A cyclic adenosine 3',5'-monophosphate-dependent histone kinase from pig brain. Purification and some properties of the enzyme.

A cyclic adenosine 3',5'-monophosphate-dependent histone kinase (ATP: protein phosphotransferase, EC 2.7.1.37) was isolated from pig brain. The enzyme has been purified 1140-fold; it is homogeneous on polyacrylamide gel electrophoresis and gel filtration. The estimated molecular weight of the enzyme is 120 000. Histone kinase dissociates into a catalytic subunit and a regulatory one (molecular weights 40 000 and 90 000, respectively). The catalytic subunit has been obtained in homogeneous state as evidenced by sodium dodecylsulphate-polyacrylamide gel electrophoresis. At all purification steps, enzymatic activity is stimulated 5-fold by cyclic AMP. An apparent Km value for cyclic AMP is about 3.3 - 10- minus 7 M. In the presence of cyclic AMP(5 - 10- minus 6 M), the Km value for ATP and F1 histone were 1.2 - 10- minus five and 3 - 10- minus 5 M, respectively. Optimum pH value for histone kinase is 6.5, its isoelectric point is situated at pH 4.6. The purified enzyme displays high specificity for the lysine-rich and moderately lysine-rich histones F1, F2a2 and F2b. Arginine-rich histones and other known protein substrates for cyclic AMP-dependent protein kinases (casein, Escherichia coli RNA polymerase, etc.) are extremely poor substrates for this enzyme.


Biosynthesis of acid phosphatase of baker's yeast. Characterization of a protoplast-bound fraction containing precursors of the exo-enzyme.

1. Yeast protoplasts, secreting acid phosphatase (orthophosphoric-monoester phosphohydrolase (acid optimum) EC 3.1.3.2) contain a small amount of firmly bound enzyme, even after lysis (Van Rijn, H.J.M., Boer, P. and Steyn-Parvé, E.P. (1972) Biochim. Biophys. Acta 268, 431-441). The major part (70%) of this protoplast-bound acid phosphatase can be solubilized by nonionic detergents, such as Triton X-100. 2. The kinetics of radioactive amino acid incorporation in the solubilized and in the secreted enzyme has been estimated by pulse-chase labelling of secreting protoplasts, followed by fractionation and counting radioactivity in the enzyme band in polyacrylamide gels after electrophoresis at pH 5.0. A precursor-product relationship between the Triton X-100-extractable fraction of the protoplast-bound acid phosphatase and the secreted enzyme is apparent. 3. The solubilized acid phosphatase is essentially indistinguishable from the secreted enzyme with regard to a number of enzymatic properties and its stability towards pH and temperature. Both enzymes also behave alike on polyacrylamide-gel electrophoresis, producing a single acid phosphatase band with glycoprotein character and comparable mobility. 4. A striking difference is seen in isopycnic equilibrium sedimentation in CsCl: the secreted acid phosphatase is homogeneous, with a buoyant density of p equals 1.47 g/cm3, while the Triton X-100-extractable part of the protoplast-bound acid phosphatase is heterogeneous; besides heavier material a major component with buoyant density of p equals 1.37 g/cm3 is always visible.


Phosphoprotein phosphatase in the central nervous system of Manduca sexta.

The existence and some enzymological properties of phosphoprotein phosphatase (EC 3.1.3.16) have been established in the larval central nervous system of the tobacco hornworm, Manduca sexta (Lepidoptera: Sphingidae). A simple, sensitive and reproducible assay employing 32-P-labeled protamine as a phosphoprotein substrate was employed to measure phosphatase activity in both soluble and particulate fractions of the insect nerve cord. The specific activity of soluble phosphatase in the Manduca sexta central nervous system is of the same order of magnitude as that in mammalian brain. Nerve cord phosphoprotamine phosphatase activity may be stimulated by a variety of monovalent salts, the optimal concentration of NaCl or KCl being 0.2 molar. Activity does not appear to be dependent on bivalent metals and is stimulated by EDTA. A reduced sulfhydryl group is obligatory for maximum activity. Phosphatase could be greatly inhibited by sodium fluoride, ATP and GTP. Cyclic AMP and cyclic GMP are without effect on enzyme activity. Although most of the phosphatase activity in the insect nerve cord appears to be of cytosolic origin, much latent activity can be unmasked by incubating membranous fractions with Triton X-100. In contrast to soluble phosphatase, the detergent-solubilized activity is moderately stimulated by Mn-2+.?


Purification and properties of a beta-1,6-clucosidase from Flavobacterium.

An intracellular beta-1,6-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) was produced semiconstitutively by Flavobacterium M64. This enzyme was purified 180-fold by fractionation with ammonium sulfate followed by chromatographies on carboxymethylcellulose, hydroxyapatite and Sephadex G-100. The final preparation appeared homogeneous on disc electrophoresis on polyacrylamide gel. The molecular weight of the enzyme was determined to be ca. 59 000 by Sephadex G-100 gel filtration and sodium dodecylsulfate-polyacrylamide gel electrophoresis. The optimum pH of the enzyme was 5.8 and the optimum temperature was 40 degrees C. The enzyme readily hydrolyzed oligomers with beta-a,6-glucosidic linkages, converting them to glucose. The Km values for gentio-biose, -triose, -tetraose and -pentaose were 2.8, 3.0, 4.2 and 4.6 times 10- minus 4 M, respectively. The rates of their hydrolyses decreased with increase in their chain lengths. The enzyme was concluded to be a beta-1,6-glucosidase from its substrate specificity, production of glucose, transferring ability and inhibition by glucono-delta-lactone. The enzyme activity was inhibited by Hg-2+, Cu-2+, Ag-+, Fe-3+, p-chloromercuribenzoate, N-ethylmaleimide, glucose and trishydroxyaminomethane (Tris) but not by ethylenediaminetetraacetic acid.


Proteolytic degradation of insulin and glucagon.

The degradation of insulin and glucagon by a highly purified enzyme isolated from rat skeletal muscle was investigated. A sensitive assay for proteolytic degradation of insulin and glucagon using fluorescamine to detect an increase in primary amine groups was established. As measured by an increase in fluorescamine reactive materials, insulin was rapidly degraded by this highly purified enzyme without requiring initial disulfide cleavage. Associated with the increase in fluorescamine reactive materials was a decrease in immunoassayable insulinmglucagon wal also proteolytically degraded by this enzyme but a number of other peptides and proteins including proinsulin, and A and B chains of insulin were not degraded. Thus, we have demonstrated that insulin (and glucagon) can be proteolytically degraded by an enzyme isolated from an insulin sensitive tissue, skeletal muscle. Proteolytic degradation by this enzyme requires the intact insulin molecule rather than separate A and B chains.


Purification and properties of a constitutive beta-lactamase from Pseudomonas aeruginosa strain Dalgleish.

1. The beta-lactamase (penicillin amido-beta-lactamhydrolase EC 3.5.2.6) appeared to be periplasmic rather than truly intracellular, since it was released by freeze-thawing without gross morphological changes in the cell. 2. The partially purified enzyme had pI between 5.0 and 5.5, mol. wt 32 000 and a broad pH vs activity profile with a maximum at pH 8. 3. The cephalosporins tested were hydrolysed less rapidly than most of the penicillins, and the Km values for penicillins were lower than for cephalosporins. However cloxacillin was hydrolysed very slowly although it was strongly bound. The substrate-induced inactivation common to many beta-lactamases was particularly marked with cephaloridine and cloxacillinmthe cloxacillin-induced inactivation was shown to be reversible.


Catalytic and thermodynamic properties of the urocanate hydratase reaction.

Urocanate hydratase (4-imidazolone-5-propionate hydro-lyase, EC 4.2.1.49) isolated from Pseudomonas putida contains covalently bound alpha-ketobutyrate as its cofactor. In the process of examining the mechanism by which alpha-ketobutyrate serves in this capacity, various thermodynamic parameters and temperature effects on urocanate hydratase activity were determined. As the equilibrium constant at 15 degrees C for imidazooone propionate formation from urocanate is approximately 69, regardless of whether urocanic acid or chemically synthesized imidazolone propionate is used as the initial substrate, it is concluded that the reaction is freely reversible. DeltaG degrees ', deltaH degrees ' and deltaS degrees ' were --2.5 kcal/mole, +5.2 kcal/mole and +26 cal/deg mole, respectively. Measurement of first-order reaction rates at various temperatures, in order to calculate the Arrhenius activation energy, showed a sharp break in the Arrhenius plot at 29 degrees C. Further examination of this phenomenon by determining s20,w values of urocanate hydratase as a function of temperature revealed a dramatic change at 31 degrees C. Since the enzyme in both experiments reverts to its original state when the temperature is lowered back below the transition point, it is proposed that urocanate hydratase undergoes a reversible conformational change or partial dissociation which affects its catalytic properties in the range of 29--31 degrees C.


