Chicken (Gallus gallus) hemolytic complement: optimal conditions for its titration. The effect of pH, ionic strength, cation concentration, ethylenediaminetetraacetic acid trisodium salt (Na3EDTA), time and temperature were studied to determine the optimal conditions for titrating the hemolytic complement (C) activity in sera of chicken (Gallus gallus). Swine erythrocytes (E) sensitized with rabbit antibodies were the most sensitive, while chicken serum had a smaller amount of "natural" antibody against them than against red blood cells from other five species tested. The highest titers of chicken C, when tested with swine sensitized E, were detected when isotonic NaCl-barbital buffer was used as diluent, having the ionic strength of 0.15, conductance of 11 millimhos/cm at 20 degrees C. However, maximal chicken C titers detected with sensitized rabbit E were obtained at ionic strength of 0.07 to 0.11 depending on pH. A final concentration of 1 X 10(-3) M of Mg2+ and 3 X 10(-4) M of Ca2+ and pH 8 were optimal in both cases. The temperature of 30 degrees C and time of 60 minutes were appropriate to reveal the maximal titers..


Purification of soluble complement-fixing antigens from two Burkitt's lymphoma cell lines. Conditions affecting the stability and recovery of antigens and antibodies. Soluble Epstein--Barr virus (EBV) associated antigens of P3HR-1 and Raji cell lines, which fix complement in the presence of human sera containing antibody against EBV--VCA antigens, were partially purified by fractionating centrifuged cell lysates by the method of immunoadsorbents or by precipitation with ammonium sulphate or glycine--HCl. Purification conditions were limited by the instability of both antigen and antibody activity. The antibody was inactivated under all the conditions commonly used to dissociate antigen--antibody complexes. The antigen was inactivated at pH 2-3, and was irreversibly bound to Sephadex G-200 conjugates which had not been exposed to large excesses of serum.


[Changes in the acid-base equilibrium in hypokalemic patients during potassium infusions with various anions]. Based on the parameters of the acid-base status, the effect of different anions in potassium and magnesium substitution was studied. A mixture of chloride with the metabolisable anions acetate and malate does not cause any changes in the acid-base balance. The exclusive supply of metabolisable anions in the form of aspartate always results in a change of blood pH values towards an alkalosis.


[Acid-base equilibrium- -disorders and therapy]. The purpose of this work is to give a brief resume, with figures and tables, of the knowledge concerning the acid-base balance which is indispensable in intensive therapy. The effects of disturbances of the acid-base balance on organic functions are shown and, therefore, the importance of a situation-adapted therapy is particularly stressed. In this connection, the role of infusion therapy in the treatment of disturbances of the acid-base balance is especially discussed.


[Systematic and random errors in the determination of parameters of the acid-base equilibrium]. A review is given of the most common errors made in the estimation of the parameters of acid-base balance. Especially important are the systematic errors, for instance in handling the blood before estimation, by use of incorrect standard solutions or poor temperature control during the measurement of the pH. By paying attention to these details, it is possible to obtain excellent precision in final measurement in the standard apparatus.


Barriers to the nurse practitioner movement: problems of women in a woman's field. Barriers to the nurse practitioner movement are explored in this article. Historically, the subordination of women and the sex segregation of nursing and medicine have helped to establish interaction patterns between the two professions which included a subordination of nurses as well as informal doctor-nurse games. These patterns were reinforced by hospital training schools and state laws which restricted the roles of nurses. Recent changes in state movement, but the stereotyped communication patterns between nurses and physicians remain as a barrier to the full use of the talents of nurses in practitioner roles.


The methylsulfonylethyloxycarbonyl group, a new and versatile amino protective function. A new amino protecting group, the methylsulfonylethyloxycarbonyl (Msc) group, is described which combines well with other familiar groups (benzyloxycarbonyl, t-butyloxycarbonyl) in peptide syntheses. Its main characteristics are an extreme acid stability, a high base lability and a high polarity which enhances solubility in polar solvents including water. The group resists catalytic hydrogenolysis but does not poison the catalyst. It has good crystallizing properties. Application in peptide synthesis is exemplified in the synthesis of Msc-Phe-Arg-Trp-Gly-OMe.HCl. Deprotection of the masked tetrapeptide was accomplished within 5 sec with a 1.0 N solution of base (OH- and OCH3-). A reaction scheme for the cleavage of the group is suggested.


Radiation inactivation of rabbit muscle aldolase. Pulse radiolysis and steady-state X-radiolysis have been used to investigate the radiation inactivation of aldolase from rabbit muscle. Both eaq-and OH readily react with aldolase, and contribute to inactivation. The radical anions (CNS)2-and (Br)2-react with aldolase at neutral pH. The progressive addition of alkali results in an increase in the second-order rate constants, with an apparent pK approximately 10 +/- 0-3, and with the formation of an unstable intermediate, lambdamax approximately 400 nm resembling a phenoxyl radical. Steady-state radiolysis in the presence of (CNS)2-and (Br)2- at alkaline pH results in increased aldolase inactivation, with a pK of enzyme inactivation similar to that observed for reaction of the radical anions. We propose that a reaction of the radical anoins with tyrosine residues accounts for the resultant inactivation.


Folic acid conjugase from plasma. III. Use of the enzyme in the estimation of folate activity in foods. A convenient method for the estimation of food folates using human plasma as a source of folic acid conjugase has been standardized. Bovine, goat and dog plasma could not be used for the purpose because of low enzyme activity and slow reaction rates. Human plasma conjugase released food folates from conjugated forms over a pH range of 4.5 to 6.0. The food folate values obtained by employing rat liver and chicken pancreas conjugase closely agreed with the values obtained by using human plasma conjugase. The advantages in using human plasma as a source of conjugase were (1) easy availability (2) non-essentiality of preliminary processing (3) stability of the enzyme on storing plasma in cold (4) low enzyme blank folate activity and (5) low amount of plasma needed for assay. Using this method, folic acid activity of some common Indian foods of plant and animal origin has been estimated. There were wide variations in the folate activity of different classes of foods. In general legumes and green leafy vegetables were found to be rich in folate activity.


Standardized aerobic and anaerobic exercise: differential effects on intraocular tension, blood pH, and lactate. The effects of standardized aerobic and anaerobic exercise intensities on intraocular tension, blood lactate, and pH were studied. Intraocular tension decreased rapidly at all exercise intensities. The absolute lowest level of intraocular tension reached with aerobic and anaerobic exercise levels varied by only 1.5 mm. Hg and this difference was not statistically significant. Blood lactate and pH changes correlated with intraocular tension changes at anaerobic exercise levels, but not at aerobic exercise levels. These findings associated with aerobic exercise have not been previously reported. It is suggested that parameters other than the decrease in blood pH and the increase in blood lactate are responsible for most of the decrease in intraocular tension associated with dynamic exercise.


Reversal of oliguria and renal cortical ischemia of hemorrhagic shock in the dog with tris (hydroxymethyl) amino methane (THAM). Hemodynamic studies. We investigated the functional and hemodynamic effects on the kidney of tris (hydroxymethyl) amino methane (THAM) infusion during sustained hemorrhagic hypotension in dogs. The large osmotic load introduced into the vascular compartment during hemorrhagic hypotension resulted in a marked reduction in systemic vascular resistance and a return of cardiac output, total renal blood flow, and the renal arteriographic pattern to normal range. The observed diuresis is probably due to an osmotic effect, because the glomerular filtration rate remains significantly depressed. Anatomical assignment of the mathematical components of the xenon washout curve led to an assessment of intrarenal blood flow distribution that was at variance with the arteriographic pattern and the intrarenal distribution of tagged microspheres.


The use of hexazonium-p-rosanilin in the histochemical demonstration of peptidases. The suitability of hexazonium-p-rosanilin (HP) in the histochemical demonstration of peptidases was investigated. The detection was carried out in cold mictrotome sections adherent to slides or semipermeable membranes. Alanyl-1-naphthylamide, alanyl-2-naphthylamide, leucyl-2-naphthylamide, leucyl-4-methoxy-2-naphthylamide (all substrates in concentration of 0.4 mg/1 ml of citrate phosphate buffer pH 6.5), gamma-L-glutamyl-1-naphthylamide, gamma-L-glutamyl-2-naphthylamide (both substances in concentration of 0.24 mg/1 ml of acetate buffer pH 6.5) were used as the substrates. Results were compared with those obtained with Fast Blue B and Fast Garnet GBC. In comparison with Fast Blue B and Fast Garnet GBC HP is a faster coupler, furnishes azodyes which are stable, amorphous (even without lipid extractions from sections), more substantive and in the case of 1-naphthylamine almost insoluble in ordinary lipid solvents used for the dehydration and clearing of sections before mounting. The molecular extinction coefficient of azodyes furnished by HP is 1.5X higher for 1-naphthylamine than for 2-naphthylamine. It is higher than that of Fast Garnet GBC, however, lower than that of Fast Blue B. The inhibitory influence of individual diazonium salts on enzyme activity (activities) splitting leucyl-2-naphthylamide amounts to 36% (Fast Garnet GBC), 37% (Fast Blue B), 52% (HP, 0.03 ml/1 ml) and 63% (HP, 0.09 ml/1 ml) at pH 6.5. For gamma-glutamyl-transpeptidase the corresponding values are 50%, 59%, 62% and 67%. The higher inhibitory influence of HP is compensated by the possibility of its using in the technic of semipermeable membranes. HP improves greatly the localization of peptidases in cold microtome sections from which lipids were not extracted. The best results are furnished by 1-naphthylamine dervatives. In the case of 4-methoxy-2-naphthylamine derivatives the localization is very sharp, however, the azodye is less distinct than that of 2-naphthylamine. The localization as obtained with HP in combination with substrates derived of simple naphthylamines is similar or even better than with 4-methoxy-2-naphthylamine derivatives applied with Fast Blue B. Typical examples are shown.


Contrast and electrolyte dynamics of the intravenous pyelogram. I. Urinary pH and volume changes in the canine I. V. P. Canine peripheral venous or suprarenal aortic injections of sodium or meglumine iothalamate produced a significant swing towards an alkaline urine only in individual dogs injected with meglumine salts. When mean values were compared for the sodium or meglumine groups as a whole, however, no significant differences could be established for pH change or induced diuresis. With both salts, ATPase inhibition could play a role in urinary electrolyte excretion. Carbonic anhydrase inhibition does not seem to play a significant role in urinary pH changes induced by contrast media.


