifreeze activity. Alkaline borate also decreased hemagglutinating activity of Osage orange lectin and decreased the inhibition of the activity by the antifreeze glycoproteins.


Binding of (3H)prostaglandin E1 to putative receptors linked to adenylate cyclase of cultured cell clones. A method for assessing the binding of 3H-labeled prostaglandin E1 ([3H]PGE1) to cell membranes has been developed and used to study the interaction of [3H]PGE1 with membranes from cultured mammalian cells. Receptor sites were identified by correlation of the potency of a series of compounds to compete for [3H]PGE1 binding sites and to stimulate adenylate cyclase activity, by correlation of rates of binding and change in enzyme activity, and by the correspondence of [3H]PGE1-binding activity with the presence or absence of PGE1-sensitive adenylate cyclase in several clones. In clone B82, a murine L-cell, [3H]PGE1 binds with an activation energy of 14 kcal/mol to a class of sites with an affinity of 0.5 X 10(8) M-1 and a capacity of 150 fmol/mg of protein. Concentration dependence of adenylate cyclase activation by PGE1 (KD =30 nM) and kinetic analysis of [3H]PGE1 binding (k1 = 4 X 10(6) liters/mol/min, k-1 0.15/min) verify this affinity. Concentration dependence and specificity of binding and activation of adenylate cyclases in neuroblastoma clone N4TG1 and N18TG2 substantiate the method. In several clones that lack PGE1-responsive adenylate cyclase, no specific [3H]PGE1 binding is detectable.


The mechanism of superoxide anion generation by the interaction of phenylhydrazine with hemoglobin. The mechanism by which superoxide anion is generated by the interaction of phenylhydrazine with either oxy- or methemoglobin was investigated. Rather than superoxide anion generation resulting from an accelerated autooxidation of oxyhemoglobin, it was found that both oxy- and methemoglobin function as peroxidases toward phenylhydrazine with the resultant oxidation of this compound to phenyldiazine. Generation of phenyldiazine from the oxidation of phenylhydrazine by hemoglobin or by the hydrolysis and subsequent decarboxylation of methyl phenylazoformate (C6H5N=NCOOCH3) resulted in the production of superoxide anion. It is suggested that under certain conditions hemoglobin may function as a drug-metabolizing peroxidase.


Affinity labeling of a previously undetected essential lysyl residue in class I fructose bisphosphate aldolase. The affinity label N-bromoacetylethanolamine phosphate (BrAcNHEtOP) has been used previously at pH 6.5 to identify His-359 of rabbit muscle aldolase as an active site residue. We now find that the specificity of the reagent is pH-dependent. At pH 8.5, alkylation with 14C-labeled BrAcNHEtOP abolishes both fructose-1,6-P2 cleavage activity and transaldolase activity. The stoichiometry of incorporation, the kinetics of inactivation, and the protection against inactivation afforded by a competitive inhibitor or dihydroxyacetone phosphate are consistent with the involvement of an active site residue. A comparison of 14C profiles obtained from chromatography on the amino acid analyzer of acid hydrolysates of inactivated and protected samples reveals that inactivation results from the alkylation of lysyl residues. The major peptide in tryptic digests of the inactivated enzyme has been isolated. Based on its amino acid composition and the known sequence of aldolase, Lys-146 is the residue preferentially alkylated by the reagent. Aldolase modified at His-359 is still subject to alkylation of lysine; thus Lys-146 and His-359 are not mutually exclusive sites. However, aldolase modified at Lys-146 is not subject to alkylation of histidine. One explanation of these observations is that modification of Lys-146 abolishes the binding capacity of aldolase for substrates and substrate analogs (BrAcNHEtOP), whereas modification of his-359 does not. Consistent with this explanation is the ability of aldolase modified at His-359 to form a Schiff base with substrate and the inability of aldolase modified at Lys-146 to do so. Therefore, Lys-146 could be one of the cationic groups that functions in electrostatic binding of the substrate's phosphate groups.


Purification and characterization of a repressible alkaline phosphatase from Thermus aquaticus. A repressible alkaline phosphatase has been isolated from the extreme bacterial thermophile, Thermus aquaticus. The enzyme can be derepressed more than 1,000-fold by starving the cells for phosphate. In derepressed cells, nearly 6% of the total protein in a cell-free enzyme preparation is alkaline phosphatase. The enzyme was purified to homogeneity as judged by disc acrylamide electrophoresis and sodium dodecyl sulfate electrophoresis. By sucrose gradient centrifugation it was established that the enzyme has an approximate molecular weight of 143,000 and consists of three subunits, each with a molecular weight of 51,000. Tris buffer stimulates the activity of the enzyme, which has a pH optimum of 9.2. The enzyme has a broad temperature range with an optimum of 75-80 degrees. The enzyme catalyzes the hydrolysis of a wide variety of phosphorylated compounds as do many of the mesophilic alkaline phosphatases. The Michaelis constant(Km) for the enzyme is 8.0 X 10(-4) M. Amino acid analysis of the protein revealed little in the amino acid composition to separate it from other mesophilic enzymes which have been previously studied.


Association of gylcogenolysis with cardiac sarcoplasmic reticulum. Sarcoplasmic reticulum fragments isolated from dog cardiac muscle possess a calcium-accumulating system associated with a series of enzymes linked to glycogenolysis. These enzymes include: adenylate cyclase, cyclic AMP-dependent protein kinase, phosphorylase b kinase, phosphorylase (b/a, 30/1),"debrancher" enzyme, and glycogen (0.3 to 0.7 mg/mg of protein). The sarcoplasmic reticulum preparation produced glucose 1-phosphate and glucose from either endogenous or exogenous glycogen. Both the calcium-accumulating and glycogenolytic enzymes sediment in a single peak at 33% sucrose on a linear continous sucrose density gradient, and the complex remains intact throughout repeated washing. Glycogen particles appear to be associated with the sarcoplasmic reticulum in situ as well as in the isolated microsomal fraction. The sarcoplasmic reticulum-glycogenolytic complex, monitored by a linked enzyme spectrophotometric assay, shows several features: (a) activation of phosphorylase activity to peak rate occurs over a very rapid time course which cannot be duplicated using combinations of purified enzymes; (b) activation is inhibited by protein kinase inhibitor; (c) phosphorylase b functions as in the purified form with respect to AMP (Km, 0.3 mM); (d) in the presence of limiting amounts of glycogen, optimal phosphorylase b activity in the sarcoplasmic reticulum requires the presence of debrancher, and the activity is sensitive to inhibitors of that enzyme such as Tris, which suggests the possiblity that the enzymes bear a specific structual relationship to the glycogen present. Phosphorylase b leads to a activation in the sarcoplasmic reticulum was completely resistant to ethylene glycol bis(beta-aminoethyl either)-N,N'-tetraacetic acid (EGTA). Inhibition of calcium accumulation by or release of bound calcium from sarcoplasmic reticulum by X537A (RO 2-2985) did not alter the EGTA resistance. These results suggest that cardiac sarcoplasmic reticulum is a complex organelle containing functions that may be related to excitation-contraction coupling and intermediary metabolism.


Purification and properties of a virion protein kinase. The protein kinase associated with virions of frog virus 3 was purified to apparent homogeneity by ion exchange chromatography and gel filtration. The enzyme protein appeared as a single polypeptide of molecular weight 50,000 to 55,000 as determined by gel filtration, glycerol gradient sedimentation, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and comprised approximately 0.4% of the total virion protein. The activity was classified as a cyclic nucleotide-independent protein kinase as it was not effected by cyclic adenosine 3':5'-monophosphate, cyclic guanosine 3':5'-monophosphate, or inhibited by a cyclic nucleotide-dependent protein kinase inhibitor protein, and utilized GTP as well as ATP as a phosphate donor. The greatest rates of phosphorylation were obtained with acidic phosphoprotein substrates such as casein or phosvitin, although potential physiological substrates for this activity included specific virion polypeptides of frog virus.