Renal cortex guanylate cyclase. Preferential enrichment in glomerular membranes.

1. The localisation and some of the properties of rabbit kidney cortex guanylate cyclase (GTP pyrophosphatase lyase (cyclizing) EC 4.6.1.2) have been studied. Upon fractionation of dissociated renal cortex, guanylate cyclase activity was preferentially enriched in fractions of pure glomeruli, where its specific activity was 44.5 times that measured in tubular fragments. Most, if not all, of the glomerular activity was found to be firmly membrane-bound, whereas the guanylate cyclase activity of the tubules was mainly soluble. Therefore, particulate guanylate cyclase activity could serve as marker enzyme for kidney glomeruli. 2. All hormones or hormone-like agents tested were without effect on kidney guanylate cyclase activity. Triton X-100 stimulated both glomerular and tubular activity. 3. Considering the high cyclic GMP forming capacity of kidney glomeruli, part of the cyclic GMP found in urine might be synthetized locally in these structures.


An acridine probe into the physiological state of the cell.

Acridine orange was used as a probe to look into the physiological state of the yeast cell, particularly as regards the change in the properties of the membrane (which acts as a barrier against the incoming acridine orange) and the availability of binding sites for acridine orange in chromosomal DNA during growth. After acridine orange had been introduced into the cell, the genetic change at a specific locus with incubation time was measured photodynamically. A three-fold increase in the rate of penetration of acridine orange into the cell was observed, for instance, in going from the resting phase to the dividing phase. A five-fold increase was observed in the number of binding sites in chromosomal DNA under the same transition of the cell. These two parameters may be useful as a measure of the physiological changes in the cell. Some environmental factors such as pH and temperature were also demonstrated to affect the parameters.


On the molecular weight of thiosulfate sulfurtransferase.

Bovine liver thiosulfate sulfurtransferase (rhodanese) (EC 2.8.1.1) HAS BEEN REPORTED TO EXIST IN SOLUTION IN A RAPID, PH-dependent equilibrium between monomeric and dimeric forms of molecular weights 18 500 and 37 000 (Volini, M., DeToma, F. and Westley, J. (1967), J. Biol. Chem. 242, 5220). We have reinvestigated the proposed dissociation using sodium dodecylsulfate-polyacrylamide gel electrophoresis. The smallest rhodanese species observed has a molecular weight around 35 000, which is not reduced by severe denaturing conditions, including alkylation in 8 M guanidine-HCl or dialysis against 2% sodium dodecylsulfate and 5% mercaptoethanol. After limited CNBr cleavage, intermediate products of greater than 18 500 molecular weight are formed. The apparent molecular weight of these intermediate fragments is not changed by addition of mercaptoethanol. The total apparent molecular weights of the CNBr fragments after exhaustive cleavage is approx. 45 000 plus or minus 15 000. These results are not consistent with a monomer molecular weight of approx. 18 500 for thiosulfate sulfurtransferase.


Raman studies of the conformation of the basic pancreatic trypsin inhibitor.

The vibrational Raman spectra of the basic pancreatic trypsin inhibitor in aqueous solution, as lyophilized powder and in a single crystal and presented. The thermal stability of this protein is demonstrated by the fact that minor alterations in the spectrum, mainly in the amide III band near 1260 cm-1, occur in the solution spectrum only at temperatures above 75 degrees C. No significant spectral changes appear when the pH value of the solution is varied in the range from 1.5 to 8.7. The distinct differences of the powder spectrum compared to that of the solution, show that lyophilization causes appreciable conformational changes both in the main-chain and in the side-chains. A difference in main chain conformation of the basic pancreatic trypsin inhibitor in single crystal and in solution is suggested by different amide III frequencies.


Structural and functional determinants of Mucor miehei protease. V. Enthalpy changes upon thermal denaturation in solution of varying pH.

The thermal denaturation of Mucor meihei protease was studied as a function of pH by differential scanning calorimetry. In both citric acid-Na2HPO4 and in acetic acid-sodium acetate buffers, maximum thermal stability was at pH 4.0-4.2. However, the maximum enthalpy changes associated with the denaturation process were buffer-dependent and occurred between pH values of 4.7 and 5.7.


Analysis of bacterial biotin-proteins.

The biotin-protein populations in several bacterial strains were analyzed by solubilization of [3H]biotin-labeled cells with sodium dodecylsulfate followed by electrophoresis on polyacrylamide gels containing the detergent. A variety of patterns of biotin-labeled polypeptide chains was seen, ranging from a single biotin-protein in Escherichia coli, corresponding to the biotin carboxyl carrier protein component of acetyl-CoA carboxylase, to multiple species in Enterobacter aerogenes, Pseudomonas citronellolis, Bacillus cereus, Propionibacterium shermanii, Lactobacillus plantarum, and Mycobacterium phlei, which probably represent subunits of multiple biotin-dependent enzymes present in these organisms. In the case of Pseudomonas citronellolis two major biotin-containing polypeptides with approximate molecular weights of 65 000 and 25 000 were shown to correspond to the biotin carboxyl carrier components of pyruvate carboxylase and acetyl-CoA carboxylase, respectively. Thus in the case of Pseudomonas citronellolis two different biotin-dependent enzymes in the same cell do not share common biotin carboxyl carrier subunits.


The location of protein in serum lipoproteins: a fluorescence quenching study.

Quenching of the tryptophan fluorescence of pig serum HDL3 and LDL2 lipoproteins by iodide and succinimide has been used to estimate the accessibility of the fluorophores to the solvent and, by inference, the location of the protein in the macromolecular complexes. At least 80% of the protein is thought to be located at or near the surface in both lipoproteins but its accessibility is hindered especially in LDL2. A difference in surface topography in the two lipoproteins is suggested with the protein in LDL2 more buried in lipid and further away from the charged phospholipid polar groups than in HDL3. A refined treatment of the quenching data has been developed to take account of the heterogeneity of quenching sites found in the lipoproteins.


Cationic proteins from human neutrophil granulocytes. Evidence for their chymotrypsin-like properties.

Three cationic proteins from the granules of human neutrophil granulocytes were obtained in a high degree of purity be means of affinity chromatography on 4-phenylbutylamine-Sepharose. Together with lysozyme, the three cationic proteins exhibit the highest electrophoretic mobility toward the cathode in acrylamide gels at moderately acid pH, among the granule constituents that are solubilized in 0.1 M phosphate buffer, pH 7.0, containing 1 M NaCl. The three cationic proteins represent a group of "neutral proteases" distinct from elastase and collagenase. They hydrolyze casein, azocasein and the chymotrypsin substrate N-acetyl-L-tyrosine ethyl ester. Optimal activity is found at pH 7.4-7;5. The enzymes are inhibited by the specific chymotrypsin inhibitor N-tosyl-L-phenylalanylchloromethane and by the naturally occurring inhibitors alpha-antichymotrypsin, alpha-1-antitrypsin, as well as by the trypsin inhibitors from soy beans and limabeans.


A conformational change in bovine beta-lactoglobulin at low pH.

A conformational change at low pH in bovine beta-lactoglobulin A has been studied by intrinsic fluorescence and fluorescence of the bound dye 8-anilinonaphthalene-1-sulphonate. Both studies show that when the pH of beta-lactoglobulin solutions is altered between 6.5 and 2.0, a rapid change in protein conformation occurs, followed by a slower conformational change. It seems likely that the rapid changes are linked with the predominance of protein dimer at pH 6.5 and monomer at pH 2.0. The slow changes involve shifts in protein conformation of the region that includes one of the protein tryptophan residues.


Lipoprotein lipase in rat lung. The effect of fasting.

We measured lipoprotein lipase activity in dried defatted preparations of rat lung using doubly labeled chylomicron triglyceride as substrate. The enzyme activity was linear for the first hour of incubation at 37 degrees C, had a pH optimum of 8.1 and was completely inhibited by 0.5 M NaC1. Lungs from fed rats hydrolyzed chylomicron triglyceride at a rate of 13.00 mumoles/g per h; the activity rate was unchanged by fasting 8-72 h. Heparin infusion into isolated lungs caused immediate release of lipoprotein lipase to the venous effluent. The activity released was equivalent to about 10% of total lung lipoprotein lipase activity in both fed and fasted rats. Since the ability to remove blood triglyceride is directly related to the level of lipoprotein lipase activity, these findings indicate that the lung is one of the few tissues able to remove efficiently blood triglyceride during fasting.