The UV fading of hydrocarbon fluorescence and its prevention for observations in single living cells. Excitation intensities used for standard microspectrofluorometric observations of natural cell fluorescence, i.e. NAD(P)H, lead to fading of hydrocarbon (polycyclic aromatic, heterocyclic) fluorescence in EL2 cells incubated with such compounds. The disappearance of hydrocarbon fluorescence under excitation at 366 nm seems to be an exponential function of time. The fading prevents studies on hydrocarbon metabolization in correlation with intracellular microelectrophoretic injection of substrate, e.g. glucose-6-P. A return to 8-10 times less intense excitation conditions used in an earlier prototype microspectrofluorometer, has allowed the observation of sequential changes in the difference spectra (after glucose-6-P minus before) of hydrocarbon-treated cells (e.g. benzo(a)pyrene, dibenzocarbazols). The possible relative contributions of NAD(P)H and hydrocarbon metabolites (or alterations) to such sequential spectra are still under consideration, but the main obstacle to their observation, fading, is removed by less intense excitation.


Mutational biosynthesis of a new antibiotic, streptomutin A, by an idiotroph of Streptomyces griseus. Microorganisms producing antibiotics have been genetically converted by earlier workers to mutants which cannot produce antibiotic without supplementation with a moiety of the antibiotic. These antibiotics include neomycin, kanamycin, paromomycin, butirosin, sisomicin, ribostamycin and novobiocin. Success has not been reported for organisms producing guanidinocyclitol antibiotics such as streptomycin. We mutagenized conidia of the streptomycin-producing Streptomyces griseus strain 7-455F3 with nitrosoguanidine at pH 7.0. Non-producers of streptomycin were visually selected by the agar-plug technique using Bacillus subtilis. We successfully isolated mutant MIT-A5 which produces no streptomycin unless streptidine is added to the agar medium. The streptidine-dependent phenotype was confirmed in submerged culture in flasks. Attempts to produce new antibiotics by feeding aminocyclitols to mutant MIT-A5 failed. However a new antibiotic (streptomutin A) was produced by supplementation with the guanidinocyclitol, 2-deoxystreptidine. We propose the term "mutational biosynthesis" for the production of new metabolites by the use of mutants blocked in the biosynthetic pathway to the secondary metabolite. We further propose the term "idiotroph" to properly describe such mutants.


Biological effects of lipoteichoic acids. Pneumococcal lipoteichoic acid (Forssman antigen) added to the medium of growing pneumococcal cultures caused chain formation, prevented culture lysis in the stationary phase of growth, and inhibited lysis by penicillin and by the pneumococcal bacteriophage Dp-1.


Peptidoglycan synthesis in L-phase variants of Bacillus licheniformis and Bacillus subtilis. Stable L-phase variants isolated from Bacillus licheniformis and Bacillus subtilis, when grown in osmotically stabilized media, do not synthesize peptidoglycan but have been found to accumulate the nucleotide precursors of this polymer. The enzymes involved in the synthesis of these precursors and the later membrane-bound stages of peptidoglycan synthesis have been investigated, and the L-phase variants have been shown to contain lesions, which provide a rational explanation for the absence of peptidoglycan and for the nature of the precursor accumulated. The majority of the L-phase variants contained a single enzymic defect, but two strains were isolated with double lesions. Five out of seven strains examined accumulated uridine 5'-diphosphate (UDP)-MurAc-L-ala-D-glu and were unable to synthesize diaminopimelic acid as a consequence of a defect in aspartate-beta-semialdehyde dehydrogenase activity. Two strains were deficient in UDP-MurAc: L-alanine ligase and accumulated UDP-MurAc. One strain accumulated the complete nucleotide precursor UDP-MurAc-L-ala-D-glu-mA2pm-D-ala-D-ala and was deficient in phospho-N-acetylmuramyl pentapeptide translocase. A second strain also had this lesion, together with defective aspartate-beta-semialdehyde dehydrogenase activity. The other enzymes of peptidoglycan synthesis were present in the L-phase variants, with activities similar to those found in the parent bacilli grown under identical conditions. Membrane preparations from certain of the L-phase variants were also capable of synthesizing the secondary polymers poly(glycerol phosphate) teichoic acid and teichuronic acid and also a polymer of N-acetylglucosamine.


Enzymology and genetic regulation of a cyclic nucleotide-binding phosphodiesterase-phosphomonoesterase from Aspergillus nidulans. A cyclic nucleotide-binding phosphohydrolase that possesses both a phosphomonoesterase and a phosphodiesterase catalytic function has been partially purified from Aspergillus nidulans. The enzyme hydrolyzes both p-nitrophenylphosphate and bis-(p-nitrophenyl)-phosphate. o'-Nucleoside monophosphates are the best physiological phosphomonesterase substrates but 5'- and 2'-nucleoside monophosphates are also hydrolyzed. The enzyme catalyzes the hydrolysis of adenosine 5'-triphosphate, adenosine 5'-diphosphate, and 2',3'- and 3'5'-cyclic nucleotides, but not of ribonucleic acid, deoxyribonucleic acid, or nicotinamide adenine dinucleotide. The enzyme has acid pH optima and is not activated by divalent cations. Nucleosides and nucleotides inhibit the enzyme. Cyclic nucleotides are competitive inhibitors of the phosphodiesterase-phosphomonoesterase. The enzyme can occur extracellularly. The phosphodiesterase-phosphomonoesterase is present at high levels in nitrogen-starved mycelium, and it is strongly repressed during growth in media containing ammonium or glutamine and weakly repressed during growth in glutamate-containing medium. Experiments with various area mutants show that this regulatory gene is involved in the control of the enzyme. No evidence for regulation of the enzyme by carbon or phosphorus starvation has been found.


Glutamate dehydrogenase from Escherichia coli: purification and properties. Glutamate dehydrogenase (L-glutamate:NADP+ oxidoreductase [deaminating], EC 1.4.1.4) has been purified from Escherichia coli B/r. The purity of the enzyme preparation has been established by polyacrylamide gel electrophoresis, ultracentrifugation, and gel filtration. A molecular weight of 300,000 +/- 20,000 has been calculated for the enzyme from sedimentation equilibrium measurements. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate and sedimentation equilibrium measurements in guanidine hydrochloride have revealed that glutamate dehydrogenase consists of polypeptide chains with the identical molecular weight of 50,000 +/- 5,000. The results of molecular weight determination lead us to propose that glutamate dehydrogenase is a hexamer of subunits with identical molecular weight. We also have studied the stability and kinetics of purified glutamate dehydrogenase. The enzyme remains active when heat treated or when left at room temperature for several months but is inactivated by freezing. The Michaelis constants of glutamate dehydrogenase are 1,100,640, and 40 muM for ammonia, 2-oxoglutarate, and reduced nicotinamide adenine dinucleotide phosphate, respectively.


Isolation and characterization of the newly evolved ebg beta-galactosidase of Escherichia coli K-12. The ebg beta-galactosidase of Escherichia coli K-12 strain LC110 has been purified and characterized. Strain LC110 is a Lac+ revertant of a mutant with a deletion of the lacZ beta-galactosidase gene. Its new ebg beta-galactosidase activity was shown to be due to a discrete protein, immunologically unrelated to lacZ beta-galactosidase. Its kinetics of action conformed to those of a simple conventional enzyme. With o-nitrophenyl-beta-D-galactoside as substrate, the Vmax was 11,200 nmol/min per mg of enzyme, the Km was 5 mM, and the activation energy was 12,400 cal/mol. Corresponding values for lacZ beta-galactosidase of wild-type E. coli K-12 were 350,000 nmol/min per mg of enzyme, 1.3 mM, and 8,000 cal/mol. A series of sugars has been examined as competitive inhibitors of ebg beta-galactosidase. Kinetic analyses suggest that ebg beta-galactosidase has a particularly high affinity for galactosamine and gamma-galactonolactone, binds galatose more tightly than lactose, and shows a general preference for monosaccharides rather than beta-galactosides. We conclude that the ebg beta-galactosidase may have arisen by modification of a gene involved with the metabolism of a monosaccharide, possibly a 2-amino sugar.


Selective release of a deoxyribonucleic acid-binding factor from the surface of competent pneumococci. Methods are described that resulted in the selective release of deoxyribonucleic acid (DNA)-binding factor from the surface of competent pneumococci. The same methods caused a parallel inactivation of the DNA-binding capacity of the extracted bacteria. Genetically or physiologically incompetent pneumococci did not yield binding factor upon exposure to the same methods. The solubilized binding factor appeared to be a protein; it could be assayed by a membrane filter binding procedure. The binding factor had properties reminiscent of those of the DNA receptors of transformable pneumococci (Seto et al., 1975).


Serine transhydroxymethylase isoenzymes from a facultative methylotroph. Two serine transhydroxymethylase activities have been purified from a facultative methylotrophic bacterium. One enzyme predominates when the organism is grown on methane or methanol as the sole carbon and energy source, whereas the second enzyme is the major isoenzyme found when succinate is used as the sole carbon and energy source. The enzyme from methanol-grown cells is activated by glyoxylate, is not stimulated by Mg2+, Mn2+, or Zn2+, and has four subunits of 50,000 molecular weight each. The enzyme from succinate-grown cells is not activated by glyoxylate and is stimulated by Mg2+, Mn2+, and Zn2+, and sodium dodecyl sulfate-acrylamide gel electrophoresis indicates that this enzyme has subunit molecular weight of 100,000, the same as the molecular weight obtained for the active enzyme. Cells grown in the presence of both methanol and succinate incorporate less methanol carbon per unit time than cells grown on methanol and have a lower specific activity of the glyoxylate-activated enzyme than methanol-grown cells. Adenine, glyoxylate, or trimethoprim in the growth medium causes an increased level of serine transhydroxymethylase in both methanol- and succinate-grown cells by stimulating the synthesis of the glyoxylate-activated enzyme.


Ethanolaminephosphate cytidylyltransferase. Purification and characterization of the enzyme from rat liver. Ethanolaminephosphate cytidylyltransferase (EC 2.7.7.14), which catalyzes a central step in phosphatidylethanolamine synthesis, has been purified 1000-fold from a postmicrosomal supernatant from rat liver. The enzyme, which requires a reducing agent, like dithiothreitol, for activity, is stable for weeks at 0-4 degrees when stored in the presence of dithiothreitol and in the pH range 7.5 to 9.0. A molecular weight of 100 to 120 X 10(3) was estimated by gel chromatography on Sephadex G-200. Gel electrophoresis in the presence of sodium dodecyl sulfate gave only one protein band with an apparent molecular weight of 49 to 50 X 10(3). The reaction catalyzed by the enzyme is reversible with a Keq for the forward reaction of 0.46 under the assay conditions. Michaelis constants of 53 and 65 muM were determined for CTP and ethanolaminephosphate, respectively. From the product inhibition pattern an ordered sequential reaction mechanism is proposed, in which CTP is the first substrate to add to the enzyme and CDP-ethanolamine is the last product to be released. The possible role of this reaction in the regulation of phosphatidylethanolamine synthesis in liver is discussed.