The interaction of macromolecular solutions with macromolecular monolayers adsorbed on a hydrophobic surface. In order to elucidate the general patterns of intermacromolecular surface interactions that may be involved in hemocompatibility phenomena, monolayers of representative macromolecules on an octadecylsilylated glass surface were exposed to solutions of other macromolecules, and the changes in interfacial composition were characterized by zeta potential-pH titration curves, as measured by alternating flow streaming current analysis and, in some cases, by radiotracer labeling. Experiments with poly(vinylpyrrolidone) (PVP), a blood-compatible linear polymer; bovine serum albumin (BSA), a representative serum protein; whole human serum (HS), a complex mixture of proteins; and erythrocyte surface glycoprotein (GP), an extended-chain macromolecular amphiphile, showed the following: 1) Penetration of the original monolayer occurred within 24 hr in 9 of the 12 possible cases; it did not occur for BSA or HS monolayers exposed to PVP, and probably not for PVP exposed to GP. 2) In all cases, penetration was accompanied by no more than partial displacement of the original monolayer, thereby generating a mixed monolayer. Each of the six possible binary mixed monolayers could be obtained by at least one of the two possible mixing sequences. 3) In the three binary systems containing BSA, the formation of the mixed monolayer could be related to increased adsorption in the two-component system. 4) The two components of the mixed monolayers were not equally distributed across their thicknesses: thus, the outer surfaces of the PVP-BSA and (at neutral pH) the PVP-HS mixed monolayers contained only PVP; that of the BSA-HS mixtures only HS. In the PVP-HS, and probably the GP-BSA and GP-HS mixed monolayers, the composition of the outer surface appeared pH-dependent. The resultant zeta potential versus pH profiles in the latter two cases resembled those of intact blood cells. The results suggest that neither the compact monolayers of globular proteins nor the diffuse monolayers of randomly coiled water-soluble polymers can, by their prior adsorption on a synthetic surface, prevent the subsequent adsorption of other globular macromolecules. It is possible that the randomly coiled polymers may impede the adhesion of platelets to the substrate since the results indicate that the adsorption of such polymers causes a displacement of the shear plane.


Stimulation of ornithine decarboxylase activity in chick fibroblasts by non-suppressible insulin-like activity (NSILA), insulin and serum. A factor isolated from human serum (nonsuppressible insulin-like activity, NSILA) stimulates multiplication of serum-starved chick embryo fibroblasts and stimulates activity of ornithine decarboxylase (ODC). Physiological doses of NSILA (200 muU/ml) and pharmacological doses of insulin (200 mU/ml) stimulate ODC 4-5-fold, 10% fetal calf serum about 18-fold. Combined addition of NSILA and insulin does not result in higher activities, suggesting a common mechanism of action. The increase in cell number obtained with NSILA, insulin or serum parallels the degree of ODC stimulation. Treatment of cells with pronase also stimulates ODC activity. A sharp increase in ODC activity occurs between 2, 5 and 5.0 hours after addition of the growth factors with a peak at 4.0-4.5 hours ("activation period"). As cells leave G1 phase, ODC activity decreases rapidly. To achieve maximal activity of ODC, the growth factors have to be present during the entire "activation period." The potential to reactivate ODC decreases as cells pass through S phase. Results obtained using cycloheximide suggest that ODC is translated only in the second half of the "activation period." Data on effects of dbcAMP and dbcGMP on ODC activation by serum are discussed.


Gas-liquid chromotographic determination of perazine, thioridazine and thioridazine metabolites in human plasma. A gas-liquid chromatographic method for the detection of perazine, thioridazine and its major metabolites in human plasma is presented. Repeated extraction, an internal standard and a temperature program with flame ionization detection make possible accurate and reproducible results with patients on therapeutic doses of these drugs. Examples of chromatograms after extraction of plasma are given.


A reversed-phase thin-layer chromotographic method for the determination of relative partition coefficients of very lipophilic compounds. A reversed-phase thin-layer chromatographic method has been developed for the determination of partition coefficients. A support phase has been chosen, following investigation of the lack of adsorptive properties, which has a minimal effect on the pH of the buffer system. A stationary phase has been chosen to give deltaRm values of the same magnitude as Hansch pi values for a series of phenothiazines. The method can be applied to molecules of a wide range of lipophilicity following preliminary investigations of suitable phase-volume ratios and of the pH and composition of the binary mobile phase, providing adsorption on the support phase is excluded.


High-pressure liquid chromatography of drugs. II. An evaluation of a microparticulate cation-exchange column. A microparticulate cation-exchange column has been evaluated for the chromatography of thirty compounds selected as representative of a wide variety of drug substances. Although the column exhibited a strong partition effect besides the expected ion-exchange mechanism, the retention of drugs could be predictably influenced by variation of the eluent ionic strength and organic solvent content. For acidic drugs the column showed little selectivity (although a salting-out effect increased retention at high eluent ionic strengths), but for basic substances Partisil SCX may afford a useful separative medium offering reasonable chromatographic efficiency (HETP is approximately 0.1 mm). The column longevity, however, is at present questionable.


Resolution in affinity chromatography. The effect of the heterogeneity of immobilized soybean trypsin inhibitor on the separation of pancreatic proteases. By affinity chromatography, trypsins and chymotrypsins from mouse pancreas homogenates have been separated using soybean trypsin inhibitor immobilized on Sepharose. The effects of the functional heterogeneity of the adsorbent have been investigated in terms of the resolution obtained. Heterogeneity of the adsorbent have been investigated in terms of the resolution obtained. Heterogeneity has been found to originate from the following sources: heterogeneity of the ligand before immobilization; alteration of the ligand by immobilization; and modification of the ligand after immobilization by molecules to be fractionated. Only when the heterogeneity of the adsorbent was minimized could the resolution of closely related enzyme species be achieved. The elution conditions for different enzymes depended on the amount of enzyme applied, as no complete homogeneity could be obtained. In addition, it was found that the adsorbent was partly degraded by the pancreas extract, reducing its fractionating capacity.


A thin-layer chromatographic method for the determination of acebutolol and its major metabolite in serum. A sensitive and specific thin-layer chromatographic method for the simultaneous determination of acebutolol [DL-1-(2-acetyl-4-n-butyramidophenoxy)-2-hydroxy-3-isopropylaminopropane] and its major metabolite [DL-1-(2-acetyl-4-acetamidophenoxy)-2-hydroxy-3-isopropylaminopropane] is described. A 2-ml volume of serum with 350 ng of quinidine as internal standard was extracted at pH 10, the solvent was evaporated off and the residue was dissolved in 50 mul of methanol. A 10-mul volume of the solution was spotted on a thin-layer plate and after elution (ethyl acetate-methanol-ammonia, 75:20:5) the plate was dried at 90 for 15 min and, after cooling, dipped in a 10% paraffin wax solution. The fluorescence was measured using a spectrofluorimeter with a thin-layer scanning attachment. The peak-height ratios of acebutolol to internal standard and metabolite to internal standard were used to quantitate acebutolol and the metabolite, respectively.


Influence of L-prolyl-L-leucyl-glycine amide on growth hormone secretion in normal and acromegalic subjects. Melanocyte Release-Inhibiting Peptide (MRIP-I) did not affect circulating levels of ACTH, LH, FSH, TSH,ORL, betaMSH and insulin when iv infused (5.0 mg in 5 min plus 0.4 mg/min for 70-115 min), while it significantly reduced serum GH response to hypoglycemia in normal subjects and lowered serum GH levels in acromegalics. There was no correlation between the fall in serum GH after MRIP and after dopaminergic drugs in acromegaly. These data are compatible with either a direct suppressive action exerted by MRIP-I at pituitary level or an extra-pituitary effect not involving dopaminergic pathways. It can be spec-lated that since labelled MRIP-I accumualtes in the pineal and melatonin blunts GH response to hypoglycemia, the pineal gland might be involved in the MRIP-I-induced suppression of GH secretion.


Use of Counter and rocket immunoelectrophoresis in acute respiratory infections due to Streptococcus pneumoniae. The use of Counter immunoelectrophoresis (CIE) for the detection of pneumococcal capsular antigen in the sputum and serum of patients suffering from acute respiratory infections is described. The CIE of sputum gave positive results in 224 (99%) out of 225 samples in which Streptococcus pneumoniae was isolated by cultural techniques, and in 23 (9%) out of 262 samples in which no or other potential pathogens had been isolated. In the detection of capsular antigen in serum, CIE was positive in 32 (35%) out of 92 pneumonia cases and was associated with an increase in mortality.


Requirement for a macromolecular factor for sodium azide activation of guanulate cyclase. Sodium azide, a highly nucleophilic agent and a potent metabolic inhibitor, markedly increased guanylate cyclase activity from supernatant fractions of rat liver homogenates. The effect of sodium azide was not observed with partially purified guanulate cyclase from liver or crude soluble guanylate cyclase from cerebral cortex. However, the effect of sodium azide could be restored by the readdition of a fraction isolated from rat liver homogenates. The macromolecular factor required for the sodium azide effect was separated from soluble guanylate cyclase of rat liver with DEAE-cellulose column chromatography, and some of its properties were examined. The factor was nondialyzable and heat labile.