A possible mechanism for the peroxidation of lipids due to chronic ethanol ingestion.

The effects of chronic ethanol ingestion on NADPH-oxidase and on the NADPH-catalyzed peroxidation of lipids in rat liver microsomes have been studied. It was demonstrated that the rates of NADPH oxidation, of oxygen consumption, and of malondialdehyde formation increased significantly above control values after one month of ethanol ingestion. Further, the fatty acid composition of these microsomes revealed a decrease in arachidonate and in the C22 polyenes. Also, the energies of activation for the formation of malondialdehyde increased in the microsomes from the ethanol-treated animals. These results were interpreted to mean that ethanol ingestion had induced changes in the microsomal membranes such that additional or alternate, possibly abnormal, pathways for lipid peroxidation were functional. Finally, these data suggest a mechanism whereby chronic ethanol ingestion inhances the production of lipid peroxides via the microsomal-catalyzed oxidation of NADPH.


The biosynthesis of plasmalogens by rat brain: involvement of the microsomal electron transport system.

The biosynthesis of 1-alk-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine (ethanolamine plasmalogens) was studied using 1-[1-14C]hexadecyl-sn-glycero-3-phosphoethanolamine as the substrate and EDTA-washed microsomes from brains of 14-day-old rats. It was found that the 1-E11-14C]hexadecyl-sn-glycero-3-phosphoethanolamine was first acylated to form 1-[1-14C]hexadecyl-2-acyl-sn-glycero-3-phosphoethanolamine, then was desaturated to form 1-[1-14C]hexadec-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine. The desaturation required O2 and NADH or NADPH and was inhibited by KCN but not by CO. The data indicated that the desaturation is carried out by a mixed-function oxidase system similar to that involved in the desaturation of fatty acids and that the pathway for the biosynthesis of plasmalogens in brain is similar to that previously found in other tissues. The desaturase was not stimulated by ATP and Mg2plus nor inhibited by EDTA. The specific activity of microsomes from brains of rats of different ages was determined; the activity decreased with age until in adults the activity was only 15% that of the 12--14-day-old rats.


The inhibition by bromothymol blue of anion translocation across the mitochondrial membrane.

1. In rat liver mitochondria bromothymol blue inhibited the exchange of [14C]succinate for succinate, malonate, L-malate and inorganic phosphate; the [14C]citrate/citrate and [14C]citrate/malate exchange reactions and the phosphate/hydroxyl exchange were also inhibited by this dye. The inhibition of the rate of succinate, citrate and phosphate uptake by bromothymol blue is found to be competitive. 2. The degree of inhibition by bromothymol blue of the ]14C]succinate/malonate exchange reaction was pH dependent. It has been shown that the inhibition increased linearly while the pH was increased from 6.0 to 8.2. However, the binding rate of bromothymol blue to the mitochondria decreased with the rising pH of the medium. It is concluded that the binding of acidic bromothymol blue was not essential for the inhibitory effect. 3. Other sulfonephthalein derivatives also inhibited [14C]succinate/malonate exchange reaction. At pH 7.2 the relative order of the strength of the inhibitory action of the sulfonephthalein compounds tested was: thymol blue greater than bronocresol green greater than bromothymol blue greater than phenol red greater than bromocresol purple. The results do not indicate any correlation between the pK values of pH values of pH indicators and their extents of inhibition. 4. It is suggested that the negatively charged bromothymol blue interacts with the positively charged centers of the anion carrier systems causing inhibition of membrane permeability for anions.


Electromechanical stresses and the effect of pH on membrane structure.

The presence of an electric field in a membrane creates stresses which lead to a mechanical compression of the membrane material. It is shown that considerations of this effect yield results consistent with the observed differential effect of pH on the plasma membrane substructure if the central electron-lucent layer in OsO4-fixed membranes is identified with the ionic depletion in the double-fixed charge model of cell membranes.


Enzymes of the human intestinal brush border membrane. Identification after gel electrophoretic separation.

The postition of a number of human intestine brush border membrane enzyme activities in polyacrylamide gels after electrophoresis has been determined. These activities are, in order from the origin, maltase/glucoamylase, lactase/phlorizin hydrolase, maltase/sucrase/isomaltase, enteropeptidase, trehalase and gamma-glutamyl-transferase. Leucylnaphthylamide hydrolyzing activity was inactivated by sodium dodecylsulfate and its position was not determined. The positions of the activities have been correlated with the positions of protein bands previously determined. One such band situated between enteropeptidase and alkaline phosphatase has not been identified.


Bicarbonate ion-ATPase in rat liver cell fractions.

The distribution of HCO3MINUS-ATPase activity was studied in cell fractions prepared from homogenates of rat liver. The level of mitochondrial contamination in the microsomal fraction depended on the fractionation procedure and on the method of homogenization. With proper care, microsomes with undetectable mitochondrial contamination could be prepared. These microsomes had no detectable HCO3MINUS-ATPase activity. Approximately 85% of the total HCO3minus-ATPase activity of the post 6000 times g-min supernatant was recovered in the mitochondrial fraction. The properties of this mitochondrial HCO3minus-ATPase were not distinguishable from those of the various microsomal HCO3minus-ATPases previously described by other investigators.


A calorimetric study of the helix-coil transition of poly(cytidylic acid) in acid solution.

In this study we show how the thermal stability of the helical structure of poly(C) is dependent on the degree of protonation. deltaH values for the thermal denaturation are measured directly by means of an adiabatic microcalorimeter in the pH range from pH 5.8 to 3.7. The maximum transition temperature T-m is found at pH 4.5 where poly(C) is semiprotonated. At the same pH value a maximum value of deltaH (deltaH equals 5.27 Kcal/mol base pairs) was obtained. deltaH decreases slightly if the pH changes to lower or higher pH values. No transition was observed either at pH 6.0 or at pH 3.4. The deltaGtheta(25 degrees C) values show a similar dependence on the degree of protonation of poly(C).


[A calorimetric study of the triple helix formation: interaction of poly(C) - guanosine - protonated poly(C)].

The triple helix formation of poly(C) - guanosine - poly(C+) was investigated by the help of an LKB scanning micro-calorimeter. The existence of the triple helix could also be shown by recording the melting curves. The ultraviolet absorption at different wave lengths namely 275 nm, 260 nm, and 245 nm was plotted as a function of the temperature. Furthermore formation of the triple helix was shown by plotting the ultraviolet absorption at 245 nm during the increasing addition of guanosine solution to a fixed amount of poly(C) in the solution. Finally the formation of the triple helix was demonstrated by plotting the ultraviolet absorption at 245 nm of a certain mixture of the components while the pH value of the solution was continuously lowered. All these methods show that the monomer interacts with the polymer double helix to form a triple helix. The calorimetric measurements show that the reaction enthalpy is concentration dependent. Above a threshold concentration a rapid increase of the reaction enthalpy is observed. This increase occurs in a very narrow concentration interval. Above this interval a final value of the reaction enthalpy is reached. The amount of the reaction enthalpy for the interaction of guanosine with poly(C) - poly(C+) double helix is 5.5 Kcal (mol base triplet)-1.


Near-ultraviolet light-induced strand breaks in DNA pretreated with the carcinogen, N-acetoxy-2-acetylaminofluorene.

Neutral sucrose gradients of supercoiled DNA (phiX-174 RF I) were used to measure the in vitro production of strand breaks by the carcinogen, N-acetoxy-2-acetylaminofluorene (AcO-AAF). Treatment with AcO-AAF in 10% dimethyl sulfoxide did not directly yield strand breaks. Breaks in relatively low yield appeared after alkali treatment (pH 13, 60 min) of the RF I previously reacted with AcO-AAF. The DNA treated with AcO-AAF was sensitive to single-strand breakage by 303 nm near-ultraviolet light under neutral conditions. The greater the prior AcO-AAF treatment, the greater the sensitivity to 303 nm light. Post-irradiation alkali treatment greatly enhanced the light-induced strand breakage.


Viscoelastic analysis of high molecular weight, alkali-denatured DNA from mouse 3T3 cells.