Refolding of reduced, denatured trypsinogen and trypsin immobilized on Agarose beads. The reoxidation of fully reduced and denatured bovine trypsinogen and the regeneration of the native structure can be accomplished if the protein is initially attached to Agarose beads. Reoxidation was performed under aerobic conditions, in the presence of mercaptoethanol and dehydroascorbate or with a mixture of reduced and oxidized glutathione. In 24 hours, the yields of regenerated trypsinogen were 60 to 70% with 0.2 to 0.6 mg of protein bound/ml of gel but 30% or less if greater than 1.7 mg of protein were bound. Rapid reoxidation, with dehydroascorbate as catalyst, gave molecules which could not be converted to active trypsin. However, if the incorrectly folded structures were placed in a mixture of reduced and oxidized glutathione, the molecules underwent disulfide interchange and could continue to refold. The rapidly reoxidized molecules regained their native structure with the same rate and to the same extent as they did initially in the absence of rapid reoxidation. Therefore, the rate-limiting step in the refolding of trypsinogen was disulfide interchange. The regenerated Agarose-bound trypsinogen displayed the usual properties of the native molecule in (a) its conversion to active trypsin by a process of limited proteolysis, (b) the kinetic constants of the activated product toward typical trypsin substrates, and (c) the limited cleavage of 1 disulfide bond with sodium borohydride. Refoldind of immobilized trypsin was also observed with an overall yield of 50%. Trypsin can fold spontaneously to its native structure even though it lacks the NH2-terminal hexapeptide of its precursor.


Factors contributing to the inhibition of aspartate aminotransferase by dicarboxylic acids. At pH 8.0 aspartate aminotransferase (L-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1) reacts with the modified substrate, erythro-beta-hydroxy-L-aspartate, to form a mixture of enzyme-substrate complexes absorbing at 492 nm. A variety of dicarboxylic acids were studied spectrophotometrically as competitive inhibitors of this reaction. All of the inhibitory dicarboxylic acids form a complex with the enzyme, absorbing at 362 nm. In addition, some of the dicarboxylic acids form a protonated complex absorbing at about 435 nm. This complex, which is the conjugate acid of that absorbing at 362 nm, is formed only by those dicarboxylic acids which can assume a configuration in which the two carboxyl groups are positioned as in maleic acid. Bulky substituents, such as aromatic rings or even methyl groups, prevent the formation of the protonated complex, presumably because of steric restrictions at the active site. Substitution of the central carbon atom of glutaric acid by heteroatoms of increasing charge density results in a progressive decrease in inhibitory effectiveness, at pH 8, primarily due to a loss of this pH-dependent stabilization of the enzyme-dicarboxylic acid complex. Acids with an aromatic ring are among the most potent dicarboxylic acid inhibitors of this enzyme in spite of the fact that they do not undergo the pH-dependent stabilization of their enzyme complexes. From these observations it was concluded that the affinity of aspartate aminotransferase for dicarboxylic acids is determined as much by the mechanism of binding as by the solvation and steric effects.


A DNA polymerase from embryos of Drosophila melanogaster. Purification and properties. The more than 2,300-fold purification of a DNA polymerase from the embryos of Drosophila melanogaster is described. The enzyme, which forms a single band on gel electrophoresis, has a molecular weight of about 87,000 and a pH optimum of 8.5. A divalent metal is required for activity, Mg2+ being preferred with activated DNA, Mn2+ with homopolymer template-primers. The enzyme is inactivated completely by mercurials; polyamines are also inhibitory with certain templates. The most efficient template-primer is activated DNA, but homopolymers such as poly(dA)-oligo(dT), poly(A)-oligo(dT), and poly(A)-oligo(U) are also utilized with high efficiency. The purified enzyme preparations appear to be devoid of nuclease activity when assayed directly with suitable substrates. In addition, neither primer nor product is degraded after prolonged incubation with the enzyme. In accordance with previous observations on other DNA polymerases, the Drosophila enzyme can replicate single-stranded DNA only under conditions of simultaneous transcription by RNA polymerase.


Purification and properties of Bacillus subtilis aspartate transcarbamylase. Aspartate transcarbamylase from Bacillus subtilis has been purified to apparent homogeneity. A subunit molecular weight of 33,500 +/- 1,000 was obtained from electrophoresis in polyarcylamide gels containing sodium dodecyl sulfate and from sedimentation equilibrium analysis of the protein dissolved in 6 M guanidine hydrochloride. The molecular weight of the native enzyme was determined to be 102,000 +/- 2,000 by sedimentation velocity and sedimentation equilibrium analysis. Aspartate transcarbamylase thus appears to be a trimeric protein; cross-linking with dimethyl suberimidate and electrophoretic analysis confirmed this structure. B. subtilis aspartate transcarbamylase has an amino acid composition quite similar to that of the catalytic subunit from Escherichia coli aspartate transcarbamylase; only the content of four amino acids is substantially different. The denaturated enzyme has one free sulfhydryl group. Aspartate transcarbamylase exhibited Michaelis-Menten kinetics and was neither inhibited nor activated by nucleotides. Several anions stimulated activity 2- to 5-fold. Immunochemical studies indicated very little similarity between B. subtilis and E. coli aspartate transcarbamylase or E. coli aspartate transcarbamylase catalytic subunit.


Escherichia coli enterotoxin. Purification and partial characterization. Enterotoxin, a diarrheagenic protein elaborated by pathogenic Escherichia coli strains has been isolated from the supernatant of fermenter cultures of E. coli strain P263, a porcine enteropathogen. Purification steps involving Bio-Gel agarose A-5m, Sephadex G-75 chromatography, and preparative isotachophoresis were used in the isolation. The resulting product appears to be pure according to immunoelectrophoretic, disc electrophoretic, ultracentrifugal, and immunologic criteria. The entertoxin has an apparent molecular weight of 102,000 as judged by gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis, and its isoelectric point is 6.90. The isolated product is highly active in inducing experimental diarrhea in adult rabbits and piglets. It also elicits, in small dosage, a marked increase in adenylate cyclase activity in broken cell preparations of cat heart tissue. The enterotoxin activity is acid-labile and is destroyed by heating at 65 degrees for 30 min. It is suggested that the heat-stable enterotoxin material is derived from heat-labile enterotoxin by forming a complex with endotoxin or capsular material present in the culture supernatant.


Affinity column purification of amylases on protein inhibitors from wheat kernel. An affinity column was devised for the purification of a large number of amylases inhibited by the albumin from wheat kernel. The procedure involved linking the protein inhibitors from wheat to Sepharose and then specifically eluting the amylase adsorbed to the gel with a high concentration of maltose. By this procedure, the amylases from Tenebrio molitor L. (yellow mealworm) larvae and chicken pancreas were purified to homogeneity with good yields for the first time, as shown by both alkaline and acidic electrophoresis. Human saliva alpha-amylase, purified by the same procedure, showed specific activity and electrophoretic patterns similar to those obtained by other workers with different techniques.


Gas-chromatographic analysis of 4-prenyl-1,2-diphenyl-3-5-pyrazolidinedione (feprazone) at therapeutic levles in human plasma. A specific and sensitive procedure for the determination of prenazone at therapeutic levels in human plasma has been developed. The method involves extraction of the drug from acidified plasma into chloroform. After further purification by back extraction the residue is examined on a gas-liquid chromatograph fitted with a flame ionisation detector. Tetraphenylethylene is used as an internal standard for quantitation by the relative peak height technique. Plasma levels encountered after oral ingestion of therapeutic doses are reported.


Analysis and quantification of ether lipids by chromatographic methods. Chromatographic methods, especially thin-layer chromatography (TLC) and gas-liquid chromatography (GLC) are widely used in investigations of the occurrence, molecular structure and metabolism of ether lipids. The application of such techniques to structural analysis and quantification, in combination with methods for the degradation and derivatization of ether lipids, is discussed.


Antibacterial activity of cationic proteins from human granulocytes. Human granulocytes contain several cationic proteins with a molecular weight of approximately 25,000, almost identical amino acid composition, and complete immunologic identity. These proteins possess a chymotrypsin-like protease activity at a neutral pH. The antibacterial activity of the cationic proteins has been studied. Bactericidal activities are found against both Gram-positive (Streptococcus faecalis and Staphylococcus aureus) and Gram-negative (Escherichia coli and Pseudomonas aeruginosa) organisms. Gram-positive bacteria are, however, the most sensitive. The pH-optimum is near neutrality, and the microbicidal activity shows an inverse relationship to the ionic strength, indicating an ionic interaction between the cationic  proteins and the bacterial surface. The microbicidal effect of the cationic proteins is generally independent of the chymotrypsin-like activity of the same proteins since the activity against several bacterial species is heat stable while the chymotrypsin-like activity is heat labile. The surface properties of S. aureus that are determined by protein A do not seem to influence the susceptibility to cationic proteins. The properties of the Gram-negative envelope of E. coli that determine the susceptibility to the lytic action of serum do not influence the sensitivity to the action of cationic proteins. The present study shows that cationic proteins of human granulocytes represent one potential microbicidal mechanism that is independent of hydrogen peroxide and myeloperoxidase.


Radioactive labeling of proteins with an iodinated amidination reagent. An iodinated phenolic imidoester has been synthesized for the labeling of proteins to high specific activities with radioactive iodine. The main advantage of this two-step labeling method is that it obviates direct contact of the protein with deleterious oxidizing agents, such as chloramine-T, pressent in direct methods of iodinating proteins.


Cutaneous immediate hypersensitivity in man: effects of systemically administered adrenergic drugs. In in vitro models of hypersensitivity, beta adrenergic drugs inhibit the antigen-induced release of histamine and other mediators from mast cells and basophils. Epinephrine, an agent with both beta and alpha adrenergic properties, is clinically useful for treating immediate hypersensitivity reactions. We examined the effects of intravenously administered adrenergic drugs on cutaneous wheal-and-flare reactions to antigens and histamine in 7 normal men. Both epinephrine and isoproterenol, a beta agonist, inhibited skin reactions produced by antigen or histamine. Phenylephrine, an alpha adrenergic agonist, produced no effect. These results suggest that epinephrine's inhibition of cutaneous immediate hypersensitivity in vivo is mediated by beta, rather than alpha, receptors. This inhibitory effect in vivo is probably not mediated solely in preventing allergic release of inflammatory mediators since the skin reactions to histamine were inhibited as well.


Release of bound immunoglobulin from SSPE brain by acid elution. SSPE brain homogenate extracted at pH 7.4 yields immunoglobulin with a 4- to 5-fold greater hemagglutination inhibition activity per microgram of IgG than serum from the same patient. Serial washing of the homogenate results in a low level steady-state release of IgG. Elution of the washed sediment with pH 2.5, 0.1 M glycine buffer results in a 2- to 3-fold increase in recovery of hemagglutination inhibition activity with a greater hemagglutination inhibition activity per milligram of IgG than the IgG recovered by phosphate-saline extraction at pH 7.4.