A rapid method for the assay of guanylate cyclase. An extremely rapid and sensitive assay for guanylate cyclase utilizing [alpha-32P]-GTP has been developed. It involves incubation of 5-100 mug of enzyme protein with 1 mM [alpha-32P]-GTP in 40 mM Tris HC1 buffer (pH 7.4) containing 3-3 mM MnSO2, 10 mM theophylline and 1 mM cyclic GMP. The reaction is terminated by addition of EDTA, and [32P]-cyclic GMP formed is isolated by sequential chromatography on Dowex-50-H+ and alumina. Recovery of 75-85% of [3H]-cyclic GMP and a blank of 0.001-0.003% of added [32P]-GTP was routinely obtained. The [32P] radioactivity isolated was shown to be cyclic GMP by a variety of techniques. The assay has also been shown to be applicable for a variety of tissues.


Lysosomal enzyme secretion from human neutrophils mediated by cyclic CMP: inhibition of cyclic GMP accumulation and neutrophil function by glucocorticosteroids. The effects of several glucocorticosteroids on cyclic GMP accumulation, guanylate cyclase activity, calcium influx, lysosomal enzyme secretion, and phagocytosis were studied in human neutrophils. Contact between neutrophils and serum-treated zymosan particles, in the presence of calcium at pH 7.4, triggered these cellular events within five minutes. Each of these neutrophil functions was markedly inhibited by methylprednisolone sodium succinate, triamcinolone acetonide hemisuccinate and paramethasone acetate but was unaffected by two mineralo-corticosteroids. Human neutrophil soluble guanylate cyclase activity was not changed by the glucocorticoids. Inhibition of phagocytosis by, and lysosomal enzyme secretion from, neutrophils by glucocorticosteroids may be the result of a reduction in cyclic GMP accumulation within these cells. The data suggest that glucocorticosteroids inhibit cyclic GMP accumulation in neutrophils by reducing the influx of extracellular calcium into the cells, thereby limiting the availability of intracellular calcium for metabolic processes associated with the accumulation of cyclic GMP.


Cyclic AMP content and regulation of tyrosine-3-mono-oxygenase in rat striatum. The unilateral transection of nigro striatal dopaminergic axons produced a short lasting (15-30 min) increase in the affinity of striatal tyrosine 3-mono-oxygenase (TH), for a synthetic pteridine cofactor (DMPH4). The kinetic changes of striatal TH were paralleled by an increase of striatal cAMP content. Apomorphine (10 mg/kg i.p.) failed to block the activation of TH by cerebral hemisection. Haloperidol (5 mg/kg i.p.) and reserpine (5 mg/kg i.p.) also elicited a sudden and similar change in striatal TH activity but this response occurred without an increase in the cAMP content and lasted for longer than 2 hrs. If the hemisection of nigrostriatal pathway was performed after haloperidol or reserpine injection the activation of TH produced by these two drugs was reversed rapidly (about 30 minutes). Pretreatment with haloperidol or reserpine prevented the increase of striatal cAMP following cerebral hemisection. Moreover, haloperidol injected 30 min after cerebral hemisection failed to change the TH kinetic properties in the striatum ipsilateral to the lesion but it changed striatal TH in the side contralateral to the lesion. These results suggest that the increase in the affinity of striatal TH for the pteridine cofactor elicited by cerebral hemisection is not related to a lack of stimulation  of DA autoreceptors. Moreover these experiments provide an  evidence that postsynaptic dopamine receptors play a role in the activation of striatal TH elicited by haloperidol.


Properties of guanylate cyclase from rat kidney cortex and transplantable kidney tumors. The subcellular distribution and properties of guanylate cyclase was examined in preparations of normal rat renal cortex and Morris renal tumors MK2 and MK3. In normal kidney cortex about two-thirds of guanylate cyclase activity of homogenates was found in soluble fractions. With renal tumors the homogenate activity was less and the enzyme was equally divided between particulate and soluble fractions. The particulate enzyme in kidney cortex and tumors was associated with all particulate fractions. Triton X-100 increased the activity of all preparations. All preparations preferred Mn2+ as the sole cation. The stimulatory effects of Ca2+ on soluble enzyme and inhibitory effects on particulate activity were similar with preparations of renal cortex and tumors. ATP inhibited all preparations. Soluble and particulate guanylate cyclases from renal cortex were activated several-fold with 1 mM NaN3. Preparations of tumor enzymes did not respond to NaN3. Thus, compared to normal renal cortex the subcellular distribution of guanylate cyclase and some of its properties are altered in preparations of renal tumors.


Reduction of enamel solubility by sodium monophosphate. The preponderance of evidence indicates that sodium monofluorophosphate exerts a highly beneficial effect on dental caries incidence and enamel solubility. Optimal effects are obtained with a concentration of 4 X 10(3) ppm fluoride, by a four-minute application and by adjusting the pH to 4.0. The reaction of sodium monofluorophosphate with enamel is not temperature dependent. Sodium monofluorophosphate is significantly more protective than sodium fluoride in aqueous solutions at all equivalent fluoride concentrations and in pastes at concentrations exceeding 8 X 10(3) ppm fluoride.


Proxemics, locus of control, anxiety, and type of movement in emotionally disturbed and normal boys. In order to determine the interpersonal distancing requirements for emotionally disturbed and normal children and in order to investigate the relationship of locus of control and anxiety to interpersonal space, 20 emotionally disturbed and 20 normal boys were randomly required to approach an object person and to let the object person approach them until they felt uncomfortable. Results indicated that emotionally disturbed boys required more space than normals; that subjects would approach closer than they would allow the object person to approach them; and that externals required more space than internals. There were no significant differences between high and low anxious subjects, nor between emotionally disturbed children diagnostically classified as overanxious reaction and those with other diagnosis. Finally, neither anxiety nor locus of control explained the significant normal-emotionally disturbed differences in space requirements. Theoretical and practical implications were discussed as well as the relationship between the present and previous research.


Pharmacotherapy in older depressed patients. Treatment of depression in elderly patients is not generically different from treatment of depression in younger age cohorts. Because of certain age-related physical, physiological, and biochemical factors, however, drug prescription for geriatric patients must be modified in several respects. Tricyclic antidepressants are the principal agents in treatment, but their side effects tend to be magnified in the elderly. Dosage should initially be lower than with younger patients and increased in gradual increments. Lithium, MAO inhibitors, and neuroleptics are appropriate in some cases, but additional precautions are necessary. Because the elderly are liable to multiple system decompensation, they are likely to be prescribed multiple pharmacological agents. Drug-drug interactions involving antidepressant medication present a variety of therapeutic problems and can threaten life. Depression in late life can be treated pharmacologically, but both the therapeutic and deleterious activities of the drugs can be altered by compromised organ systems.


The binding of complement by complexes formed between a rabbit antibody and oligosaccharides of increasing size. Immune complexes formed between a homogeneous rabbit antibody to type III pneumococcal polysaccharide and a series of oligosaccharides of varying size derived from it were prepared and tested for their ability to fix guinea pig hemolytic complement. Antibody and either tetra-, hexa-, or octasaccharide formed only monomeric antibody-hapten complexes and did not show any complement binding. A dodecasaccharide and a 16-sugar residues oligomer formed dimer and trimer immune complexes. These complexes were also unable to fix complement. However, as the size of the sugar oligomers was increased to about 21 sugar residues per oligosaccharide molecule or more, the resulting complexes exhibited substantial complement binding, concomitant with the formation of antigen-antibody aggregates higher than trimers. On the other hand, an independent study carried out with the same material suggested changes in the conformation of the Fc moiety in the antibody molecule upon addition of oligosaccharide ligands as small as a 16-residue unit. Since the resulting complexes hardly ehibited any complement binding, ligand-induced conformational changes in the Fc part of the antibody molecule appears to be an insufficient condition per se for triggering complement fixation.


Neonatal infection with mouse thymic virus: effects on cells regulating the antibody response to type III pneumococcal polysaccharide. Mice infected neonatally with mouse thymic virus (TA) were evaluated at different ages with respect to their ability to give a plaque-forming cell (PFC) response to type III pneumococcal polysaccharide (SSS-III), as well as the degree of amplifier and suppressor thymus-derived (T) cell activity present. B cell activity matured rapidly from 2 to 4 weeks of age and was not affected by TA infection. Amplifier T cell activity matured progressively over the first 8 weeks of life and was transiently suppressed in TA-infected mice at 4 weeks of age. Suppressor T cell activity measured at 2,4, and 6 weeks of age was unaffected by TA. The findings suggest that TA is highly tropic for T cells and has selective effects on subpopulations of T cells.