Alkaline lysates of mouse 3T3 cells showed viscoelastic properties characteristic of very large molecules of single-stranded DNA. The viscoelastic retardation time and the sensitivity to low doses of nitrogen mustard and of X-irradiation suggest a molecular weight in excess of 10-10 daltons. Contact-inhibited cells yielded larger single strands than actively growing cells.


Long-term remission from acute myelogenous leukemia after bone marrow transplantation and recovery from acute graft-versus-host reaction and prolonged immunoincompetence.

A 19-yr-old boy has been in continuous complete remission from acute myelogenous leukemia for 3 yr after allogeneic bone marrow transplantation prepared with combination chemotherapy. During the first year post-transplant, however, the patient developed near-fatal graft-versus-host reaction followed by 11 severe viral and bacterial infections. Immune evaluation during this period revealed multiple defects which were not present prior to transplantation, nor present in the transplant donor: diminution of lymphoid tissue, decline of all immunoglobulin subtypes, deletion of secretory immunoglobulin, disappearance of isohemagglutinins, loss of antibody to diptheria and tetanus toxoids, cessation of cutaneous hypersensitivity to mumps antigen, and inhibition of serum opsonizing activity. The patient was also unable to develop normal humoral or cellular reactivity to brucella antigen, keyhole limpet hemocyanin, or dinitrochlorobenzene. This patient's course illustrates the severity and chronicity of immunoincompetence associated with allogeneic marrow grafting, the importance of early detection and rigorous treatment of infectious disease in these patients, and the need for improved immunologic reconstitution in human marrow transplantation. It also indicates that complete recovery from the immune defects is possible, and that long-term remission from acute myelogenous leukemia can be achieved with allogeneic marrow transplantation.


Abnormal red cell metabolism in patients with chronic uremia: Nature of the defect and its persistence despite adequate hemodialysis.

A red cell metabolic abnormality, which diminishes the maximum activity of the pentose phosphate shunt, occurs in some uremic patients, even those adequately dialyzed with fluids prepared from distilled or charcoal-filtered water. Within individual patients the severity of this abnormality does not change even after 9 mo of consecutive hemodialyses. However, between patients it does correlate inversely with hematocrit. When erythrocytes from patients with the abnormality are stressed with oxidant compounds, such as ascorbate, erythrocyte glucose consumption and lactate formation are abnormally increased, while lactate/pyruvate ratios abnormally diminish. Concomitantly, red cell glycolytic intermediates, including fructose-1,6-diphosphate, glyceraldehyde-3-phosphate, 3-phosphoglycerate, phosphoenol pyruvate, and pyruvate, markedly accumulate. Surprisingly, no increase of 2-phosphoglycerate occurs, which suggests that inefficient phosphoglyceromutase activity underlies this perturbation of erythrocyte metabolism and its associated hemolytic process.


Fibrinogen ""New York"--an abnormal fibrinogen associated with thromboembolism: functional evaluation.

A 54-yr-old woman presented with a 23-yr history of repeated life-threatening thromboembolism. The presence of a qualitatively abnormal fibrinogen was suggested by the demonstration of delayed and incomplete coagulation of plasma or partially purified fibrinogen by thrombin or Reptilase. Two brothers showed a similar in vitro defect but were clinically not affected. The plasma fibrinogen concentration was 0.50-1.64 mg/ml when estimated by heat turbidity, clottability, or immunologic techniques. The serum contained 80-820 mug/ml of unclottable fibrinogen-related materials even after 24 hr exposure to thrombin. The fibrinogen-related material in the serum showed faster anodal mobility an immunoelectrophoresis than that of normal plasma. Immunodiffusion studies with rabbit antihuman fibrinogen antiserum showed lines of identity between control plasma and the patient's plasma and serum. Studies of the kinetics of thrombin action on fibrinogen demonstrated impaired release of fibrinopeptide A and B and defective polymerization of preformed fibrin monomers. The maximum amount of fibrinopeptide A released by exhaustive treatment with thrombin was similar (per milligram protein) for both the patient's and control fibrinogen. This abnormal fibrinogen varient is tentatively designated fibrinogen "New York"; its possible identity with one of the previously described abnormal fibrinogens has not been excluded.


Pharmacological receptors of the cerebral arteries of the goat.

5-Hydroxytryptamine (5-HT), norepinephrine (NE), histamine (H) and potassium (K+) chloride induce dose-dependent changes in tension of the isolated middle crerbral artery of the goat. Vasopressin produces highly variable responses followed by tachyphylaxis; angiotensin II is ineffective over a wide dose range. The order of potencies of these vasoactive agents is 5-HT greater than NE greater than H greater than K+. With regard to their ability to induce maximal contractile responses, the order is: H greater than 5-HT, K+ greater than NE. Lysergic acid diethylamide (LSD) antagonizes the actions of 5-HT in a manner which progresses from surmountability to unsurmountability of the blockade depending on the concentration of LSD. The blockade exerted by LSD is reversed by washing. Phentolamine and diphenhydramine competitively antagonize the actions of NE and H, respectively. The potency of phentolamine and diphenhydramine in the cerebral arteries of the goat is similar to that determined in different tissues obtained from a variety of animal species. It is concluded that the cerebral arteries of the goat possess receptors for biogenic amines, the most effective of which is 5-HT; receptors for vasoactive peptides are ill defined.?25


Gamma-glutamyl transpeptidase, triglycerides, and enzyme induction.

A study conducted on 109 consecutive patients submitted for routine lipid and lipoprotein screening has shown a significant positive association between serum gamma-glutamyl transpeptidase (gamma-GT) activity and the serum triglyceride concentration and between serum gamma-GT activity and the serum pre-beta-lipoprotein concentration. We suggest that these associations may reflect hepatic microsomal enzyme induction in hyperlipidaemic subjects which increases the hepatic content of the rate-limiting enzyme(s) for triglyceride synthesis.


Gamma-aminobutric acid and glutamate decarboxylas (l-glutamate 1-carboxy-lyase e.c. 4.1.1.15) in the nervous system of the cockroach, periplaneta americana.i.regional distribution and properties of the enzyme.

Both the central and peripheral nervous system of the cockroach Periplaneta americana contain gamma-aminobutyric acid (GABA) and glutamate decarboxylase (GAD). In the central ganglia of the cockroach, an average of more that 60 mumoles of GABA are formed from glutamate (Glu) per gram wet weight of tissue per hour. This activity level of the GAD apoenzyme is considerably higher than that found in the central nervous system of crustaceans, amphibians, avians and mammals but is similar to that reported for nervous system tissues from other insect species. A comparison of properties of the crude cockroach enzyme with GAD from crustacean and mammalian origin revealed both similarities and differences: whereas crude cockroach GAD has cofactor requirements and an affinity for Glu substrate (Km 2.8 X 10-2) which are similar to GAD from lobster and mouse, it is uniquely inhibited by both Cl-and by GABA. The GAD from cockroach nervous tissues has two apparent pH optima of which the lower one is preferentially inhibited by a compound which is found in the nerve sheath and the fat body tissue adjacent to ganglia and axons.


Studies on  bursting pacemaker potential activity in molluscan neurons. II. Regulations by divalanet cations.

Identified cells in Aplysia (R15, R2, LPG) and cell 11 in Otala have been used to investigate the effects of divalent cations, temperature, pH and ouabain on neuronal activity. Divalent cations act primarily to regulate the appearance of bursting pacemaker potential (BPP) activity in these cells. These ions are necessary for B generation and will inhibit its appearance at high concentrations (Ca greater than Mg greater that greater than Sr). In addition, Ca is involved in the seasonal modulation of BPP activity in a neurosecretory cell in Otala. Monovalent cations play secondary roles as regulators of BPP activity by competing with divalent cations for the sites involved in the regulation of the (probable) monovalent conductances underlying BPPs. The effects of pH, temperature and ouabain on membrane properties and BPP activity are partly related to their interaction with divalent cations. The results described indicate important roles for divalent cations in the regulation of the expression of BPP activity and its underlying membrane properties both in different nerve cells and in the same cell during dormancy and activity of the snails.


A simple chromatographic method for the determination of the methylated basic amino acids in proteins.