EAC4 and EAC14 production without purified Ci. EAC4 and EAC14 of high activity were prepared by treating sensitized red cells with human or guinea pig serum in the presence of TTHA, a chelating agent whid at low pH to maximize C1 and C4 activity and to minimize C3 contamination. Cells prepared with guinea pig complement were contaminated with C2, which could be decayed away at 37 degrees C. Cells sensitized with IgG antibody were more reactive than those sensitized with whole serum or with IgM, and preparations made with TTHA-complement were more reactive than those prepared with purified C1 plus EDTA-complement. EAC14 stored at 0 degrees C for 3 weeks lost very little activity.


Physical separation of "suppressor" from "helper" thymocytes. Thymocytes from 1-month-old NZB/W mice were separated at unit gravity and then studied for helper or suppressor effects in a GVH assay. Thymocytes with a settling rate of 4 to 6 mm/hr suppressed but did not help. In contrast, help but no suppression characterized thymocytes settling at 6 to 10 mm/hr. Thus helper and suppressor thymocytes in this GVH assay were physically separated.


Pharmacokinetic characteristics of doxycycline accumulation in normal and severely diseased kidneys. Intrarenal concentrations of doxycycline were measured in 48 normal canine kidneys during wide variations in urine pH and the systemic state of hydration of the experimental animals. The results were compared with the renal parenchymal levels achieved in 10 severely diseased human kidneys. Although the state of hydration, the urine pH, and renal disease all significantly influenced the urinary concentrations and rate of renal clearance of doxycycline, there was no detectable difference between concentrations in normal renal tissue and those in severely diseased kidneys. This result is in contradistinction of the findings for previously evaluated antibiotics. The levels of doxycycline measured in renal tissue averaged twice the concentration in serum without notable difference among levels in renal cortex, medulla, and papilla. Prospective clinical trials will be necessary for identification of any solid correlation between the remarkable characteristics of accumulation of doxycycline in diseased renal parenchymal tissue and the clinical, therapeutic importance of such an observation.


In vitro and in vivo evaluation of change of local pH on bacteria in C.S.O.M. An in vitro study has shown a high bactericidal activity of 2 per cent acetic acid. An in vitro study in 400 cases of C.S.O.M. has shown that conservative treatment of C.S.O.M. by pH change with 2 per cent acetic acid is better than antibiotic ear drops alone. However, combined therapy (pH change plus antibiotic ear drops) is definitely superior to single treatment by either of the two methods. Two per cent acetic acid is well tolerated by the middle ear mucosa except in 1 per cent cases, where its use had to be discontinued because of irritation. The use of 2 per cent acetic acid is highly recommended, particularly for poor nations, because of its negligible cost as compared to antibiotic ear drops.


Common dental emergencies which may be encountered by the family physician. Dental emergencies are common in everyday practice, and the family physician may frequently be called upon to evaluate and, at times, to provide initial care for such problems. This article acquaints the family physician with diagnosis and treatment of the most common dental emergencies: disorders secondary to trauma, including soft tissue laceration, fracture or displacement of teeth, and bone fractures; and severe pain related to acute alveolar infection.


Neurobiology of Stomotoca. I. Action systems. The layout of nerves, muscles, and conducting epithelia is described for the simple hydrozoan medusa Stomotoca. Comparisons are drawn with Sarsia and other recently studied forms. The major action systems are those responsible for swimming, crumpling (protective involution), tentacle posture, pointing (unilateral reciprocal flexions of the manubrium and margin), and visceral movements (barely mentioned). Crumpling is a simple summating response in this species. Crumpling and pointing are considered to use the same effectors but different conduction pathways. New histological results include the demonstration of a nerve plexus running through the endodermal canal system and a nerve plexus in the ectoderm encircling the peduncle. Special attention is given to the distribution of synapses and gap junctions, as possible trasmission pathways in behavioral responses. Some details are included on organization within the marginal nerve rings.


Neurobiology of Stomotoca. II. Pacemakers and conduction pathways. Evidence is presented for separate conduction pathways for swimming and for tentacle coordination in the marginal nerves of the jellyfish Stomotoca. The effector muscles are fired through junctions sensitive to excess Mg++, probably represented by the neuromuscular synapses observed by electron microscopy. The swimming effector (striated muscle) fires one-to-one with nerve input signals and myoid conduction occurs. Tentacle responses (smooth muscle contractions) involve facilitation, presumably at the neuro-effector junction; responses are graded and nonpropagating. Electrical correlates of two further conducting systems using the marginal nerves have been recorded. Their functions are unknown. One, the bridge system, extends up the four radii and encircles the peduncle; the other (ring system) is confined to the margin. A fifth conducting system is inferred in the case of the pointing response and its distribution is plotted. Signals have not been obtained from it. Pointing is accompanied by a burst of muscle potentials in the radial smooth muscles and is exhibited after a lengthy latency, indicating a local pacemaker. A sixth conducting pathway is the epithelial system, which mediates crumpling, a response involving the radial muscles without pacemaker intervention. Characteristic conduction velocities and wave forms are noted for the first four systems and for epithelial pulses. All systems, except perhaps the pointing conduction system, through-conduct under excess Mg++. Spontaneous activity patterns are described for the swimming, tentacle pulse, and ring systems. Abrupt increases in light intensity inhibit spontaneous activity, sudden decreases augmenting it. In the absence of specialized photoreceptors, light is presumed to act directly on central neurons. Epithelial pulses inhibit swimming, apparently by blocking the generation or conduction of the primary nervous events. This observation, taken in conjunction with evidence of feedback inhibition of the primary swimming system by the cells it fires, is discussed in relation to possible mechanisms whereby the output of nerve cells might be altered by activity in the excitable epithelial cells which envelop them.


Occurrence of Diplococcus pneumoniae in the upper respiratory tract of children. The patterns of colonization of D. pneumoniae were studied over a 46-month period in a group of young children in a day-care center. Forty-four percent of nasal cultures yielded D. pneumoniae. The most frequently isolated serotypes--6, 19, and 23--accounted for 49% of the isolates; the nine most common serotypes included 80% of the isolates. Individual serotypes frequently were carried for several months. Reacquisition of a serotype previously carried occurred frequently. There was limited spread of serotypes among the children despite prolonged contact.


Intestine of Ascaris: oxygen consumption, fermentation acids, and anaerobic synthesis of protein. The oxygen uptakes by pieces of gut from the porcine roundworm, Ascaris suum, under Po2 10 to 710 torr were from 0.6 to 3.0 mul/mg dry wt/hr (Qo2). Increasing concentrations of tissue, 30 to 90 mg (dry wt/3 ml) decreased Qo2 in air from 1.9 to 1.1 and only technical grade catalase was found to relieve this inhibition. Conversely aerobic fermentation, judged by acid production, was directly proportional to the amount of tissue present: the acids from this fermentation were 2-methyl-butyric (1% of the total), succinic (8%), propionic (40%), and acetic (51%). Glucose did not increase Qo2 but it doubled the incorporation of labeled carbon into protein from glycine-1-14C. Judged by this, protein synthesis proceeded at the same rate under low Po2 (0.01 torr) as under air.


Sympathetic neurotransmitter metabolism in Hirschsprung's disease. Tyrosine hydroxylase activity was measured in high speed supernatants obtained from full thickness segments of aganglionic and ganglionic colon of three children with Hirschsprung's disease. Tyrosine hydroxylase activity expressed as pmole DOPA/mg protein/min was 0.93 +/- 0.16 in ganglionic and 2.67 +/- 0.21 in aganglionic colon. Tyrosine hydroxylase activity in ganglionic colon rose to 2.29 +/- 0.11 following calcium stimulation (100 muM) but could not be further increased in aganglionic colon. Addition of norepinephrine (2 X 10(-4) M) to tissue homogenates inhibited tyrosine hydroxylase activity in ganglionic colon by 57 +/- 8% but only by 14 +/- 3% in aganglionic colon, suggesting that the enzyme present in aganglionic colon is insensitive to feedback inhibition by endogenous norepinephrine. The elevation of tyrosine hydroxylase activity in aganglionic colon and its insensitivity to calcium stimulation and norepinephrine inhibition is further evidence of sympathetic overactivity in the aganglionic colon and suggests a basic enzymatic abnormality in the pathogenesis of Hirschsprung's disease.


Accelerated reattachment with cementogenesis to dentin, demineralized in situ. I. Optimum range. This study confirms an original report describing accelerated reattachment with cementogenesis to root dentin, surgically exposed and demineralized in situ. It additionally describes results of 250 experiments on over 1000 teeth in mongrel dogs and cats designed to identify an optimum range of demineralization related to type of agent, pH, and time of application. This optimum range consistently induces flap reattachment with cementogenesis, while demineralization rates above and below this range enhance reattachment relative to undermineralized controls, but do not consistently induce new cementum. Although most controls demonstrated some reattachment with partial cementogenesis, none produced complete repair as did properly demineralized root surfaces and approximately one-third showed spithelial migration to the apical borders of the wound. No demineralized teeth demonstrated this control result. Small species differences in the response to root demineralization and the degree of hypermineralization of roots adjacent to chronic periodontal pockets, may make optimum ranges determined in this animal study slightly low for human pocket reaattachment techniques.


The difference in activity between (+)- and (-)-methadone is intrinsic and not due to a difference in metabolism. The disposition and metabolism of (+)- and (-)-methadone has been compared in rats. At equal molecular doses, somewhat higher plasma levels of (-)-isomer were observed. At equal analgesic doses, brain and plasma concentrations of (+)-methadone were at least 25 times greater than those of (-)-methadone. No qualitative differences were observed between isomers with respect to in vivo metabolic pattern or in vitro N-demethylation rates. The results strongly support the conclusion that the large differences in analgesic potency between the isomers is due to an intrinsic difference in pharmacologic properties and is not related to a difference in disposition or metabolism.


Binding of chlorpromazine and imipramine to red cells, albumin, lipoproteins and other blood components. The binding of model drugs to human blood and individual blood components has been determined by equilibrium dialysis and expressed in terms of classes of binding sites, association constants and binding capacities. Chlorpromazine and imipramine are bound to three major components: membranes of red cells, albumin, and lipoproteins. The affinity and capacity of lipoprotein binding is at least as high as that of albumin and is equally distributed on HDL, LDL, VLDL, and on the chylomicrons. White blood cells and platelets are of minor importance in terms of binding capacity. No binding was detected with gamma-globulins or alpha- or beta-globulins other than lipoproteins. In contrast, salicylic acid was not bound to red cells or lipoproteins.