The chromosomes of four species of the nasuta complex of Drosophila. I. Chromosome maps and inversion polymorphism. The salivary chromosomes of four species of the nasuta complex of Drosophila, D. sulfurigaster albostrigata, D, kohkoa, D. albomicans, and D. kepulauana were studied and chromosome maps of each species are presented; the maps of the latter three species are based on the map of D. sulfurigaster albostrigata. Three of the species D. sulfurigaster albostrigata, D. albomicans, and D. kohkoa were shown to be highly polymorphic for chromosomal inversions while the available evidence indicated that D. kepulauana is much less polymorphic. These facts are correlated with the geographic distribution of the species. Transitional homoselection has not been complete in the evolution of three of the species since D. sulfurigaster albostrigata, D. kohkoa, and D. albomicans have a number of naturally occurring polymorphisms in common.


Graft-vs-host reactions in F1 mice induced by parental lymphoid cells: nature of recruited F1 cells. Graft versus host (GVH) reactivity of parental lymph node (LN) cells was assayed by measurements of 3H-thymidine incorporation in vivo. Mitomycin (Mit.) treatment of parental cells abolished their proliferative activity but the combination of such Mit.-treated parental cells with F1 LN cells resulted in much higher proliferation than either one population alone. This recruitment into proliferation of F1 cells was prominent on days 3 and 4 after cell injection and amounted to 35 to 51% of the total activity seen after injection of untreated parental cells alone. The F1 cell sensitive to recruitment was resistant to anti-Thy 1.2 treatment, was not removed by carbonyl iron-magnet separation; and was not present in thymus. The parental cell inducing recruitment was, however, sensitive to anti-Thy 1.2. When spleen cells from hapten immune F1 donors were injected together with Mit.-treated parental LN cells and boosted with hapten on another carrier, a typical "allogeneic effect" was observed in the anti-hapten immune response. It was concluded that Mit.-treated parental T cells exerted a mitogenic effect on F1 B cells resulting in extensive recruitment similar to that seen in murine mixed lymphocyte reactions.


Determinants of red cell sickling. Effects of varying pH and of increasing intracellular hemoglobin concentration by osmotic shrinkage. The effects of varying pH and of increasing intracellular hemoglobin (Hb) concentration on red cell sickling and oxygen affinity were studied in whole blood from persons with sickle cell anemia (SS) and sickle cell trait (SA). Small increases in SS blood pH inhibited sickling, and small reductions in both SS and SA blood pH promoted sickling far more than accounted for by the Bohr effect. Sickling behavior correlated with minimum gelling concentrations (MGC) of deoxygenated hemolysates without 2,3-diphosphoglycerate. MGC values fell sharply when pH was lowered from 7.25 to 7.15 for HbS and from 7.15 to 6.90 for SA hemolysates, suggesting effects on specific ionic interactions involved in Hb gelation. Possible clinical counterparts are acute metabolic acidosis and alkalosis (prior to change in red cell 2,3-diphosphoglycerate), where the Bohr effect and oxygen affinity-independent effects of pH alterations on sickling would be additive. Osmotic shrinkage of HbS-containing red cells produced a large fall in oxygen affinity and a marked increase in sickling independent of that fall. The oxygen affinity and sickling properties of SA cells whose MCHC was raised to 40 per cent resembled those of unaltered SS cells, supporting a relationship between molecular aggregation of Hb and low oxygen affinity. Sickling of aerated SS cells in hypertonic saline depended upon partial Hb desaturation due to lowered oxygen affinity. Thus osmotic shrinkage of HbS-containing cells acts synergistically with partial deoxygenation to promote sickling. These conditions are present in the renal medulla, but may occur elsewhere in severe hyperosmolar states.


Bacterial interference as a factor in renal infection. These experiments have investigated the role of bacterial interference as a determinant in the epidemiology of renal infection. Two unrelated strains of Escherichia coli, E. coli 08 and 075, isolated from cases of clinical pyelonephritis were used. Although both strains had identical morphology on conventional media they could be differentiated using genetically stable markers for streptomycin resistance and arabinose utilization. When the 2 strains of E. coli were introduced into the kidney simultaneously by direct inoculation mixed infections were readily established. On the other hand, although both strains of E. coli were equally invasive as individual pathogens, pyelonephritis, when induced using a retrograde challenge with a mixed culture of the same organisms was almost invariably caused by the 08 strain alone. Further experiments showed that bacterial interference occurred within the kidney and determined the pattern of infection. When unilateral renal infections were established with E. coli 08 and the animals subsequently challenged with E. coli 075, it was found that E. coli 075 infection never occurred in kidneys infected with E. coli 08 but infection was established in the contralateral kidney. The experiments have shown that mixed renal infection with E. coli are uncommon even when both pathogens are equally nephropathogenic and are introduced simultaneously into the bladder.


The effect of the plasma bicarbonate level on proximal tubule sodium reabsorption in NH4Cl-loaded dogs. In an attempt to examine the effects of mild and severe chronic metabolic acidosis on proximal tubule sodium reabsorption, 6 dogs were given 10 mEq. per kilogram per day and 5 dogs were given 20mEq. per kilogram per day of ammonium chloride for 3 days and compared to 12 normal dogs during a steady-state water diuresis and following the administration of ethacrynic acid (EA) intravenously (2 mg. per kilogram) utilizing standard clearance methodology, In the severely acidotic group (pH decrease is greater tthan 0.2) plasma pH was 7.08 +/- 0.06 and plasma bicarbonate was 6.3 +/- 1.0 Eq. per liter compared to a pH of 7.33 +/-0.02 and bicarbonate of 13.4 +/- 0.7 in mild acidosis (pH decrease is less than 0.2). During a steady-state water diuresis urine flow was 14.2 +/- 0.9 in severely acidotic compared to 10.5 +/-0.7 ml. per minute per 100 ml. glomerular filtration rate (GFR) in normal dogs (p is less than 0.01). Following EA sodium clearance increased 38.4 +/- 3.5 in severely acidotic dogs and 27.6 +/- 2.0 ml. per minute per 100 ml. GFR in normal dogs (p is less than 0.02). In mild acidosis, steady-state fractional urine flow and the increase in fractional sodium clearance following EA were not significantly different than normal dogs. We conclude that chronic metabolic acidosis leads to an increase in distal solute load and enhanced natriuretic effect of EA secondary to a decrease in proximal tubule sodium reabsorption which may be dependent upon the degree of reduction in the plasma bicarbonate level.


Lethal midline granuloma: a pathological spectrum. Lethal midline granuloma is a non-specific clinical term used for different pathological entities often exhibiting some semblance to each other as they lie along a histological spectrum. Nevertheless, each entity has a specific diagnostic pattern. The histological picture with distinguishing features of each specific type is pointed out. Stress is made on the use of repeated biopsy until the specific histological diagnosis can be made so that specific treatment as breifly outlined, can be carried out to insure the best prognosis.


Ganglioside biosynthesis. Characterization of uridine diphosphate galactose: GM2 galactosyltransferase in golgi apparatus from rat liver. An enzyme that transfers galactose from UDP-Gal to ganglioside GM2 (Tay-Sachs ganglioside) was concentrated 50 times in Golgi apparatus from rat liver relative to total homogenates. This enzyme required detergents or phospholipids as dispersing agents. Of the numerous detergents tested, sodium taurocholate and Triton CF-54 were most effective in stimulating the reaction. Cardiolipin alone was more effective than any of the detergents tested in stimulating enzyme activity. The pH optimum for the reaction varied with the nature of the dispersing agent. With sodium taurocholate, Triton CF-54 and cardiolipin, the pH optima were 6.2, 5.9, and 5.6, respectively. The enzyme had a nearly absolute requirement for Mn2+, with maximum activity being attained at a concentration of 15 mM Mn2+. Other divalent or trivalent cations were either less effective than Mn2+ or inhibited the transferase reaction. The Km values calculated for UDP-Gal and GM2 were 1.1 X 10(-4) M and 9.9 X 10(-5) M, respectively. The enzyme could not be dissociated from Golgi apparatus fractions by treatment with ultrasound, indicating that it is tightly associated with the membrane and not part of the luminal contents. The newly synthesized GM2, the product of the reaction, was incorporated into or became tightly associated with the membranes of the Golgi apparatus.