A chromatographic method is described for the determination of epsilon-N-monomethyl-epsilon-N, epsilon-N-dimethyl-, and epsilon-N, epsilon-N, epsilon-N-trimethyllysines, 1-methyl-, and 3-methylhistidines, N-G, N-G-dimethyl-, and N-G, N'G-dimethylarginines, ornithine, the diastereoisomers of hydroxylysine, basic amino acids and related compounds, using Durrum type DC-6A 11.0 plus or minus 1.0 mum spherical resin. The complete separation of all of these amino acids as discrete peaks in the Beckman 120B amino acid analyzer was achieved for the first time using a single column (60 times 0.9 cm), one buffer system (0.35 N sodium citrate) adjusted at either pH 5.734 or 5.657 plus or minus 0.002, one buffer flow rate (30 ml/h), and one temperature (28 degrees C). This procedure was used for the analysis of these unusual amino acids in protein hydrolysates of histones and collagen and be applied to the determination of these compounds in hydrolysates of plant and animal tissues.


Lipase and esterase formation by psychrophilic and mesophilic Acinetobacter species.

Acinetobacter O16, a psychrophilic species, produced extracellular lipase (measured by hydrolysis of olive oil, tributyrin, or beta-naphthyl laurate) when grown on a complex medium (peptone plus yeast extract). Most lipase was produced during the logarithmic phase of growth. Very little cell-bound lipase was formed. These cells also produced an esterase (measured by the hydrolysis of beta-naphthyl acetate). At first, all esterase was cell bound; significant amounts appeared in the external medium late in growth. Breaking the cells did not increase cell-bound lipase activity. After breaking of the cells, most of the cell-bound lipase and esterase activity was solubilized, even after very high speed centrifugation. No appreciable amounts of these enzymes were released by osmotic shock. Lipase formation was greatly affected by nutrient conditions. Lowering either the yeast extract of the peptone content of the normal complex medium lowered or abolished lipase formation. Esterase activity was lowered to a lesser extent. Cells growing in synthetic amino acid plus vitamin medium or in acid-hydrolyzed casein produced substantial amounts of esterase but no cell-free or cell-bound lipase. However, if sodium taurocholate was added to these media, lipase was produced. Greatest production occurred if a mixture of di- and poly-peptides was also present. Taurocholate also stimulated lipase production in the normal complex medium. Adding Tween 80 or ethanol to the normal complex medium inhibited lipase production. Sodium acetate, oleic acid, olive oil, or Tween 20 added to synthetic media did not affect lipase production. The psychrophile grew more quickly at 30 degrees C than at 15 or 20 degrees C but produced more lipase at the lower temperatures. Esterase production was about the same at 20 and 30 degrees C. A mesophilic Acinetobacter species produced the same amount of lipase and esterase at 20 and 30 degrees C. The best production of lipase by the psychrophile occurred in standing cultures.


Partial purification and characterization of the lipase of a facultatively psychrophilic bacterium (Acinetobacter O16).

The extracellular lipase(s) of the psychrophile Acinetobacter O16 was studied. When the enzyme was precipitated by (NH4)2SO4 and passed through a Sephadex G200 column, two peaks of lipase activity appeared. The larger peak, which behaved like a substance of high molecular weight, being eluted in the void volume, was purified 250-fold over the crude enzyme (culture supernatant) by passage through a DEAE-Sephadex column. When the enzyme was applied to a DEAE-cellulose column it could not be eluted unless it had first been treated with the detergent Titon X 100. It is suggested that lipids or phospholipids make up an important part of the molecule. The activity of the crude and partly purified enzymes was studied in relation to pH and temperature optima. Lipases from the psychrophilic Acinetobacter O16 and from the mesophilic Acinetobacter O4 reacted in the same way to temperature. The crude enzyme from Acinetobacter O16 was more temperature-stable than the purified enzyme.


California encephalitis virus development in mosquitoes as revealed by transmission studies, immunoperoxidase staining, and electron microscopy.

Isolates of the snowshoe hare subtype of California encephalitis (CE) virus from Yukon mosquitoes during 1972 and 1973 were transmitted by bites of Aedes aegypti mosquitoes after 4 to 5 weeks of extrinsic incubation at 55 degrees F after intrathoracic injection, and the 1973 strain was transmitted after mosquitoes were fed virus and held for 3 to 4 weeks at 75 degrees F. Antigen of a 1971 isolate of CE virus (Marsh Lake 23) was detected in salivary glands of infected mosquitoes by the immunoperoxidase technique, using highly purified antiserum before and after conjugation with horseradish peroxidase, plus the use of orthotolidine as a substitute for benzidine. enveloped virions 45 nm in diameter were observed in thin sections of salivary glands of Culiseta inornata mosquitoes 59 days after intrathoracic injection with the 1971 isolate, afterincubation at 55 degrees F.


Rsistance to streptomycin in a producing strain of Streptomyces griseus.

Streptomyces griseus S 104 was sensitive to streptomycin during exponential growth in a medium which, in the subsequent stationary phase, supported production of the antibiotic in yields above 200 mug/ml. When antibiotic production began cultures developed a tolerance toward their lethal metabolite. This was not due to an increase in pH associated with antibiotic production, since pH effects on streptomycin sensitivity in S. griseus were in the reverse direction. However, the degree of tolerance was directly related to the amount of cell material present. Streptomycin production caused no change in the proportion of resistant variants in the population, nor did it cause the severe inhibition of protein synthesis observed in non-producing cultures exposed to the antibiotic. The lack of an effect on protein synthesis is attributed to the absence of streptomycin with in the cytoplasm since soluble extracts from mycelium harvested in the production phase were inactive when bioassayed immediately after cell disruption. However, they developed antibacterial activity rapidly when heated, and more slowly when incubated at 25 degrees C. The addition of phosphatase inhibitors during incubation prevented the appearance of antibiotic activity, and it was concluded that a small amount of streptomycin phosphate is present in the mycelium during antibiotic production. Differences in (14C) streptomycin uptake suggested that the mycelium was appreciably less permeable to the antibiotic in the production phase than during exponential growth. However, a small amount was taken up and much of it was in the soluble fraction of disrupted cells. Bioassays showed that this 14C-labeled antibiotic within the cells had been partially inactivated, suggesting that conversion of streptomycin to an inactive derivative is involved in the mechanism which protects the organism from its metabolite.


Dual effects of glucose on dicarboxylic acid transport in Kluyveromyces lactis.

The properties of succinate uptake in succinate-grown Kluyveromyces cells were examined. The rate of succinate transport at 15C exhibits an approximate V-max of 1.2 mumol times h-1 times mg-1 dry weight of cells and an apparent K-m of 18 muM. The uptake process appears to be tightly coupled to metabolism. L-Malate, fumarate, and alpha-ketoglutarate were the only other dicarboxylates tested, which were found to inhibit succinate transport. The aggreement between the order of inhibition of succinate transport by these dicarboxylates and their rates of uptake, as well as the competitive nature of the inhibition are all consistent with the existence of a common carrier system showing specificity for dicarboxylates of the TCA cycle. Cells transferred from succinate to glucose medium rapidly lose their ability to transport succinate. Glucose-grown cells also exhibit an inability to oxidize dicarboxylates or to use them for growth without a very long lag. The dicarboxylate uptake system, therefore, appears to be subject to a strong catabolite repression. The depression of the succinate transport system requires the presence of succinate, as well as low concentrations of glucose.


Bacteriostasis in soils sterilized by gamma irradiation and in reinoculated sterilized soils.

An agar-disc method was used to compare the bacteriostatic properties of five soils with those of samples of the same soils sterilized by gamma irradiation. For three of the soils, bacteriostasis was removed either partially or entirely by sterilization. Bacteriostasis was completely restored to sterile soil by reinoculating it with natural soil. All the reinoculation treatments restored the level of bacteriostasis equally effectively, whatever the origin of the soil used as inoculum.


Partial purification and properties of a beta-glucosidase from Erwinia herbicola Y46.