Mechanism of induction of hepatic microsomal drug metabolizing enzymes by a series of barbiturates. The inducing effect of certain barbiturates (secobarbitone, thiopentone, pentobarbitone, allobarbitone, phenobarbitone and barbitone) on the levels of the hepatic microsomal drug-metabolizing enzymes has been studied in the rat both in vivo and in vitro. The extent of induction was related to the plasma half-lives of the barbiturates; compounds with low rates of metabolism and long half-lives were the most potent inducing agents. The latter (phenobarbitone, pentobarbitone and allobarbitone) were shown by spectral technique to interact with cytochrome P-450 suggesting that their mechanism of enzyme induction was 'substrate induction' in type. Barbiturates containing an allyl group (secobarbitone and allobarbitone) had a weaker inducing effect than expected, possibly due to their destruction of cytochrome P-450. Despite its short plasma half-life of 0-5 h thiopentone was a relatively potent inducer probably due to its metabolism to pentobarbitone, which has a much longer plasma half-life (1-3 h). Barbitone is an effective inducer of the drug-metabolizing enzymes, yet does not interact spectrally with cytochrome P-450; this is in accord with the observations that although there are increases in NADPH-cytochrome c reductase and cytochrome b5, following administration of barbitone there is no increase in cytochrome P-450. Barbiturate pretreatment does not affect the activities of glucose-6-phosphatase, glucose-6-phosphate dehydrogenase or 6-phosphogluconate dehydrogenase.


Effects of a human plasma fraction on the release of chemotactic factors and anaphylatoxin from complement. A fraction prepared from normal human plasma inhibits the release of chemotactic factors and anaphylatoxin when complement is activated by the alternate but not by the classical pathway. The fraction gave no interference with the actions of the released substances on either leucocytes or guinea-pig isolated ileum. The possible relation of this effect to the mechanism of the anti-inflammatory actions of the plasma fraction is discussed.


The surface activity of some antihistamines at the air-solution interface. The surface activity of some antihistamines at the air-solution interface has been examined. Change of the counterion associated with the drug from chloride to maleate or chloro-theophyllinate considerably decreased the critical micelle concentration and increased the surface activity. The effect on surface activity of changes in the nature of the hydrophobic and hydrophilic groups have also been evaluated.


The influence of polyvinylpyrrolidone on the dissolution properties of hydroflumethiazide. The incorporation of hydroflumethiazide with polyvinylpyrrolidone (PVP) was found to retard and to enhance the dissolution of the drug from compressed discs, the magnitude of the effect being dependent on the proportion of PVP present and its method of incorporation. The most active system dissolved sixteen times faster than pure hydroflumethiazide. Low concentrations of PVP were also found to decrease the apparent solubility of hydroflumethiazide while at high concentrations solubility was enhanced. X-ray and infrared analysis of systems suggested the presence of an amorphous form of hydroflumethiazide in coprecipitate systems. The dissolution data were consistent with a physical model which takes account of the roles played by crystalline and amorphous hydroflumethiazide together with the complexing and crystal growth inhibiting effect of PVP on hydroflumethiazide.


Effects of hardness on the disintegration time and the dissolution rate of uncoated caffeine tablets. The effects of hardness on disintegration and dissolution characteristics of uncoated caffeine tablets made at eight different pressure levels were studied. The disintegration times were determined using the J.P. VIII procedure with disks and the dissolution rate measurements were performed with the U.S.P.XVIII procedure (U.S.P. method) and the J.P. VIII disintegration test apparatus (J.P. method). A good correlation between the hardness and the disintegration times was obtained. The dissolution rate constants were determined from the equation of Noyes & Whitney (1897) and a good correlation between the hardness and the dissolution rate constants was obtained. The hardness governed the dissolution over all the stages from tablet to the smallest particles after the breakage by disintegration. The dissolution rates of the J.P. method were greater than those of the U.S.P. method.


Evidence for a clorgyline-resistant monoamine metabolizing activity in the rat heart. When benzylamine was used as substrate, a component of the total monoamine oxidase (MAO) activity in the rat heart was found to be resistant to inhibition by clorgyline. The proportion of the total activity represented by this component, decreased as the rat grew. It was also inhibited by both semicarbazide and isoniazid but not by potassium cyanide. Inhibitor studies with MAO in subcellular fractions showed that this component was more concentrated in the microsomal and soluble fractions. However, it could not be concluded that the activity was entirely a soluble enzyme. Determination of quasi-Michaelis constants ("Km") for total benzylamine oxidizing activity revealed a high ("Km" of approximately 10(-5)M) and low ("Km" of approximately 5 X 10(-4)M) affinity component. The high affinity component was inhibited by semicarbazide and the low affinity component by clorgyline. In the presence of 10(-3)M clorgyline, the high affinity component showed substrate inhibition at higher substrate concentrations. The possibility is discussed that the clorgyline-resistant activity is due to an amine-oxidizing activity distinct from mitochondrial MAO.


Enhancement of permeability of ethyl cellulose films for drug penetration. Permeability constants of salicylic acid, caffeine and benzoic acid have been measured at 37 degrees for heterogeneous films of ethyl cellulose containing up to 50% PEG 4000, the latter component undergoing leaching out. For the first two compounds, steady state constants were independent of film thickness and solute concentration but increased linearly and sharply with PEG content and were reproducible. The films were impermeable to NaOH and permeation rates were independent of receiver compartment pH. Solubility coefficients and diffusion coefficients of the substances in the films were measured using sorption and/or time lag methods and were low as compared with polyethylene films. High solubility seemed to be associated with the presence of a free activated hydrogen, shown by sorption studies on other substances. From the evidence, it appeared that mass transfer was controlled by the solubility diffusion process in the ethyl cellulose of the membranes with all three substances. Enhancement of permeability by PEG thus seemed due to increased porosity, equivalent to reduction in the effective thickness of the matrix, which nevertheless retained its barrier properties. Enhancement coefficients calculated from the slopes of PEG concentration plots may be useful for predicting the increased mass transfer of drugs through such membranes and could enable the porosity and thickness factors to be balanced against each other for formulation of coated products.


Some relations between dissolution rates and physical parameters of a drug in aqueous micellar solutions of a non-ionic surfactant. Dissolution rates of salicylic acid from a constant surface area into a series of aqueous micellar polysorbate 20 solutions at pH 1-0 to 4-0 have been measured using two different methods; a stirred beaker and a rotating disc technique. The micellar molecular weight of polysorbate 20 has been obtained from light scattering and differential refractometry data and used with other independently determined physical data to calculate diffusion coefficients of the diffusing species. Linear multiple regression analysis was used to assess the dependence of drug dissolution rate on the diffusion coefficient and the viscosity of the dissolution medium.


The effect of ageing on the rheological properties of gelatin gels. The effect of ageing at 4 degrees on the viscoelastic properties of 1-5 and 2-0% acid and alkaline processed gelatin gels has been investigated. Viscoelastic properties were not markedly affected by ageing time although evidence of a decrease in gel structure with increasing ageing time was apparent. This has been tentatively attributed to hydrolysis of amide links. 1-5% alkaline gels were shown to be more rigid than the corresponding acid gels and this has been discussed in terms of differences in molecular weight, polydispersity, charge and charge distribution.


The metabolism of dibenzylamine: identification of NN-dibenzylhydroxylamine as the major in vitro metabolic product from rabbit fortified hepatic homogenates. A study of the in vitro metabolism of dibenzylamine with fortified rabbit liver 9000 g supernatant fractions has shown that N-oxidation is the major metabolic process. Of the total amount of dibenzylamine metabolized, 90% is converted to NN-dibenzylhydroxylamine. The primary amine, benzylamine, is formed to the extent of only 6% of total substrate metabolized. The identity of the major metabolite, NN-dibenzylhydroxylamine has been established by thin-layer chromatography, gas-liquid chromatography and combined gas-liquid chromatography-mass spectrometry.


Locomotor activity stimulation in rats produced by dopamine in the nucleus accumbens: potentiation by caffeine. The increased motor activity of reserpine-nialamide pretreated rats given dopamine into the nucleus accumbens was potentiated in a dose-dependent manner by systemically administered caffeine. Similarly, the increase in motor activity seen when the endogenous dopamine was released by intraperitoneally administered amphetamine was potentiated by systemically given caffeine. These effects might be due to an increase in the dopamine-induced accumulation of cyclic AMP in the nucleus accumbens after inhibition of the phosphodiesterase by caffeine.


Tyrosine hydroxylase activity in rat brain regions after chronic treatment with +/--propranolol. Rats were injected twice daily with +/--propranolol (6 mg kg-1 day-1) for 14 days and killed 16 h after the final injection. Tyrosine hydroxylase activity was measured in both soluble and particle-bound forms in various brain regions. The activity of the soluble enzyme was not significantly altered by propranolol treatment in any of the brain regions studied. The tyrosine hydroxylase activity in the particulate fraction was significantly increased in corpus striatum and unchanged in other brain regions. The propranolol concentrations in the various brain regions in this chronic study were far lower than necessary to produce a significant change in tyrosine hydroxylase activity in acute experiments. It was concluded that chronic propranolol treatment produces a persistent increase in bound tyrosine hydroxylase activity in rat corpus striatum.


Oxotremorine- and atropine-induced changes of dopamine metabolism in the rat striatum. The effects of various doses of oxotremorine and of atropine (30 min, i.p.) on the metabolism of dopamine were examined in the striatum of the rat. Changes in striatal dopamine metabolism were estimated by following either the accumulation of [3H] dopamine 15 min after intravenous injection of [3H] tyrosine or the accumulation of dopa (taken as an index of dopamine synthesis) in animals pretreated with a dopa decarboxylase inhibitor Ro4-4602. Oxotremorine, 0-1 mg kg-1, and atropine, 1 mg kg-1, did not affect dopamine metabolism. Oxotremorine, 0-5 and 1-5 mg kg-1, did not modify dopamine concentrations but increased the accumulation of [3H]dopamine. The drug enhanced dopa formation in animals pretreated with Ro4-4602. Atropine, 5 and 20 mg kg-1, increased the accumulation of [3H]dopamine but did not affect dopamine concentrations. The accumulation of dopa was not modified there being no difference from the saline value in animals pretreated with the dopa decarboxylase inhibitor at high dopamine metabolism and atropine reduced dopamine utilization.