Effect on various putative neurotransmitters on the secretion of corticotrophin-releasing hormone from the rat hypothalamus in vitro-a model of the neurotransmitters involved. The effect of incubating the hypothalamus of adult male rats with various neurotransmitters upon the release of corticotrophin-releasing hormone (CRH) was studied. The CRH activity in the incubation medium was assayed in 48 h median eminence-lesioned rats and the corticosteroidogenesis of excised adrenals in vitro was used as the end-point. 5-Hydroxytryptamine (100 pg/ml-10ng/ml) caused a dose-dependent release of CRH which was antagonized by methysergide (30-100 ng/ml). The response to 5-hydroxytryptamine was also inhibited by hexamethonium and atropine which indicated that it was acting through a cholinergic interneurone. Melatonin (10 ng) did not alter the basal release of CRH but inhibited the action of both 5-hydroxytryptamine (10 ng) and acetylcholine (3 pg). Thus it appears that both 5-hydroxytryptamine and melatonin play a role in the control of CRH release. Noradrenaline blocked the release of CRH induced by both acetylcholine and 5-hydroxytryptamine and presumably this inhibition was caused by direct action on the CRH neurone. gamma-Aminobutyric acid (GABA) also inhibited the release of CRH and may also be involved in the regulation of CRH secretion. The inhibitory neurotransmitters, noradrenaline, GABA and melatonin, act via independent receptor mechanisms. A model based on the above data is presented.


Purification of canine prolactin by preparative isotachophoresis. Preparative isotachophoresis in polyacrylamide gel using carrier ampholytes as 'spacers' has been used to purify prolactin from canine pituitary extracts. Using Ampholine pH 5-8 as spacers, a prolactin fraction was obtained which was essentially homogeneous as judged by the criteria of disc electrophoresis and isoelectric focusing. Amino acid analysis indicated a close similarity between canine and ovine prolactin. A growth hormone fraction, identified by its electrophoretic mobility, was also obtained although this was heterogeneous in disc electrophoresis at alkaline pH.


Acid-base balance in rainbow trout (Salmo gairdneri) subjected to acid stresses. 1. The respiratory properties of rainbow-trout blood were investigated in acid-stressed fish. In the first group acid was introduced into the bloodstream and in the second the carbon dioxide content of the ambient water was increased. 2. Initially the introduction of acid to the blood caused a decrease in blood pH and bicarbonate, and increases in oxygen uptake and ventilation volume. After 2-3 h these values had returned to the control levels. 3. Trout subjected to high ambient CO2 (about 10 mmHg) showed a decrease in blood pH while PCO2 and bicarbonate increased. After 8 h the trout began to show signs of compensation to the acidosis. 4. In each experiment the blood PO2 was little changed but blood O2 content was decreased and tended not to resume the control value even after several hours. 5. The results are discussed in terms of the various acid-base mechanisms thought to be available to the fish. These include branchial ion exchanges and the possible buffering roles of the extracellular and intracellular fluids.


Connexions between hair-plate afferents and motoneurones in the cockroach leg. 1. The trochanteral hair-plate afferents in the metathoracic leg of the cockroach, Periplaneta americana, were stimulated electrically and at the same time intracellular recordings were made from either motoneurones, interneurones or afferent terminals within the methathoracic ganglion. 2. Activity in the hair-plate afferents evoked short latency excitatory postsynaptic potentials (EPSPs) in femur flexor motoneurones. The latency of the IPSPs was on average 1-8 ms longer than the latency ofthe EPSPs. 3. Intracellular recordings from terminal branches of the hair-plate afferents showed that the delay between the peak of the afferent terminal spike and the beginning of the EPSPs is about 0.4 ms. This finding, together with the observations that the amplitude of the EPSPs is increased by the passage of hyperpolarizing current and decreased following high-frequency stimulation, indicates that the EPpSPs are evoked via-monosynaptic chemical synaptic junctions. 4. The observations of the long latency of the IPSPs, the need for a number of afferents to be simultaneously acive for them to be evoked and the occasional variability in latency, all indicate that the IPSPs are evoked via a disynaptic pathway...


Physiological properties of eel haemoglobin: hypoxic acclimation, phosphate effects and multiplicity. Unlike the whole body oxygen affinity, which adapts readily to environmental oxygen tensions, haemoglobins, prepared from normoxic- and hypoxic-accimated eels (Anguilla anguilla) show no adaptive changes in oxygenation properties or in multiplicity. Hypoxic acclimation is, howeveer, accompanied by a strong decrease in red cell nucleoside triphosphates, particularly guanosine triphospphate (GTP), which depresses oxygen affinity of the composite and component haemoglobins more strongly than does the concurring ATP. The effects of pH, temperature and salts on the oxygenation properties of the (isolated) haemoglobins are reported, discussed in relation to the varying environmetal conditions encountered by eels, and compared with data on American and Japanese eels (A. rostrata and A. juponica, respectively.


[LP-X in newborns: increased incidence of positive tests without cholestasis (author's transl)]. The investigation of 194 newborns has shown that during the first weeks of life the abnormal lipoprotein-X (LP-X) was present in the serum of nearly 50% of the infants, with no clinical chemical evidence of cholestasis. The percentage of LP-X positive tests was even higher in the group of immature newborns (65%). There was no correlation between the bilirubin concentration and the detection of LP-X. The activities of leucine arylamidase (EC 3.4.1.1) and gamma-glutamyltransferase (EC 2.3.2.2) as well as the concentrations of total and free cholesterol did not differ in the LP-X positive and negative infants. Except in one case, LP-X was never detectable on the first day of life. The earliest date of appearance was the second day. In the serum of some infants, who were LP-X positive shortly after birth, the lipoprotein could still be found at the age of 2--3 months. The incidence of LP-X was not higher in newborns with blood group incompatibility than in newborns with unspecific hyperbilirubinaemia. After exchange transfusions LP-X disappeared in most cases, but it could later often be detected again. In some newborns, who were LP-X negative a few days after birth LP-X was first detected at the age of 2-3 months. The LP-X test is of no use for th diagnosis of cholestasis in newborn infants. The test is specific for cholestasis only after the first year of life. The increased incidence of positive LP-X tests in newborns is discussed as a consequence of immature liver function.


Neurotrophic activity of brain extracts in forelimb regeneration of the urodele, Triturus. The loss in protein synthesis which the regenerating forelimb of the newt suffers after denervation can be recovered by infusing into it an extract of newt soluble brain protein. Moreover, the synthesis of basic protein shows a greater response to the active brain principle than does that of acidic protein. The active agent of the nervous tissue is destroyed by heat and trypsin digestion. Extracts of liver and spleen, similarly prepared, do not evoke recovery of lost protein synthesis. Synaptosomal extracts of the frog brain also cause recovery of protein synthesis in the denervated regenerate, demonstrating the likelihood that the active agent is not species-specific within these amphibians, that it is a constituent of the neuronal fraction of nervous tissue, and that it is present in axonal terminals. Additional experiments showed that the nervous agent is likely a basic protein, and that the amount of protein infused is of the order of only 1.0% of the total regenerate protein. The significance of the findings is discussed in relation to the nature of the effect on protein synthesis and the nature of the active principle.


Interaction between phloretin and the red blood cell membrane. Phloretin binding to red blood cell components has been characterized at pH6, where binding and inhibitory potency are maximal. Binding to intact red cells and to purified hemoglobin are nonsaturated processes approximately equal in magnitude, which strongly suggests that most of the red cell binding may be ascribed to hemoglobin. This conclusion is supported by the fact that homoglobin-free red cell ghosts can bind only 10% as much phloretin as an equivalent number of red cells. The permeability of the red cell membrane to phloretin has been determined by a direct measurement at the time-course of the phloretin uptake. At a 2% hematocrit, the half time for phloretin uptake is 8.7s, corresponding to a permeability coefficient of 2 x 10(-4) cm/s. The concentration dependence of the binding to ghosts reveals two saturable components. Phloretin binds with high affinity (K diss = 1.5 muM) to about 2.5 x 10(6) sites per cell; it also binds with lower affinity (Kdiss = 54 muM) to a second (5.5 x 10(7) per cell) set of sites. In sonicated total lipid extracts of red cell ghosts, phloretin binding consists of a single, saturable component. Its affinity and total number of sites are not significantly different from those of the low affinity binding process in ghosts. No high affinity binding of phloretin is exhibited by the red cell lipid extracts. Therefore, the high affinity phloretin binding sites are related to membrane proteins, and the low affinity sites result from phloretin binding to lipid. The identification of these two types of binding sites allows phloretin effects on protein-mediated transport processes to be distinguished from effects on the lipid region of the membrane.


Effect of synthetic substance P on monoaminergic mechanisms in brain. Synthetic substance P has been discovered to stimulate significantly the formation of dopa in the limbic, striatum, hemisphere and diencephalon regions of the brain and the lower brain stem. There was no effect upon 5-hydroxytryptophan formation or on tryptophan or tyrosine levels. After inhibition of monoamine synthesis by N'-(DL-SERYL)-N2-(2, 3, 4-trihydroxybenzyl)hydrazine, substance P significantly accelerated the disappearance of dopamine, noradrenaline and 5-hydroxytryptamine. Substance P appears to stimulate monoaminergic neurons in the brain and to serve as an excitatory transmitter in nerve terminals impinging upon dopaminergic cell bodies. A similar stimulation of noradrenaline and 5-hydroxytryptamine indicate a similar transmitter role for noradrenergic and serotonergic neurons. These data strengthen questions about the possible clinical influence of substance P in disease states involving monoaminergic mechanisms including Parkinsonism and schizophrenia.