A constitutive beta-glucosidase of Erwinia herbicola Y46 was studied as a prerequisite to an assessment of its significance in the release of bacteriotoxic aglycones from plant beta-glucosides, and the possible effects of the aglycones on the course of such plant diseases as "fire-blight". The enzyme was purified 86.5-fold from crude extracts of cells grown on yeast beef broth. Ammonium sulfate precipitation, DEAE-cellulose fractionation, and gel filtration through Sephadex G-100 resulted in a preparation having one peak of activity on isoelectrofocussing, on gel filtration through Sephadex G-200, and on polyacrylamide gel electrophoresis. The latter techniques demonstrated, in addition to the major protein band associated with activity, a single minor impurity. The enzyme was active against p-nitrophenyl-beta-glucoside (p-NPG) and phloridzin, but showed only very slight activity against salicin and arbutin, and no detectable activity against beta-methyl-D-glucoside, cellobiose, lactose, and esculin. The production of beta-glucosidase was maximum at the late log phase of growth on yeast beef broth medium and declined somewhat thereafter. The incorporation of inducers (carbohydrates) in defined basal medium resulted in only small variations in specific activity in the resulting cells; The activity (p-NPG substrate) was not inhibited by D-glucose, phloretin, esculin, salicin, arbutin, lactose, or cellobiose, but was slightly inhibited by 1.0 mM phloridzin. Slight inhibition was observed in the presence of sulfhydryl reagents (iodoacetamide, p-chloromercuribenzoate), but sodium azide, ethylene-diaminetetraacetic acid, Cu2+, and Zn2+ ions produced no effect. The activity was stable, in both crude and purified preparations, over the pH ranges 6.0-7.5 (100% activity) and 4.5-greater than 8.5 (50% activity). The enzyme retained 80% activity after 30 min at 50 degrees C, but only 25% after 30 min at 60 degrees C. The enzyme had a mean K-m value (phloridzin) of 1.35 times 10-4 M, an isoelectric point of 4.75, a molecular weight, determined by Sephadex G-200 gel filtration, of about 122 000, and an optimum pH for activity of 6.5-7.0.


Successful orthotopic free autogenous joint grafts in the dog.

Thirty free autogenous metacarpal and 26 phalangeal articular cartilages were transplanted in the forefoot of dogs. The graft in each case included the articular surface and a minimal amount of supporting subchondral bone. The majority of the metacarpal grafts retained a normal gross, microscopic, and radioautographic appearance, some for up to 1 year after transplantation. In contradistinction, all of the phalangeal grafts failed, probably because their flatt surface made it impossible to maintain adequate fixation to the host bed. As a result the majority of these grafts became displaced. Analysis of the incorporation of the metacarpal grafts into the host bed showed that this occurred when there was no significant period during which the articular cartilage was without subchondral support. The situation is different in the case of joint grafts with a larger amount of subchondral bone. In these, during the time that this necrotic bone is being resorbed and replaced by normal viable bone, the articular cartilage has extremely limited subchondral support.


Treatment of acute osteomyelitis in children by closed-tube irrigation: a reassessment.

The addition of closed-tube irrigation to incision and drainage in the treatment of children with acute osteomyelitis has been found to be associated with: (a) a rate of technical complications of 60%, the most common complications being leaking and plugging of the tube; (b) a 30% rate of superinfection at the site of the wound, the most common organism being Pseudomonas; and (c) an average postoperative hospital stay of 30 days compared with only 16 days for the patients treated by incision and drainage alone. Suction-irrigation in the treatment of acute osteomyelitis in children does not appear to be essential for successful management, and introduces added risks of superinfection and technical complications.


Bile aspiration: an experimental study in rabbits.

It has been shown that bile injected intratracheally in rabbits produces severe pulmonary edema, atelectasis, and focal hemorrhages. The authors investigated the effect of a number of solutions, including physiological concentration of bile, hydrochloric acid pH 1.0, bile salt diluted to 1%, and bile at 100% concentrations. Whenever the bile concentration exceeded 3%, none of the test animals survived. It is not possible to apply directly the results of an experimental animal study to humans. However, the severity of the pulmonary changes produced force the conclusion that bile is a potentially dangerous aspirate in humans.


Studies on the relationship between the equilibrium curve of oxyhemoglobin and 2,3-diphosphoglycerate in open-heart surgery.

The relationship between decreased oxygen affinity of hemoglobin and intraerythrocyte 2,3-diphosphoglycerate (2,3 DPG) concentration was confirmed and its existence during the period of open-heart surgery was demonstrated. It is suggested that the trauma sustained by the erythrocytes during their extracorporeal circulation is responsible, in large measure, for the decrease in 2,3 DPG concentration.


[Hyperoxemia in extracorporeal circulation].

The authors demonstrate the effects of hyperoxemia in dogs subjected to extracorporeal circulation. Arterial Po2 was maintained between 100 and 140 mm Hg in a control group and above 200 mm Hg in another group. There was no morbidity or mortality in these experiments. Postoperative functional status as well as hematological and biochemical data were similar in both groups. However, free plasma hemoglobin was 50% higher in animals subjected to hyperoxygenation. It is thus concluded that on the basis of these results hyperoxygenation has no beneficial effect on the animals submitted to total body bypass and moreover that arterial Po2 over 200 mm Hg increases hemolysis.


Properties, inducibility, and an improved method of analysis of aryl hydrocarbon hydroxylase in cultured human lymphocytes.

The properties and inducibility of aryl hydrocarbon hydroxylase (AHH) in cultured human lymphocytes were studied, and a sensitive method of analysis has been developed. In agreement with other reports, peripheral blood lymphocytes per se had no activity and required pretreatment in culture with a mitogen for conversion to lymphoblasts to possess AHH activity. This activity had an absolute requirement for reduced nicotinamide adenine dinucleotide phosphate. Under our conditions of incubation, reduced nicotinamide adenine dinucleotide (1.3 mM) or ethylenediaminetetraacetic acid disodium salt (1 mM), when added to reduced nicotinamide adenine dinucleotide phosphate-fortified incubation mixture, caused about 30% increase in activity and MnCl2 caused an appreciable inhibition. The reaction was linear with the number of cells, with up to 16.8 X 10-6 cells contained in the incubation mixture, and had a pH optimum of 8.5. The enzyme was fairly stable at -70 degrees and retained about 90% of the original activity for 15 days. The enzyme activity in the mitogen-stimulated lymphocyte cultures appeared slowly and reached a maximum at 48 to 72 hr of incubation at 37 degrees, after which it considerably decayed with time. Viability of the cells in culture also decayed considerably after 72 hr and reached about 50% level at 120 hr. The enzyme was inducible with 3-methylcholanthrene and dibenz(a,h)anthracene. Dibenz(a,h)anthracene was a much more potent inducer than 3-methylcholanthrene and evoked a response in cells that would be considered noninducible with 3-methylcholanthrene. By using purified lymphocytes and Roswell Park Memorial Institute Medium 1640, providing larger surface area during culture, harvesting the cells at the time of minimum cell death and maximum AHH activity, and assaying AHH at its pH optimum of 8.5 instead of 7.6, it was possible to enhance the sensitivity of the assay an average of 17-fold.


Separation of myocardial cytoplasmic acidic inhibitor proteins which inhibit hepatic protein biosynthesis in vitro.

Cytoplasmic inhibitor proteins of rabbit and pig myocardium have been separated and their in-vitro effects on hepatic cell-free protein biosynthesis reported. Three protein peaks could be separated from myocardial cytoplasmic pH 5 fraction by DEAE and CM cellulose column chromatography. There was a difference in the elution pattern of the acidic inhibitor proteins obtained from pig and rabbit myocardium. The myocardial acidic inhibitor proteins inhibited a liver cell-free protein biosynthesizing system, but not a myocardial cell-free system.


Effects of bretylium and lidocaine on ventricular fibrillation in the isolated rabbit heart.

We studied the effects of bretylium tosylate and lidocaine on ventricular fibrillation in the isolated, perfused rabbit heart induced by perfusion with potassium-deficient solutions and by premature stimuli. The purpose of the study was to determine if the reported antifibrillatory effects of the drugs could be reproduced in the absence of an intact sympathetic nervous system. Lidocaine in the concentration of 5 mug/ml. prevented ventricular fibrillation induced by both methods while bretylium, in concentrations of 25 to 50 mug/ml. prevented neither type of filbrillation. These results indicate that the antifibrillatory effects of lidocaine can be attributed to drug-induced alterations in cellular electrophysiology, whereas those of bretylium are independent of such changes. Our study suggests that the reported antifibrillatory capability of bretylium is related to the drug's effects at sympathetic nerve terminals and thus is dependent upon an intact sympathetic nervous system.


Spermatogenesis in Anthozoa: differentiation of the spermatid.