Microbial kinetics of drug action against gram-positive and gram-negative organisms. II: Effect of clindamycin on Staphylococcus aureus and Escherichia coli. Clindamycin-affected Staphylococcus aureus cultures show biphasic steady-state generation curves. An initial (phase I) generation of the clindamycin-affected Staph. aureus is followed by an ultimate (phase II) generation at the same dose level. The phase I apparent generation rate constant is greater than the phase II apparent generation rate constant and suggests the development of resistant Staph. aureus mutants to clindamycin action after a finite period of drug-bacteria contact at any subcompletely inhibitory concentration level. It is rationalized that the increased resistance to drug action in mutant strains is due to a comparatively reduced ribosomal binding affinity for clindamycin. In contrast, clindamycin-affected Escherichia coli cultures show monophasic steady-state generation curves at all concentration levels; E. coli cultures do not develop resistance to clindamycin action. The dependence of the apparent generation rate constant on drug concentration yields a sigmoidal curve, which is coincident by a potency factor for the phase I and phase II generations of clindamycin-affected Staph. aureus and suggests a common mechanism of action for both generation phases. That of clindamycin-affected E. coli yields an asymptote curve, which indicates a different mechanism of action. Clindamycin possesses both a bacteriostatic and a bactericidal action on initial and mutant resistant strains of Staph. aureus, whereas its action on E. coli is only bacteriostatic. Consequently, clindamycin has a minimum inhibitory concentration (MIC) against E. coli that is about 1000 times the MIC value against Staph. aureus at 37.5 degrees. The effect of pH changes in broth media on generation inhibition of both Staph. aureus and E. coli by clindamycin action indicates that the unprotonated fraction of drug concentration contributes to the activity, possibly because of its ready penetration through cell membranes.


Effect of pKb on lipophilic binding of disopyramide derivatives to human plasma. The extent of plasma binding, the partition coefficient, and the pKb of 13 disopyramide derivatives were determined. The structural variation on the diisopropylaminoethyl group of disopyramide molecules influenced these physical parameters to varying degrees. Results demonstrated that the extent of interaction between drugs and human plasma was a linear function of their lipophilicity and inversely proportional to the magnitude of the pKb value.


Photolytic degradation of alpha-[(dibutylamino)methyl]-6,8-dichloro-2-(3',4'-dichlorophenyl)-4-quinoline methanol: an experimental antimalarial. A study of the effects of various storage conditions on the rate and products of degradation of the quinoline methanol antimalarial agent, alpha-[(dibutylamino)methyl]-6,8-dichloro-2-(3',4'-dichlorophenyl)-4-quinoline methanol, was undertaken. The degradation was followed by high-pressure liquid chromatography and TLC in oxygenated and deoxygenated methanol, ethanol, chloroform, and chloroform-heptane mixtures under UV and laboratory fluorescent lighting irradiation, as well as in the absence of light. The kinetics of degradation confirmed the major catalyzing factor to be UV irradiation. The compound was stable in the absence of light and reasonably stable under fluorescent lighting both in the presence and absence of oxygen. The degradation resulted in a major product, 6,8-dichloro-2-(3',4'-dichlorophenyl)-4-quinoline-carboxaldehyde, whose structure was confirmed by elemental analysis and IR, NMR, and mass spectral data.


GLC determination of plasma drug levels after oral administration of clorazepate potassium salts. Plasma nordiazepam levels resulting from the oral administration of clorazepate potassium salts were determined by a sensitive GLC assay. Nordiazepam and the internal standard (diazepam) were selectively extracted into ether at pH 9.2, hydrolyzed to their respective benzophenones, and quantified by electron-capture detection. The assay was used in a comparative bioavailability study of single equimolar oral doses of monopotassium and dipotassium salts of clorazepate in dogs. Both clorazepate salts were rapidly absorbed and exhibited mean peak total drug levels after 1 hr. Clorazepate levels accounted for about 50% of the total drug levels present. No statistical difference in the plasma drug levels of clorazepate mono- and dipotassium salts and the metabolite was found in dogs.


Dissolution studies with a multichannel continuous-flow apparatus. A multichannel continuous-flow apparatus for dissolution rate measurements is described. Typical data are presented to demonstrate its utility for studies with bulk drug powders as well as with tablets and capsules without any change of setup. Procedures are given for the preparation of powder samples for dissolution studies and for a simple method of changing pH for "retard" tablets. The precision in dissolution rates obtained with this apparatus and method is 1-10% mean RSD. The advantages of the method are flexibility, reproducibility, and ability to obtain data in integral or differential form.


Activity of local anesthetic agents in goldfish. The activity of procaine hydrochloride, lidocaine hydrochloride, tetracaine hydrochloride, and dibucaine hydrochloride in producing overturn in goldfish was measured in pH 8.0 buffer. Calculation of the apparent minimum effective concentration of local anesthetic necessary to result in overturn of the goldfish showed that the activity of these agents increased in the following order: procaine hydrochloride less than lidocaine hydrochloride less than tetracaine hydrochloride less than dibucaine hydrochloride. The effect of these agents on goldfish can be correlated with previous work on the minimum concentration necessary to block conduction in isolated nerve and muscle fibers. The activity of the local anesthetic agents could be explained, in part, by the relationship between the chloroform-pH 8 buffer partition coefficient and the minimum effective concentration in goldfish. Experimental results indicate that the unionized drug molecule is responsible for the observed effects.


Intestinal pH as a factor in selection of animal models for bioavailability testing. Previous in situ studies showed that intestinal drug absorption is strongly influenced by intestinal pH. Three animal species considered for bioavailability testing were studied in situ to determine intestinal pH. Results from the rat and dog correlated well with results in humans, while results from the rabbit did not. The rabbit appears to be a poor candidate for attempted animal-human bioavailability correlations.


Effects of normal alcohols on intestinal absorption of salicylic acid, sulfapyridine, and prednisolone in rats. The rates of intestinal absorption of salicyclic acid, sulfapyridine, and prednisolone from solutions containing no alcohol or 0.5% ethanol, n-butanol, or n-hexanol were determined. At the concentrations used, ethanol did not significantly affect drug absorption. Butanol reduced the rate of absorption of sulfapyridine but did not significantly affect the absorption rates of prednisolone or salicylic acid. Hexanol reduced the rates of absorption of sulfapyridine and salicylic acid and increased the rate of absorption of prednisolone. The absorption-altering effects of the alcohols were concentration dependent and rapidly reversible. Histological studies indicated that the structure of the epithelium was not altered by the alcohols. While the absorption rate of water from the drug solutions was increased by the alcohols, their absorption-altering effects could not be attributed solely to increased water flux. In addition, the absorption-altering effects of the alcohols could not be attributed to formation of drug-alcohol complexes nor to alcohol-induced alterations in the extent of binding of the drugs to nondialyzable materials in the intestinal drug solution.


High-speed liquid chromatographic analysis of sulfasalazine (salicylazosulfapyridine). A high-speed liquid chromatographic method for analysis of sulfasalazine (salicylazosulfapyridine) in bulk powder and tablet dosage form is presented. Analysis is accomplished with a reverse-phase partition column and 10% 2-propanol in pH 7.7 phosphate buffer as the mobile phase. The method of analysis utilizes a simple, one-step, solubilization procedure with dimethylformamide, addition of an internal standard, and chromatography. The method is specific for sulfasalazine in the presence of starting materials, degradation products, or by-products from its manufacture.


CO2 retention as a basis for increased toxicity of salicylate with acetazolamide: avoidance of increased toxicity with benzolamide. Two carbonic anhydrase inhibitors, acetazolamide and benzolamide, are capable of increasing the toxicity of sodium salicylate in mice. Beginning at about 2 mg/kg, each of the inhibitors, in combination with a fixed (400 mg/kg) dose of salicylate, generates a dose-mortality curve that reaches a plateau at about 60% deaths at 6 to 8 mg/kg. This effect can be duplicated by 8 to 10% inspired CO2. It appears that the respiratory acidosis secondary to the inhibition of red cell carbonic anhydrase is responsible for the increased toxicity; earlier work by others shows that acidosis increases the concentration of salicylate in the brain. In the treatment of salicylate poisoning by carbonic anhydrase inhibitors, the goal is to alkalinize the urine and increase the excretion of salicylate. With the newer inhibitor, benzolamide, it is possible to dissociate the respiratory acidosis from the renal effect. Maximal alkalinization of the urine is possible with a dose (about 1 mg/kg) below that which generates a respiratory acidosis. With this dose, there is no increase in the early toxicity of salicylate.


Antagonism of deslanoside-induced cardiotoxicity by combined nicotinic and muscarinic blockade of autonomic ganglia. The effect of ganglionic blockade on cardiotoxicity induced by deslanoside (25 mug/kg i.v. at 15-minute intervals) was evaluated in Dial-urethane anesthetized cats. Electrocardiogram, blood pressure and pre- and postganglionic cardiac sympathetic nerve recordings were monitored. When deslanoside was given to control animals, 150 +/- 8.2 and 179 +/- 11.9 mug/kg produced ventricular tachycardia and ventricular fibrillation, respectively. Pretreatment of cats with either hexamethonium or atropine alone did not influence the doses of deslanoside required to produce ventricular tachycardia or ventricular fibrillation. However, pretreatment with the combination of hexamethonium and atropine significantly increased the dose of deslanoside needed to produce ventricular tachycardia (181 +/- 12.3 mug/kg) and ventricular fibrillation (219 +/- 12.3 mug/kg). Furthermore, administration of atropine to hexamethonium-pretreated cats intoxicated with deslanoside decreased deslanoside-induced postganglionic nerve activity. These results indicate that blockade of both nicotinic and muscarinic ganglionic transmission is essential for a protective influence against cardiotoxicity induced by deslanoside.


Characterization of the angiotensin receptor in guinea-pig aorta. The binding of 14C-angiotensin to the subcellular fractions of guinea-pig aorta was compared to the known physiological data as obtained from the intact preparation. Results show that only the plasma membrane fraction possesses binding characteristics consistent with the physiological data. Equilibrium of specific binding occurs at 10(-7) M 14C-angiotensin which is the physiological maximal dose. This equilibrium is achieved after 3 minutes of incubation, the time required to develop maximal response. 8-Leu-angiotensin, a specific competitive antagonist of angiotensin, blocks the binding of 14C-angiotensin in a manner similar to nonradioactive angiotensin. Neither bradykinin nor angiotensin I has a significant effect on the binding of 14C-angiotensin to plasma membranes. The dissociation constant (Ka) as calculated from the 50% inhibition of the specific binding is 2.2 X 10(-8) M. This value corresponds to the apparent Ka estimated from both the physiological dose-response curve (6.3 X 10(-8) M) and the reversibility of binding curve (4.7 X 10(-8) M). Specific binding of 14C-angiotensin is pH dependent with maximal binding occurring at pH 7.4. The rate of dissociation of 14C-angiotensin bound to plasma membranes is compatible with the recovery of the physiological response. These results indicate that the angiotensin receptor is located in the cell membrane.