Gamma-glutamyl transpeptidase. Elevated activity in myotonic dystrophy. gamma-Glutamyl transpeptidase, a membrane-bound enzyme playing an important role in the active amino acid transport across cellular membranes, is shown to be elevated in the serum of patients with myotonic muscular dystrophy. No increase of AP, LAP, GOT and GPT activities in the sera of some of the patients studied is observed. Possible interpretations in relation to the pathogenesis of myotonic dystrophy are discussed.


Occurrence of a trypsin inhibitor in eggplant exocarps. A trypsin inhibitor was extracted from eggplant exocarps with several buffers. The 0.1 M acetate buffer, pH 5.5. extract had the highest specific activity. The crude inhibitor, obtained by heat treatment and salting-out from the acetate buffer extract, contained 4.5% nitrogen and 22.6% hexose. Isoelectrofocusing demonstrated that this crude inhibitor in the eggplant exocarps was composed of at least three  forms, one of which differed in its isoelectric point. The form at pH 4.7 had the strongest activity. The molecular weights of these inhibitors were estimated to be between 5,000-10,000 by gel filtration.


Ruptured coronary aneurysm and valvulitis in an infant with polyarteritis nodosa. This report describes a 4-mth-old infant with polyarteritis nodosa who had a rapidly fatal course with the diagnosis ultimately being made at necropsy. The pathological lesions consisted of diffuse arterial disease with severe coronary involvement, and additional unusual features included a ruptured coronary artery aneurysm and a valvulitis of the mitral and aortic valves similar to that seen in rheumatic fever. The aetiology of polyarteritis is briefly summarised and the possibility of an immune reaction to foreign antigens, such as drugs or viruses, is considered.


Mannosidosis: phenotype of a severely affected child and characterization of alpha-mannosidase activity in cultured fibroblasts from the patient and his parents. A three-year-old boy has coarse facial features, upper respiratory congestion, profound mental retardation, hepatosplenomegaly, increased height and head circumference, cataracts, a gibbus deformity, radiographic changes of dysostosis multiplex, and vacuolized peripheral lymphocytes. These findings are the most commonly reported clinical features in the previously described patients with mannosidosis. Our patient has a severe deficiency, and his parents have intermediate levels, of the acidic component of alpha-mannosidase in their cultured fibroblasts.


Maternal and fetal plasma levels of free corticosteroids in pathological deliveries. Does intrauterine acidosis induce increased steroid secretion? The concentration of free steroids (CS) increases in both fetal and maternal plasma during labor and delivery. Fetal levels are higher after vaginal than after cesarean section. These differences may indicate an important role of the fetal adrenal gland in the induction of labor or they may reflect merely the fetal response to the stress of delivery. During incrased intrauterine stress steroid secretion is increased as shown here. We examined 41 mothers and their infants during pathological labor. Pathology was assessed from fetal acidosis and/or a clinically obstetric disease of the mother or fetus. The 41 cases included 9 cesarean sections, 8 forceps deliveries; 24 spontaneous deliveries of which 7 were premature. At the time of delivery the pH and CS level were determined in maternal and umbilical vessels in all cases. During spontaneous labor blood samples were also taken during the different stages of labour. A competetive protein binding assay with transcortin without fractionation of the steroids was used. Progesteron was determined by the same assay. The level of this hormone, however, remains unchanged and hence any changes reflect changes in CS. The levels of CS were correlated with the pH values and compared to previously obtained normal values. During pathological deliveries CS levels in both mother and fetus are normal as long as there is no acidosis (Fig. 1). If acidosis is present the CS level in the umbilical artery is usually higher than normal. In 13 out of 18 vaginal deliveries the CS level was above normal, in the other 5 at the upper limit of normal (Fig. 1 and 2). At the same time the a--v difference becomes smaller and sometimes even negative. No changes were noted in maternal and umbilical venous blood (Tab. I and II). Similar dependence on the pH was found for cesarean sections (Tab. III). In premature deliveries without acidosis in the umbilical artery the CS levels were lower in both mother and fetus (Tab. I). These results indicate that the fetal adrenal gland reacts to acidosis, i.e., intrauterine stress, with increased corticosteroid secretion. This rise depends on the pH of fetal blood and not on the type of delivery (Fig. 3).


Gastric acid inactivation of erythromycin stearate in solid dosage forms. The effect of hydrochloric acid at pH 1.2-3.2 ON ERYTHROMYCIN STEARATE AND COMMERCIAL DOSAGE FORMS OF ERYTHROMYCIN STEARATE WAS STUDIED. Under all conditions examined, erythromycin was readily dissolved from the stearate as hydrochloride, and rapidly lost its biological activity in solution. The inclusion of pepsin in the test systems did not affect the results. Although formulation differences somewhat affected the rate of destruction, acid lability was exhibited by all products examined, except enteric-coated tablets. Amounts of acid considered to be normal in the fasting stomach contents of adults during the time likely for a dose to remain in the stomach caused 70-90% destruction within 15 min after the shells started to rupture. Amounts of hydrochloric acid appreciably less than 1 mEq, representing abnormally small quantities even in the fasting state, caused destruction ranging from 30 to 70% of the doses in 15 min. These results are not reconcilable with published statements that the sensitivity of erythromycin to gastric acid is overcome by providing the antibiotic in the form of stearate salt.


Synthesis and alpha-adrenolytic activity of chiral beta-haloethylamines. Several series of N,N-diaralkyl- and N-aryloxyalkyl-N-aralkyl-beta-haloethylamines were synthesized containing centers of chirality in the aralkyl substituent and in the beta-haloethyl moiety to study the stereoselectivity of alpha-adrenolytic activity of the beta-haloethylamines. These compounds were synthesized from starting materials of known absolute configuration via synthetic schemes that did not alter the stereochemical integrity of the chiral centers. Evaluation of the alpha-adrenolytic activities of these beta-haloethylamines on rat vas deferens indicated a variable degree of stereoselectivity of adrenergic blockade, which is interpreted in terms of drug-receptor interactions.


Rapid GLC determination of fusaric acid in biological fluids. A simple, sensitive GLC assay was developed for fusaric acid, the active metabolite of bupicomide, to follow the disposition of this investigational antihypertensive agent in patients undergoing therapy. Fusaric acid is efficiently extracted from biological samples, derivatized by on-column methylation, and chromatographed using flame-ionization detection. An internal standard is utilized to quantitate results. The procedure is rapid and specific for fusaric acid, and has a lower limit of sensitivity of 0.1 mug/ml. The method is suitable for supporting pharmacokinetic studies of bupicomide following therapeutic doses in animals and humans.


Conformational populations for antihistamines and antihypertensives in solution. The conformations in aqueous solution were determined for the XCH2CH2N systems of some antihistamines (H1-receptor antagonists) and some adrenergic neuron blocking agents, including the antihypertensive drugs guanethidine and guanoclor. The rotameric species thought to be the pharmacologically active one is often not the only rotamer present in solution.


Determination of hyoscyamine in BPC mixtures. The hyoscyamine contents of four BPC mixtures (containing either belladonna or hyoscyamus tincture) were determined using the acid-dye technique. A sample size of 10 ml was required. The mean percentage recovery of hyoscyamine ranged from 99.73 to 101.03 from three mixtures; from the magnesium trisilicate and belladonna mixture, it was 94.8. The effects of pH and adsorption on the extraction of the alkaloid-dye complex from the mixtures examined are discussed.


Use of 300-msec microwave irradiation for enzyme inactivation: a study of effects of sodium pentobarbital on acetylcholine concentration in mouse brain regions. Microwave irradiation of 6 kw at 2450 MHz for 300 msec was sufficient to completely inactivate mouse brain cholinesterase and choline acetyltransferase. After this method of sacrifice, the acetylcholine contents of mouse brain regions, given in nanomoles per gram, were found to be: striatum, 81; medulla-pons, 44; diencephalon-midbrain, 34; hippocampus, 31; cerebral cortex, 26; and cerebellum, 17. Sodium pentobarbital caused a dose-dependent increase in whole brain acetylcholine. A maximal increase of 81% in whole brain was seen at 15 minutes with 80 mg/kg of sodium pentobarbital. The increase in acetylcholine after sodium pentobarbital treatment was not caused by anoxia from respiratory depression or by hypothermia. All brain regions except the cerebellum exhibited an increase in acetylcholine after pentobarbital treatment. Fifteen minutes after treatment, cerebellar acetylcholine was significantly decreased. However, at the time when half of the animals had regained the righting reflex, the unconscious mice showed an increase in cerebellar acetylcholine which was statistically significant  as compared to control. The relative accumulation rate of acetylcholine calculated for cerebral cortex and hippocampus was higher than that for striatum although the absolute rate of accumulation of ACh was higher in the striatum. Thus, after sodium pentobarbital treatment, the cerebral cortex and hippocampus exhibit a greater cholinergic response than the striatum.