The fine structure of spermatids has been examined in Calliactis, Protanthea, Gonactinia and Parazoanthus (Cnidaria, Anthozoa). The sperm cells are relatively simple and lack distinct acrosomes. Their nuclei, spherical in the zoanthid, in the actinians are slendertipped cones. Condensation of the chromatin is interpreted in terms of progressive coiling of densely-stained filaments and the elimination of nucleoplasm. Nuclear elongation occurs in the absence of microtubules. A well-developed centriolar complex is attached to the nuclear envelope by fibres and in this area (that of a shallow fossa in actinian spern) the nuclear membranes seem to be thickened. The centrioles are surrounded by a mitochondrial collar, especially pronounced in Calliactis. In contact with the mitochondria and nucleus is a ring of lipid-containing vesicles 300-700 nm in diameter. A system of densely-staining vesicles 150-300 nm in size corresponds to the "pro-acrosomal vesicles" described for other coelenterates. They are scattered in the peripheral cytoplasm and are regarded as derivatives of the endoplasmic reticulum. Problems of organelle function and of differentiation during spermiogenesis are discussed.


Some physico-chemical properties of aqueous solutions of N alpha-acyl-l-histidine.

The critical micelle concentrations of aqueous solutions of N alpha-acyl-L-histidine have been determined by the spectral shift method with Rhodamine 6G and by the light scattering method. With the spectral shift method critical micelle concentrations of 40, 9.0, 1.0, 0.11, and 0.012 mM were obtained for N alpha-acyl-L-histidine containing saturated acids of 8, 10, 12, 14, and 16 carbons respectively, at 45 degrees C and pH 8.6 in the absence of added salt. For the homologs containing 10, 12, and 14 carbon acids, critical micelle concentration of 9.0, 1.0 and 0.11 mM were determined by the light scattering method. The light scattering studies yield micelle weights of 60, 66, and 84 thousand for the C-10, C-12, and C-14 homologs, respectively. N alpha-acyl-L-histidine is an unusual surfactant in that the hydrophilic portion of the molecule is relatively large and contains both an ionic group (carboxylate group) and a nonionic group (imidazole side-chain). The bulky hydrophilic group of N alpha-acyl-L-histidine causes this molecule to exhibit physico-chemical behavior which is not typical of that exhibited by most ionic surfactants. In particular, the dependence of the critical micelle concentration on the acyl chain length and on the concentration of added salt is atypical. Chemical shift measurements (by NMR) on the C-2 and C-5 protons of imidazole in micellar N alpha-dodecanoyl-L-histidine indicate that the imidazole group is, indeed, positioned at the water-micelle interface.


Studies on phosphatidylcholine model membranes. I. Iodine permeability measurement by specific ion electrode.

An iodide specific ion electrode was used to measure iodide released from egg phosphatidylcholine liposomes after passage through an ion exchange column. The permeation process was shown to be a sum of two separate first order processes. The method permits linear initial rates of iodide release to be determined for the first 5 min of permeation. Iodide permeability in the absence of Tris buffer was found to be a decreasing function of pH. In the presence of Tris, iodide permeability went through a minimum ofrom ph 7.3-8.5. the permeability was found to decrease when cholesterol was incorporated into the liposomes.


Modulation by prostaglandins of adrenergic transmission in the isolated perfused rabbit and rat kidney.

In the isolated perfused rabbit kidney prostaglandins (PGS) E1 (0.02-0-1 ng/ml), E2 (0.02-0.1 ng/ml), and A2 (1-5 ng/ml) inhibited the vasoconstrictor responses to sympathetic nerve stimulation by 21-44%, 31-39%, and 20-23%, respectively, without alerting those to injected norepinephrine. In contrast, in the rat kidney PGE1 (0.5 ng/ml), PGE2 (0.5 ng/ml), and PGA2 (5 ng/ml) enhanced the vasoconstrictor responses to sympathetic nerve stimulation by 41%, 27%, and 11%, respectively; the equiconstrictor responses to injected norepinephrine remained unaltered. Higher concentrations of these agents produced vasodilation in the rabbit kidney and vasoconstriction in the rat kidney. In both species PGF2alpha produced vasoconstriction and enhanced the response to both adrenergic stumuli. In the rabbit kidney inhibitors of PG synthesis augmented the responses to sympathetic nerve stimulation without altering those to injected norepinephrine, whereas in the rat kidney inhibition of the responses to both adrenergic stimuli occurred. Arachidonic acid inhibited the vasoconstrictor responses to sympathetic nerve stimulation in the rabbit kidney, but in the rat kidney it caused augmentation of these responses. Since these effects of arachidonic acid were reduced by indomethacin, they appear to be mediated through the acid's conversion to PGS. We conclude that PGS of the E series modulate adrenergic transmission in the kidney and that their modulatory actions are species dependent.


Cardiac performance and mortality early after intracardiac surgery in infants and young children.

One hundred thirty-nine infants and small children less than 48 months old were studied during the first 72 hours after intracardiac surgery for a variety of lesions. The hospital mortality rate was 19.4% (27 patients); 16 of those dying succumbed from acute cardiac failure. Deaths from acute cardiac failure were commonest in patients with low cardiac index (CI), and a continuous probability curve relates the two. The mean CI for all patients was 2.51 plus or minus 0.794 l-min minus 1-m minus 2, and that for individual patients varied between 0.6 and 4.9. Cardiac output normalized by surface area (cardiac index) correlated weakly with age. cardiac output normalized by weight did not correlate with age; Mean mixed venous oxygen partial pressure (P-vO2)varied between 17 and 60 mm Hg, and the weighted mean for the group of 80 patients in whom it was obtained was 33.1 plus or minus 6.57 mm Hg. CI and P-vO2 were only slightly related. Acute cardiac deaths occurred more frequently in patients with low P-vO2. Acute cardiac death was more reliably predicted using CI and P-vO2 together than either alone. The average of the mean arterial pressure was 80.5 plus or minus 2.53 mm Hg, and this did not correlate with CI. Mean average systemic vascular resistance was 30.0 plus or minus 8.395 SRU. We conclude that in such patients treatment should be directed toward keeping CI greater than 2.0 l-min minus 1-m minus 2 and P-vO2 greater than 30 mm Hg; that the possibility of maintaining adequate CI and P-vO2 is not related to the age of the patient but is related to the malformation treated; and that a therapeutic trial of reducing left ventricular afterload is indicated in some of these patients.


Use of carbon dioxide- and oxygen-tonometered phosphate-bicarbonate-chloride-glycerol-water mixtures for calibration and control of pH, pCO2, and pO2 electrode systems.

Calibration of pH, PCO2, and PO2 electrode systems of modern blood-gas analyzers, designed with one sample cuvet for measurement, is mostly performed separately with buffer solutions of known pH, PCO2, and PO2 for doing such calibrations simultaneously, containing phosphate, bicarbonate, and chloride in glycerol-water mixtures as solvent. A method is suggested for computing the relation between pH and log PCO2 of these solutions in equilibrium with carbon dioxide gas. It is demonstrated that a solution of phosphate (Na2HPO4, KH2PO4, each 25 mmol/liter), bicarbonate (NaHCO3, 30 mmol/liter), and chloride (Nacl, 30 mmol/liter) in glycerol-water mixture (3/7 by vol) and equilibrated with CO2 in air (4 vol/100 vol) and CO2 in  nitrogen (8 vol/100 vol), respectively, makes possible acurate and simultaneous calibration of the pH, PCO2, PO2 electrodes of a Corning Model 165 blood-gas analyzer. Similar solutions may also be used for quality-control of blood-gas measurement.


New, simple maltogenic assay for mechanized determination of alpha-amylase activity in serum and urine.

We report a new method for the mechanized determination of serum and urinary alpha-amylase by use of a continuous-flow system, based on the measurement of maltose formed by incubating the sample with amylodextrin at pH 7 and 40 degrees C. After dialysis, maltose is converted enzymatically to glucose, which is measured by Trinder's glucose oxidase-peroxidase method [J. Clin. Pathol. 22, 246 (1969)]. The reaction is linear for amylase activities up to 1400 Somogyi units/dl (2560 U/liter) and for maltose concentrations through 1500 mg/dl. No blank assay is required; consequently precision is improved and the automated system is simplified. Calibration with primary maltose standards increases accuracy and reliability. Common reducing substances in serum and urine do not interfere at their normal concentrations. There is a linear correlation between the results of this method and those of chromogenic and iodometric methods for normal and pathologic sera and urines. The chromogenic method yields significantly higher results and the iodometric method significantly lower results than this maltogenic method for elevated amylase activities. The normal range is 40-140 Somogyi units/dl (73-256 U/liter).


Fluorometric assay of serum gamma-glutamyltransferase in solution and on a semi-solid surface.