Histamine receptors in adipose tissue: involvement of cyclic adenosine monophosphate and the H2-receptor in the lipolytic response to histamine in isolated canine fat cells. The effects of histamine on lipolysis and associated changes in adenosine 3',5'-monophosphate (cyclic AMP) levels were examined in the isolated canine fat cell. Histamine, like norepinephrine, caused a dose-dependent increase in free fatty acid (FFA) and glycerol levels. The lipolytic response to histamine was preceded by a rise in the levels of cyclic AMP and was greatly potentiated by the addition of theophylline. In isolated canine fat cells, histamine (2 muM) caused a 7-fold increase in FFA levels. This effect was inhibited more than 50% in the presence of insulin (0.4 mmu/ml) or prostaglandin E1 (2.8 muM). In similar experiments, cyclic AMP Levels were increased 11-fold by histamine (2 muM) in the presence of 1 mM theophylline. Burimamide (0.1 mM), a histamine H2-receptor antagonist, reduced the effect of histamine (2 muM) on FFA levels as well as the effect on cyclic AMP levels greater than 95% but did not inhibit the lipolytic response to norepinephrine (2 muM). Propranolol (0.01 mM), a beta adrenergic antagonist, reduced the lipolytic response to norepinephrine by 97% but did not inhibit the effects of histamine on FFA or cyclic AMP levels. Tripelennamine and 1,5-diphenyl-3-dimethylaminopyrrolidine, histamine H1-receptor antagonists, inhibited neither the lipolytic response to histamine nor the effect on cyclic AMP levels. It was concluded that histamine induces lipolysis in canine fat cells by a mechanism involving cyclic AMP and the histamine H2-receptor.


Binding of N1-methylnicotinamide and p-aminohippuric acid to a particulate fraction from dog kidney. The active secretion of organic ions by the kidney may be described by the following models: 1)binding to a carrier protein and 2) a translocation process across the membrane. The feasibility of such a model was tested by measuring binding of either an organic cation, N1-methylnicotinamide (NMN) or an organic anion p-aminohippuric acid (PAH) to particulate material obtained from dog renal cortex tissue. The method employed was one in which the bound and free forms of the ligand were separated by centrifugation through a gel matrix. Binding of NMN and PAH was found to be tissue specific. In addition, binding was pH, time, temperature, protein-concentration and ligand-concentration dependent. Saturation of binding for either ligand was observed at concentrations greater than 50 mM, suggesting low affinity. Interestingly, a positive cooperative effect was observed for binding of either NMN or PAH to the particulate material. Although binding was associated only with particulate material, the binding proteins were released from the membrane system(s) by treatment with the nonionic detergent Lubrol WX. These studies show that NMN and PAH binding share many features in common but that the two processes are independent of each other. The results are consistent with, but do not prove, the model.


The influence of age on the distribution, metabolism and excretion of methoxyflurane in Fischer 344 rats: a possible relationship to nephrotoxicity. Age as a factor in methoxyflurane nephrotoxicity was evaluated in Fischer 344 rats of various ages by determination of: 1) serum inorganic fluoride and methoxyflurane concentrations, and urinary inorganic fluoride excretion in methoxyflurane-exposed rats; 2) liver microsomal methoxyflurane defluorinase activity; and 3) distribution of injected sodium fluoride. Only rats in the youngest age group (6 weeks) did not develop nephrotoxicity after anesthesia. Older rats had a biphasic rather than a monophasic decay in serum methoxyflurane concentration and also had increased serum inorganic fluoride concentration and urinary inorganic fluoride excretion. Older rats also excreted a greater proportion of an injected dose of sodium fluoride compared to young rats. Microsomal methoxyflurane defluorinase specific activity was similar among rats of all ages. It is likely that increased availability of methoxyflurane  due to its greater storage in fat led to more inorganic fluoride production in older compared to younger rats. Bone sequestration of inorganic fluoride in younger rats probably accounts for decreased serum inorganic fluoride levels in that group. Both factors cause significant differences in renal exposure to inorganic fluoride; thus the risk of nephrotoxicity is less in younger animals.


Functional characteristics of the renal tubular secretion of amprolium, a quaternary organic base. Amprolium [1-(4-amino-2-propyl-5-pyrimidinemethyl)-2-methyl-pyridinium chloride hydrochloridel is a basic (quaternary) organic compound. At very low plasma concentration, it is cleared by the kidney at a rate approximating renal plasma flow in the dog. Its renal clearance is not depressed by organic acids (p-aminohippurate or probenecid) but is reduced by the quaternary base, mepiperphenidol. Acetate and pantothenate may influence the clearance of amprolium but, if this is the case, the effect is less than for p-aminohippurate. Its clearance is depressed as urinary pH is increased. The clearance of amprolium was not altered over a substantial range in urine flow at either high or low urinary pH.


The mechanism by which methiothepin, a putative serotonin receptor antagonist, icnreses brain 5-hydroxyindole levels. Brain tryptophan and 5-hydroxyindole levels are elevated in rats given methiothepin, a neuroleptic that appears to block serotonin receptors. The rise in brain tryptophan probably results from a drug-induced increase in the ratio of plasma tryptophan concentration to the sum of the neutral amino acids in plasma that compete with tryptophan for uptake into the brain; this change in the plasma amino acid pattern may be mediated in part by a methiothepin-induced rise in plasma insulin. Methiothepin also decreases the proportion of circulating tryptophan that is bound to albumin. Unlike exogenous tryptophan, methiothepin fails to increase 5-hydroxyin-doles caudal to the site of a spinal cord transection. Therefore, the mechanism by which methiothepin elevates 5-hydroxyindole levels involves not only increased brain tryptophan levels but also continued impulse flow along serotonergic neurons.


Alpha adrenergic and histaminergic effects of tolazoline-like imidazolines. For eliciting contraction of rabbit aorta, the relative potency of agonists in terms of negative log molar ED50 were: oxymetazoline (8.4) greater than naphazoline (7.95) greater than phenylephrine (7.31) greater than tetrahydrozoline (6.5) greater than tolazoline (5.80). None of the imidazolines, however, produced maximal effects equal to that of phenylephrine. All agonists were directly acting agents. The interactions between oxymetazoline and phentolamine or tolazoline and phentolamine were competitive with pA2 values of 8.1 and 8.0, respectively. Phentolamine with tetrahydrozoline, naphazoline or phenylephrine produced nearly equal KB values. Thus, all the agonists and alpha adrenoceptor blockers must act at a common site in rabbit aorta. As expected, the contraction of rabbit aorta produced by an imidazole histamine was competitively antagonized by the histamine 1 antagonist, chlorpheniramine (K B 7.6 X 10(-9) M). The antagonist failed to block the contraction produced by the imidazolines studied. On guinea-pig aorta, the relative potency of the agonists varied greatly. On guinea-pig atria, tetrahydrozoline and tolazoline produced positive chronotropic effects which were not influenced by reserpine and cocaine treatment, or treatment with a beta adrenoceptor blocker, propranolol. The histamine 2 receptor antagonist, metiamide, however, selectively blocked the cardiac effects. It is concluded that oxymetazoline and naphazoline do not activate histamine 1 or histamine 2 receptors or beta adrenoceptors. Thus, the drugs are highly specific alpha adrenoceptor stimulants. On the other hand, tetrahydrozoline and tolazoline interact with histamine 2 receptors and with alpha adrenoceptors, but not with histamine 1 receptors or with beta adrenoceptors.


Gel entrapped L-asparaginase: kinetic behavior and antitumor activity. L-Asparaginase from Escherichia coli was immobilized by entrapment in a gel based on poly(2-hydroxyethyl methacrylate) with an activity as high as 730 I.U./g of dry gel. The apparent Michaelis constant for these gels was similar to that of the free enzyme. At 37 degrees C the immobilized enzyme had a half-life of more than 40 days, in vitro. The gel was freeze-dried, crushed and sieved to pass a 38 mum screen, giving a median particle size of 12 mum. C3H mice were injected intraperitoneally with 40 I.U. of L-asparaginase; the peak plasma activity after 4 hours was only 0.9 I.U. for the gel entrapped enzyme compared to a peak activity of 5.0 I.U. after 2 hours for the native L-asparaginase. Ninety percent of the plasma enzyme activity for the gel entrapped case was sedimentable at 21,000 X g, indicating a small leakage of the enzyme from the gel; the clearance for the enzyme activity in plasma had an initial half-life of 13 hours in contrast to a half-life of 2 hours for the native preparation. After intraperitineal injection of 5.0 I.U. into C3H mice, plasma L-asparagine fell to undetectable levels for 4 days and reappeared by day 8 for both the native and immobilized enzymes. Subcutaneously transplanted 6C3HED murine lymphoma was inhibited by 35, 78 and 100% after single intraperitoneal injections of immobilized L-asparaginase of 2, 4 and 8 I.U., respectively, as compared to 36, 53 and 86% for the native enzyme by the 14th day. Body weight changes after receiving immobilized L-asparaginase were essentially similar to those of animals receiving a comparable dose of native enzyme. These results indicate that while most of the immobilized L-asparaginase remains at the injection site, it produces a significant plasma L-asparagine depression and antitumor acitivity comparable to that of the native preparation without major toxicity.


Preceramic and postceramic solder joints. This investigation evaluated preceramic and postceramic soldering procedures. Three gap spaces and two soldering methods were evaluated. Wider gaps produced stronger joints. The strongest joints were achieved by using wider gaps and the oven soldering technique. Very wide gaps (0.76 mm.) are not recommended because of possible distortion through excessive solder shrinkage. However, in practice, many solder joints exceed the 0.31 mm. recommended minimum; these joints will not show decreased strength.


The interaction of pH and divalent cations at the neuromuscular junction. 1. 1. The effects of acidic pH on transmitter release were studied at the frog neuromuscular junction, using intracellular recording techniques. 2. Acid pH reduced the amplitude of the end-plate potentials (e.p.p.s) and accelerated the frequency of the miniature e.p.p.s(m.e.p.p.s). 3. At pH 6-0 the m.e.p.p. frequency was on the average 2-5 times greater than at pH 7-4. This multiplication was independent of the divalent ion concentration of the medium over a large range. 4. Reduction of the e.p.p. amplitude at low pH was the result of a decrease in m, the number of quanta of transmitter liberated by the nerve impulse. 5. The effect of low pH on m was blocked by high concentrations of Mg2+ and by lower concentrations of Mn2+ ions. This occlusion was found even when the total concentration of divalents in the bathing solution was kept constant. 6. These results indicated that H+ and Mn2+ ions bind to an acidic site which regulates Ca-mediated release of acetylcholine (ACh). The acid dissociation constant (KH) was determined using both a kinetic and a surface charge model. The pKa of the site calculated from the kinetic model was 5-7, while a pKa of 3-6 was obtained from the surface charge model. 7. It is suggested that protonation of the acidic site mentioned above reduces evoked transmitter release by blocking the influx of Ca into the nerve terminal following the nerve action potential.


Cyanogen induced phosphorylation of D-fructose. Cyanogen-induced phosphorylation of D-fructose at pH 8.8 led to the formation of a phosphorylated sugar identified as alpha-D-furcto-pyranose 2-phosphate on the basis of its chromatographic and electrophoretic properties, its lability to hydrolysis by alkaline phosphatase, the rate of its acid-catalysed hydrolysis, the results of periodate oxidation and optical rotatory measurements.