A test for antinociceptive activity of narcotic and narcotic antagonist analgesics in the guinea pig. Ethylenediamine tetraacetic acid (EDTA) was used as a nociceptive stimulus intradermally in the guinea pig. The responses evoked by EDTA included vocalization, biting and scratching at the site of injection and escape behavior. Nociception by EDTA was shown to be the result of its cation chelating activity. The suppression of EDTA-induced responses proved to be a rapid and effective antinociceptive test. The narcotic analgesics morphine, codeine, meperidine and methadone and the narcotic antagonist analgesics cyclazocine, cyclorphan, nalorphine and pentazocine were active. The slopes of the dose-response curves of the narcotic antagonist analgesics were significantly shallower than those of the narcotic analgesics. The test was highly specific for these analgesics. Antipyretic analgesics and various nonanalgesic drugs were inactive.


Pharmacological characterization of adrenergic receptors of a rabbit cerebral artery in vitro. In the light of controversy over the functional significance of the abundant sympathetic innervation of large cerebral blood vessels, an in vitro analysis of the adrenergic receptors mediating contractile effects in the rabbit basilar artery was undertaken. Beta adrenergic stimulation had no effect, and agents that block norepinephrine (NE) uptake did not alter contractile responses to l-NE. The l-NE dose-response curve could be resolved into two components S-shaped curves: the first had an ED50 OF 10(-5) M and reached a plateau at 10(-4) M; the second component continued above 10(-3) M without reaching a plateau. Phenylephrine and epinephrine dose-response curves were also biphasic; d-NE responses corresponded to the second phase of the l-NE curve. Relative potencies for the two components were different. For the first component, these were 1:0.23:0.18:0.03 for l-NE, epinephrine, phenylephrine and d-NE, respectively; relative potencies for the second component of the curve were 1:1:0.11:0.23. Phentolamine dissociation constants were analyzed separately for each component. The value for low l-NE concentrations was 5 X 10 (-8) M and, for higher concentrations, it was 3 X 10(-6) . The insensitivity of the alpha adrenergic receptor and the poor responsiveness of the muscle to its activation with agonist concentrations below 10(-4) M can probably account for the small contractile responses to nerve stimulation of large pial arteries in spite of their abundant innervation.


Cation-binding property of choroid plexus peptide IIF. Preparations of the melanotropic-lipolytic peptide IIF from bovine choroid plexus contain 3% Ca, 1% Mg and less than 0.1% Na and K. Ca and Mg were removed by gel filtration on Sephadex G-10 in 1 N acetic acid. Capacity of the resulting cation-free peptide to bind Ca++, Mg++, Na+ and K+ was examined with the method of Hummel and Dreyer (Biochim. Biophys. Acta 63: 530-532, 1962). Peptide IIF bound a maximum of 3.7 to 4.4 mEq of Ca++, Mg++, Na+ or K+ per g of peptide. For each cation, linear Scatchard plot indicated one class of binding site. Association constants (liters per mole) were Ca++, 4.3 X 10(4); Mg++, 3.6 X 10(4); Na+, 4.8 X 10(2), K+, 1.9 X 10(2). Competitive binding experiments showed that all four cations occupied the same class of binding site.


A study of the mechanism of transport of diphenylhydantoin in the rat submaxillary gland in vitro. In vitro studies performed on rat submaxillary gland slices with diphenylhydantoin (6 X 10(-6) M) showed that variations of extracellular pH (pHe) had no significant effects on uptake and only slight effects on efflux (lowering the pHe slightly decreased efflux rates, whereas increasing pHe slightly increased efflux rates). Increasing diphenylhydantoin concentration significantly decreased uptake and slightly increased the rate of efflux. Uptake of 14C-diphenylhydantoin was significantly decreased when compared to control conditions (pH 7.40, 37 degrees C, 100% O2 aeration, 6 X 10(-6) M) by the following alterations: aeration of the incubation medium with N2 instead of O2, decrease of bath temperature from 37 degrees C to 5 degrees C and addition of the following metabolic inhibitors: iodoacetic acid (10(-3)M), 2,4-dinitrophenol (10(-3)M) and cyanide (10(-3)M). Probenecid (10(-3)M) had no significant effect on diphenylhydantoin uptake when compared to control values. No evidence of salivary biotransformation of diphenylhydantoin was seen in the in vitro system using thin-layer chromatography. These in vitro data suggest an active transport process that is concentration-dependent with a possible saturable binding site on the membrane or in the interior of the cell.


Aminopyridines and sparteine as inhibitors of membrane potassium conductance: effects on Myxicola giant axons and the lobster neuromuscular junction. The effects of the compounds 2-, 3- and 4-aminopyridine and sparteine on membrane conductance changes were examined using both voltage-clamped Myxicola axons and the lobster neuromuscular junction. In Myxicola axons, the aminopyridines very specifically inhibited the potassium conductance when applied at concentrations of 0.1 mM to 5 mM without any apparent effect of resting membrane potential. Concentrations in excess of 5 mM were needed to inhibit noticeably the sodium conductance. Potassium conductance-voltage curves were shifted in the depolarized direction along the voltage axis with no significant change in shape. There were only minor changes in the kinetics of potassium activation. In high potassium solutions, both inward and outward potassium currents were equally sensitive to the aminopyridines. Sparteine was, in general, found to be a more potent, but somewhat less specific, inhibitor of the potassium conductance. In contrast to the aminopyridines, sparteine was more effective when applied at basic pH and in addition tended to produce a noticeable degree of potassium inactivation. When applied to the lobster neuromuscular junction, 2-aminopyridine and sparteine dramatically increased the amplitude of both excitatory and inhibitory postjunctional potentials, with little or no change in resting potential, resting input conductance, reversal potential, or miniature end plate potential amplitude or frequency. Quantal content per fiber was increased by approximately a factor of 3 for the excitatory responses.


Effects of calcium on the local anesthetic suppression of ionic conductances in squid axon membranes. The effects of varying the external calcium concentration on the suppression of membrane ionic conductances by procaine and benzocaine have been examined under voltage-clamped conditions. The suppression of peak conductance and steady-state conductance by procaine or benzocaine applied externally or internally was not affected by changing the external calcium concentration between 10 and 100 mM. When the calcium concentration was lowered below 10 mM (5 or 2 mM), the procaine effect was slightly potentiated. This augmentation could not be ascribed to an acceleration in the rate of penetration of procaine into the axon in low calcium solutions. The resting membrane conductance was slightly decreased by procaine in a manner independent of the external calcium concentration. The maximum effect of procaine on resting conductances was obtained at a concentration much lower than that required for maximum suppression of peak and steady-state conductances. The present results are not compatible with the hypothesis that calcium competes with local anesthetics for a negatively charged site on the membrane. It is suggested that hydrophobic interactions of local anesthetic molecules with the membrane affect the resting membrane conductance whereas coulombic interactions are responsible for the conductance changes observed during nerve activity.


Twitch potentiation of skeletal muscle by physostigmine at different pH. This report extends earlier research, done with external media at pH 7.2, to new studies at pH 6.4 and 8.4 as well as 7.2, to determine the roles of the protonated and neutral forms of physostigmine (a weak base with pKalpha = 8.2) in causing twitch potentiation of the frog sartorius muscle. Physostigmine, especially at relatively high pH (8.4) and concentration (1.5 mM), considerably blocks excitation. However, the results show in general that physostigmine potentiation increased peak contraction time and thereby indicate that potentiation is occurring in terms of prolongation of the active state. At pH 6.4 and 8.4, 1 mM physostigmine causes no change in the mechanical threshold of K depolarization contractures of toe muscles, as found previously at pH 7.2. Physostigmine increasingly prolongs the action potential as pH rises, i.e., in positive correlation with the twitch potentiation, thus indicating that this electrical change is the prime determinant of the potentiation.