Hormonal induction of tyrosine aminotransferase activity in host liver and hepatoma no. 7777 of normal and cofactor-depleted animals. Induction of tyrosine aminotransferase (TAT) (EC 2.6.1.5) by hydrocortisone was studied during cofactor (pyridoxal phosphate) depletion in hepatoma-bearing BUF strain female rats. Pairs of rats were matched for weight and age and one from each pair was fed ad libitum a diet lacking pyridoxine; the other (referred to as "pair-fed") was given the same diet supplemented with the vitamin, with the amount restricted to that consumed by the matched animal on the deficient diet. All animals were inoculated with Morris hepatoma no. 7777 cell after 21 days on the respective diets. TAT specific activity was determined weekly in host liver and hepatoma, in the presence and absence of cofactor, before and after the administration of hydrocortisone. Free and bound pyridoxal phosphate was estimated enzymatically. The average weight of hepatomas from pair-fed animals was 1.5-fold to twofold greater than that of hepatomas from animals on deficient diets. TAT activity of hepatomas was two times greater than that of host liver, and lack of dietary pyridoxine was without effect. Hormonal induction of enzymatic activity was maximal after the first week of tumor growth and subsequently reached minimal values. In pair-fed animals, tumor TAT was approximately 60% saturated with cofactor. In vitamin-deficient animals, only 6% of the tumor enzyme was saturated with the cofactor. The percent saturation of host liver TAT varied, with minimal values found in the vitamin-deficient animals. Hepatic and tumor pyridoxal phosphate content of pair-fed animals was unusually high (10 mug/g); in vitamin-deficient animals, only the coenzyme content of hepatomas was high (7.0 mug/g). The results showed that presence of the tumor altered the a) specific activity level of TAT and tissue content of cofactor, b) pattern of hormonal induction of the enzyme, and c) effects of the absence of dietary pyridoxine on TAT induction observed in animals without tumors.


Multiple endocrine adenomatosis type IIb. Diagnosis and treatment. A 29-year-old man with a marfanoid habitus, peculiar mucosal neuromas of the lips and tongue, high arched palate, hyperplastic corneal nerves, and hypertension was found at operation to have medullary carcinoma of the thyroid, parathyroid hyperplasia, and pheochromocytoma. These symptoms and findings are characteristic of multiple endocrine adenomatosis (MEA IIb) syndromes.


Mass screening for sickle cell hemoglobin. Is there an optimal method? The problem of screening large civilian and military populations for sickle cell hemoglobin and related conditions has been approached recently by three distinct methods. One method specifies hemoglobin electrophoresis as the initial screening technique, followed by a solubility (dithionite) test when a (nonspecific) "S band" is noted. Another method requires the use of the automated dithionite test, followed, when positive, by hemoglobin electrophoresis. A third method is a dual, automated technique for detecting anemia or hemoglobin S or both in the initial screening study; hemoglobin electrophoresis studies are done when indicated. All three methods provide data suitable for accurate diagnosis and genetic counseling, but substantial variations in costs and redundancy of data are found among these methods. Because it gives the "biggest diagnostic bang for the buck", the third merits widespread adoption.


Blood zinc levels in patients with arteriosclerosis obliterans, thromboangiitis obliterans and Takayasu's disease. Serum zinc concentration was measured by atomic absorption spectrophotometry in 30 normal males, 17 normal females, 20 patients with arteriosclerosis obliterans, 26 patients with thromboangiitis obliterans, 40 patients with Takayasu's disease. The mean serum zinc concentration was 93.9 +/- S.E.4.0 mug/100 ml in the normal male controls, 75.1 +/- 3.3 mug/100 ml in patients with arteriosclerosis obliterans without ulcers (P less than 0.05) and 79.2 +/- 6.5 mug/100 ml in patients with thromboangiitis obliterans without ulcers. Serum zinc concentration showed to be more decreased in patients with these diseases who had ulcers. The mean serum zinc concentration was 77.9 +/- 4.0 mug/100 ml in the normal female controls, 71.8 +/- 5.1 mug/100 ml in patients with Takayasu's disease who had never been treated with steroids and 59.1 +/- 2.7 mug/100 ml in patients with this disease who had been treated with this drug (P less than 0.01). The zinc level was significantly lower in CRP positive patients with Takayasu's disease than in CRP negative patients (P less than 0.05).


Hemodynamics of spontaneously hypertensive rats in conscious state. Cardiac output of unanesthetized spontaneously hypertensive rats (SHR) and normotensive control rats (NCR) was measured by an aortic pressure pulse contour method. A catheter was introduced into the aortic arch under ether anesthesia and aortic pressure curves were recorded after the recovery from anesthesia. Stroke volume was calculated by reading required pressure and time data on the tracings and substituting them into a special equation. Cardiac output per min per body weight was not significantly different between SHR and NCR. Arterial pressure and total peripheral resistance were higher in SHR than in NCR. On ganglion blockade with hexamethonium bromide in the conscious state, arterial pressure decreased more markedly in SHR than in NCR. Though arterial pressure was still significantly (P less than 0.05) higher in SHR after blockade, cardiac output was larger in SHR more than to account for the difference in arterial pressure. Total peripheral resistance was lower in SHR than in NCR after blockade. It is concluded that, even in the conscious state as in the anesthetized state, the major contribution to the hypertensive state in SHR is an increase in total peripheral resistance due to an elevation of the sympathetic tone.


An analysis of the drugs acting on cerebral energy metabolism. The behaviour of (a) the redox potential of the lactate/pyruvate system and the changes of the redox potential of the lactate/pyruvate system across the brain; (b) the energy charge potential of the adenylate pool, was studied in the brain of curarized beagle dogs. The influence of certain drugs (amobarbital, nicergoline, theophylline, papaverine, bamethan, dipyridamole, bemegride) on these parameters was evaluated under control conditions, during hypoxemia and during post-hypoxiemic recovery. On the whole, the action on energetic metabolism appears to be unrelated to the action believed to be exerted by drugs on cerebral vessels.


[Coronary reserve and differentiated therapy of its disorder]. The clinical course of the ischaemic heart disease was studied and compared with the ECG indices at rest, and under exercises, and with the data of selective coronary angiography in 189 patients. Three stages of decompensation of the coronary reserve were revealed. The efficiency of drug therapy and the indications for surgical management were studied with reference to the state of the coronary reserve.


[Effect of chronic beta-adrenergic blockade on blood pressure and release of renin, aldosterone and cortisol in essential hypertension (author's transl)]. Blood pressure (BP), plasma renin activity (PRA) and plasma concentration of aldosterone (PA) and cortisol (PC) were determined in essential hypertensive patients before and after beta-adrenergic blockade with propranolol and prindolol. Serial measurements of PRA, PA and PC at 30 min intervals (8 p.m. to 6 a.m.) were performed in 8 patients. Administration of propranolol (50 mg three times daily) over a period of 4 weeks was followed by a significant reduction in BP, PRA and PA. PC remained unaltered. The pattern of rhythmic secretion of renin was abolished whereas that of aldosterone persisted at a lower level. Prindolol (10 mg three times daily) had a similar effect on BP as propranolol but failed to lower PRA and PA. Rhythmic secretion of renin was also markedly altered with prindolol in the absence of any change in rhythmicity of PA and PC. The hypotensive action of prindolol was not mediated via inhibition of renin release. The dissociation between PRA and PA rhythmicity indicates that during beta-adrenergic blockade diurnal rhythm of aldosterone secretion is not regulated by the renin-angiotensin-system. Rhythmicity of PC was normal, indicating that also ACTH-secretion was unaltered. Thus, under beta-blockade unchanged diurnal rhythm of aldosterone may be due to normal ACTH-secretion.


Effect of beta-adrenoceptor blocking drugs, physostigmine, and atropine on the toxicity of doxepin in mice. Large doses of doxepin given intravenously to animals cause tachyarrhythmias, and still higher doses lead to a progressive and, finally, lethal bradycardia. The effect of pretreatment with five different beta-adrenoceptor blocking drugs (propranolol, alprenolol, practolol, metoprolol or tolamolol), p physostigmine, or atropine on these toxic actions of doxepin was investigated. Mice were sedated with diazepam. Doxepin was injected i.v. 0.1 mg every 15 sec until death. ECG was recorded at 10 sec after every injection. All five beta-blockers injected  i.p. 30 min before doxepin inhibited the doxepin-induced tachyarrhythmias. None of the drugs prevented or postponed the death of mice. Large doses of beta-blockers dose-dependently enhanced the doxepin-induced bradycardia and accelerated death. The cardioselective beta-blocking drugs practolol and metoprolol proved less active in enhancing bradycardia than the third cardio-selective drug, tolamolol, and non-selective propranolol and alprenolol. This difference may have resulted from properties other than beta-blockade since practolol and metoprolol lack the "cardiodepressant" and local anaesthetic properties. Since physostigmine and atropine did not modify the doxepin effects the anticholinergic property may not be important in the severe cardiotoxic effects of doxepin.


Effect of sodium cellulose phosphate therapy on crystallization of calcium oxalate in urine. Effects of oral sodium cellulose phosphate therapy (5 g three times a day with meals for 4 days) on renal excretion of oxalate and on the crystallization of calcium oxalate in urine were examined in six patients with absorptive hypercalciuria on a constant metabolic dietary regimen. During treatment, urinary oxalate increased by 9-50 mg/day. However, urinary calcium decreased by 138-225 mg/day (50%-70%). Thus, the state of saturation of urine with respect to calcium oxalate decreased or did not change significantly. There was no consistent or significant change in the formation product ratio (limit of metastability) or in the crystal growth of calcium oxalate in urine.


Regulation of glycolysis in Neurospora crassa. Kinetic properties of pyruvate kinase. Pyruvate kinase (ATP:pyruvate phosphotransferase, EC 2.7.1.40), extracted from the mycelium of Neurospora crassa has been purified 560-fold by precipitation with ammonium sulphate, chromatography with DEAE-Sephadex, and gel filtration with Sephadex G-200. Potassium and magnesium are required for enzyme activity. Fructose, 1,6-diphosphate is the only physiological activator found for the enzyme. In decreasing order of potency, citrate, oxalacetate, calcium, and ATP are inhibitors. Phosphoenolpyruvate is cooperatively bound by the enzyme and the cooperatively is reduced by ATP and completely eliminated by fructose-1,6-diphosphate. Lowering of pH from 7-5 to 5-5 changes the Hill coefficient from 2-7 to 1-0. Substitution of ADP by other nucleotides reduces enzyme activity. Manganese can substitute for the cofactor magnesium, but the reaction velocity is then reduced. MgADP- is cooperatively bound by the enzyme and inhibition of the enzyme occurs only when either magnesium or ADP is in excess of the other beyond the optimum concentration. These kinetics properties of pyruvate kinase are compatible with the role of a regulator of glycolysis in Neurospora crassa.