The renal blood flow and the glomerular filtration rate of anaesthetized dogs during acute changes in plasma sodium concentration. 1. The effects of acute changes in plasma Na concentration (P(Na)) on renal blood flow (RBF) and glomerular filtration rate (GFR) were studied in anaesthetized greyhounds. Saline was infused at a constant rate (0.1 ml. kg(-1) min(-1)) either into a renal artery or into a systemic vein. Plasma Na concentration was altered by varying the Na concentration of the infused saline from 0.154 to 0.077, 0.616 or 1.232 M.2. Blood pressure (B.P.), packed cell volume (PCV), concentration of plasma solids (PS) and the plasma concentration of H(+) and K (P(K)) ions were measured but no attempt was made to contain their fluctuation.3. An infusion of hypertonic saline into a renal artery usually led to an ipsilateral increase in RBF for 5-15 min, followed by a progressive fall. Over-all, mean values of RBF fell with P(Na) throughout the range studied (120-190 m-mole l.(-1)). Glomerular filtration rate rose with P(Na) to reach maximal values at P(Na) levels of 140-160 m-mole l.(-1), but fell thereafter. The combined fall in RBF and GFR, without change in filtration fraction, at P(Na) values above 160 m-mole l.(-1) is consistent with an alteration in afferent arteriolar resistance. The fall in GFR despite a rise in RBF noted when P(Na) was reduced below 140 m-mole l.(-1) requires an additional explanation.4. Renal blood flow was independent of P(K); it was inversely related to [H(+)] and directly related to PS. Glomerular filtration rate was independent of PCV and P(K). It was also inversely related to [H(+)] and directly related to PS up to a value of 6 g 100 g(-1) plasma, after which the relationship was reversed. These results suggest that the renal vascular responses to acute changes in P(Na) may be mediated in part, at least, by concurrent change in PS and [H(+)].


Hydrolytic enzymes of Euglena gracilis: characterization and activity as a function of culture age and carbon deprivation. Optimal assay conditions are described for 8 hydrolases of Euglena gracilis var. bacillaris, SM-L1 (streptomycin-bleached) strain, 7 of which have an acid pH-optimum. Acid-phosphatase, beta-galactosidase, beta-glucosidase, b-fucosidase, cathepsin D, RNase, DNase, and an esterase are active in cell homogenates. Amylase has very low activity, and beta-glucuronidase, arylsulfatase, beta, N-acetyl-glucosaminidase, alpha-fucosidase, and alpha- and beta-mannosidase are inactive.


Adrenergic sulfonanilides. 4. Centrally active beta-adrenergic agonists. The central nervous system (CNS) activities of a number of soterenol analogs have been investigated, and several of these compounds possessed potent morphine antagonistic and anorexiant properties. The CNS activity of these compounds was enhanced by certain lipophilic [e.g., 1,1-dimethyl-2-phenethyl (43) or cyclopropyl (40 and 44)] nitrogen substituents; however, minor structural changes on either the aromatic or side-chain moieties drastically reduced central activity. Toxicity in this series was related to the inherent alpha-adrenergic stimulating component (direct or indirect).


Ontogenetic development of NADH-dependent methemoglobin reductase in erythrocytes of man and rat. Ontogenetic development of NADH-dependent methemoglobin reductase was followed in humans and rats. The human kinetic profile differs from that in the rat. The low level of methemoglobin reductase in human infants at birth and for the first months of life may provide a partial explanation of the particular susceptibility to methemoglobinemic agents of this age group.


Biotransformation, sex hormones, and toxicity of two volatile anesthetics in mice. Male and female DBA 11 mice recovered from 1 hr of anesthesia with chloroform of fluoroxene apparently unharmed. However, many of the animals died within 24-48 hr after anesthesia. Pretreatment with phenobarbital increased, while pretreatment with a small dose of carbon tetrachloride decreased, this toxicity. Relatively more males than females died. Pretreatment with estradiol in males and testosterone in females reversed this ratio. We conclude that the murine toxicity of chloroform and fluoxene is dependent on biotransformation by hepatic microsomal enzymes and that the testosterone enhances postanesthetic toxicity of these agents.


Neuronal control of neurotransmitters biosynthesis during development. Studies on neuronal control mechanisms of neurotransmitters biosynthesis during the development of peripheral and central autonomic synapses are reviewed. Particular emphasis is placed on investigations of developing peripheral sympathetic ganglia and brain in chick embryo and chick. Studies on the development of autonomic neurons and synapses under different pharmacological conditions are reported. Principally the effect of a) the administration of drugs and precursors such as L-dopa, 3H-dopa, 6-OH dopa; b) the prenatal administration of reserpine; c) the blockade of cholinergic receptors; d) the nerve growth factor (NGF) is analyzed. Results of developmental studies on chick ciliary ganglia are summarized. The review particulary underlines the importance of combining the use of sensitive microchemical methods to pharmacological tools in exploring the development of regulatory mechanisms at the cellular level.


Isolation and properties of the replicase of encephalomyocarditis virus. The RNA-dependent RNA polymerase (replicase) of encephalomyocarditis (EMC) virus was found to be closely associated with the smooth membranes of infected BHK-21 cells. An RNA-dependent EMC replicase was extracted from the membranes with 0.15% sodium dodecyl sulfate (SDS) and 1,1,2-trichlorotri-fluoroethane (Genetron 113) and further purified by high-salt dextran-polyethylene glycol phase separation, sievorptive chromatography, and glycerol gradient sedimentation. The enzyme does not manifest strict specificity toward EMC RNA template. It can use also Qbeta RNA, rRNA of BHK cells, or poly(C). SDS-polyacrylamide gel electrophoresis of purified EMC replicase labeled with radioactive methionine revealed that, of all the stable EMC proteins, the enzyme contains predominantly the 56,000-dalton (E) polypeptide.


Comparative properties of bacteriophage phi6 and phi6 nucleocapsid. Nonionic detergent treatments released a nucleocapsid from the enveloped bacteriphage phi6. The nucleocapsid sedimented at nearly the same rate as the whole phage in sucrose density gradients, but the buoyant density in Cs2S04 changed from 1.22 g/cm3 for the whole phage to 1.33 g/cm3 for the nucleocapsid. The detergent completely removed the lipid and 5 of the 10 proteins from the phage. Surface labeling of the phage and nucleocapsid with 125I revealed that protein P3 was on the outer surface of the whole phage and P8 was on the surface of the nucleocapsid. Both the phage and the nucleocapsid were stable between pH 6.0 and 9.5. Low concentrations of EDTA (10-4 M) dissociated the nucleocapsid but had no effect on the whole phage. The nucleocapsid contained all three double-stranded RNA segments, as well as RNA polymerase activity.


Urologic manifestations of polyarteritis nodosa. Polyarteritis nodosa is an unusual condition that infrequently presents with signs and symptoms essentially confined to the urinary system. Such a case necessitating bilateral nephrectomy owing to hemorrhage is described. The pathologic findings, pathophysiology and etiology of the condition as well as treatment by renal transplatation are discussed.


[Effects of pH on the processes of excitation-contraction coupling of bullfrog atrium (author's transl)]. It has long been demonstrated that in cardiac muscle alterations in the extracellular pH exhibit a remarkable inotropic effect, whereas the precise mechanism is not fully clarified yet in spite of many associated thories. In recent years, however, abundant informations about the processes of excitation-contraction coupling (E-C coupling) of cardiac muscle have been obtained. Accordingly, the mechanisms of inotropic actions of pH on cardiac muscles must also be re-examined on the basis of these lately developed theories of E-C coupling. In the present experiment, therefore, we attempted to elucidate the fundamental mechanism of actions of pH upon several processes of E-C coupling using the bullfrog atrium whose processes of E-C coupling have been fairly well known...


[Gentamicin-susceptibility of various pathogens isolated from clinical materials]. We studied on the antibacterial activity of gentamicin against various pathogens isolated from clinical materials mainly isolated during 1974 and 1975, comparing with other antibiotics. Beta hemolytic streptococci, pneumococci and enterococci are less susceptible to gentamicin than staphylococci. Staph, aureus and Staph. epidermidis resistant to various antibiotics are very susceptible to gentamicin, and no resistant strain to this drug was found. Haemophilus influenzae, H. parainfluenzae and H. parahaemolyticus are very susceptible to gentamicin, and there is no resistant strain to this drug. Escherichia coli, Klebsiella, Citrobacter, Serratia and five species of Proteus are more susceptible to gentamicin and tobramycin than dibekacin and amikacin. A few resistant or less susceptible strains to gentamicin are found in E. coli, Citrobacerr, Serratia, Pr. morganii and Pr. rettgeri. Pr. inconstans is less susceptible to gentamicin than other species of Proteus. Antibacterial activity of gentamicin against Pseudomonas aeruginosa is very strong, but dibekacin and tobramycin are stronger. Gentamicin-resistant strains of Pseudomonas aeruginosa are now rather few.