We describe an enzymatic, fluorometric method for measuring the activity of serum gamma-glutamyltransferase (EC 2.3.2.2) in solution and on silicone rubber pads. N-gamma-L-Glutamyl-alpha-naphthylamide in tris(hydroxymethyl)aminomethane buffer is used as substrate. In solution, measurements are performed at 37 degrees C with use of a reaction volume of 3.0 ml, in Pyrex cuvets. Measurements on silicone rubber pads are also made at 37 degrees C, after establishing a stable substrate film by lyophilizing the reagent for the gamma-glutamyltransferase assay on the surface of the pads. Only 10-50 mul of buffered substrate and serum containing the enzyme to make a total volume of 60 mul are necessary for each assay. The rate of appearance of alpha-naphthylamine fluorescence (lambda ex equals 344 nm; lambda em equals 445 nm), liberated from N-gamma-L-glutamyl-alpha-naphthylamide by the enzymatic action of gamma-glutamyl-transferase, is measured and equated to its activity in serum. Calibration plots of the change in fluorescence per min vs. enzyme concentration for measurements in solution and on pads show a good proportionality in the range of 26.5-265 U/liter, and indicate the usefulness of these methods.


A more sensitive automated method for determination of ornithine carbamoyltransferase activity in human serum.

Ornithine carbamoyltransferase (EC 2.1.3.3) activity is a sensitive, specific indicator of hepatocellular injury. This paper describes development of an improved automated procedure for measurement of this activity. Triethanolamine-ethylenediaminetetraacetate is used as a buffer, and activity is determined by measuring the concentration of the product, citrulline. Kinetic studies have been performed to determine optimal pH and L-ornithine and carbamoyl phosphate concentrations. Recovery of citrulline was studied. The upper limit of normal obtained in a study of 106 blood-bank donors was 6 U/liter. The automated procedure developed as a result of these studies, in which optimal assay conditions are used, produces a threefold increase in sensitivity and permits use of a sample volume of 1 ml.


A new colorimetric method for the determination of NADH/NADPH dependent  glutathione reductase in erythrocytes and in plasma.

A simple and rapid colorimetric method for the assay of erythrocyte and plasma glutathione reductase (GR) activity is described. The method is based on the colorimetric measurement of reduced glutathione (GSH) (1) produced when the enzyme is incubated with oxidised glutathione (GSSG) in the presence of either reduced nicotinamide adenine dinucleotide (NADH) or reduced nicotinamide adenine dinucleotide phosphate (NADPH). Results of investigations on the effects of substrate and coenzyme concentrations, pH, EDTA, sodium/potassium chloride, and time, on enzyme activity are presented. Erythrocyte and plasma NADH-GR and NADPH-GR activities in 100 healthy blood donors, and 85 cord blood samples and plasma NADH-GR and NADPH-GR levels in patients with various disease conditions are given.


Microassay of human erythrocyte catechol-O-methyltransferase: removal of inhibitory calcium ion with chelating resin.

A radiochemical micromethod for the determination of catechol-O-methyltransferase (COMT) activity in human red blood cells (RBC) is described. The method avoids artifacts that occur with other assay procedures. Erythrocyte lysates are exposed to a particulate chelating agent (Chelex-100) to remove endogenous divalent cations that inhibit COMT. 3,4-Dihydroxybenzoic acid rather than norepinephrine is used as a substrate to increase both the sensitivity and the reproducibility of the procedure. An internal standard of purified rat liver COMT is added to lysates to detect possible variations in endogenous activators or inhibitors of the enzyme. Blood samples from 373 unselected subjects age 16-18 were assayed for RBC COMT activity with this assay procedure. The COMT activity in these blood samples ranged from 3-25 units with a mean activity of 11.3 plus or minus 4.2 (mean plus or minus S.D.).


A method for the determination of sphingomyelinase activity.

A method is presented for the determination of the activity of sphingomyelinase. It is based on the determination of sphingomyelin before and after enzymatic conversion with the aid of a quantitative paper chromatographic analysis of phospholipids. This method is used for the determination of sphingomyelinase activity in various tissues such as those of the human brain and liver and fibroblast cultures. Normal values are presented.


Mannosidosis: deficiency of a specific alpha-mannosidase component in cultured fibroblasts.

Evidence is prisented which shows that the enzymatic defect in mannosidosis, the deficiency of alpha-mannosidase, is expressed in cultured skin fibroblasts from patients with mannosidosis. Additionally, by Cellogel electrophoresis, the enzyme can be separated into two major components (a heat stable component missing in mannosidosis fibroblasts with a pH optimum of 3.6-4.0 and a heat labile component present in mannosidosis fibroblasts with a pH optimum of 5.6-6.0). The specific activity and electrophoresis of alpha-mannosidase from cultured amniotic fluid cells are also shown to be similar to cultured skin fibroblasts.


A new, highly sensitive and specific assay for chymotrypsin.

1. A simple, highly sensitive, specific fluorometric method for the determination of chymotrypsin is described. 2. The new substrate utilized in this assay, N-glutaryl-glycyl-glycyl-l-phenylalanine beta-naphthylamide (GGPNA), is readily soluble in water, stable and highly specific for chymotrypsin. It is not degraded by a large excess of carboxypeptidase B, elastase, thrombin or plasmin and is virtually resistant to trypsin. 3. GGPNA is extremely sensitive to the action of chymotrypsin and permits detection of enzyme concentrations as low as 1 ng/ml. Linearity between enzyme concentration and fluorescence produced is maintained up to at least 3000 ng/ml. 4. alpha2-Macroglobulin-bound chymotrypsin hydrolyzes GGPNA at a rate about 2/3 of that exhibited by the free enzyme. 5. Bile pigments in amounts normally found in duodenal juice or traces of blood do not interfere with the assay. 6. GG PNA which releases beta-naphthylamine upon hydrolysis is suitable also for colorimetric and histological determination of chymotrypsin.


Gd(minus)Matam, an African glucose-6-phosphate dehydrogenase variant with enzyme deficiency. Biochemical and immunological properties in various hemopoietic tissues.

Two unrelated Senegalese patients, both native of the Matam province, were found to have the same deficient G6PD variant. One has no hematological history, the other had several induced acute hemolytic episodes. The deficiency was almost complete in red blood cells and 20-30 percent of the normal level in leukocytes and platelets; in leukocytes the deficiency was due to a decrease in the molecular specific activity of the enzyme to which a molecular instability was added, explaining the greater deficiency in red blood cells. The electrophoretic mobility was slightly fast in leukocytes and platelets but normal in red blood cells. This pattern was confirmed by electrofocusing in ampholine-acrylamide gel. From a kinetic point of view, these enzymes were characterized by a lowered Km (G6P) (13 to 20 muM) a normal Km (NADP+), a Ki (NADPH) increased about twice, a thermal instability, a biphasic pH curve and an increased activation energy (15 kcal/mole). The polymorphonuclear cells were functionally strictly normal: engulfment, nitroblue tetrazolium (NBT) reduction test, induced iodination, and oxygen consumption were normal. The authors discuss the importance of post-synthetic modifications of the muted enzymes and their repercussions on the enzyme characteristics.


Changes in oxygen delivery during hemodialysis.

The effect of hemodialysis on oxygen delivery has been assessed in nine patients. During hemodialysis the arterial PO2 decreased from 76.89 mm Hg to 69.03 mm Hg. There was also an increase in pH from 7.39 to 7.49 which resulted in an increase in the affinity of the blood for oxygen. In the absence of compensatory changes this could result in a 17.9% decrease in oxygen delivery. This could be hazardous in patients who are already anemic particularly in the presence of cardiac or respiratory insufficiency.


Fetal acquisition and neonatal elimination of a large amount of salicylate. Study of a neonate whose mother regularly took therapeutic doses of aspirin during pregnancy.

The purpose of this study was to determine the amount of salicylate acquired by a newborn infant from a mother who took 6.5 gm of aspirin a day during her entire pregnancy, and to characterize the kinetics of salicylate elimination by the infant. This healthy female infant was born with a salicylic acid concentration of 25 mg/100 ml plasma and 75 mg/kg body weight. The drug was eliminated during the first 5 days of life, primarily in the form of salicyluric acid. Salicylate elimination was relatively slower than in normal adults, but more rapid than in newborn infants of mothers who had taken only one small dose of aspirin shortly before delivery. The apparent in vivo KM and Vmax for salicylurate formation, on a body weight basis, were at the adult level. The slower elimination of salicylate (relative to adults) by the infant was due to immaturity of the glucuronidation and renal excretory pathways.


Antiarrhythmic activity of four pteridine compounds in ouabain intoxication.

The antiarrhythmic effects of 4 pteridine analogues, 2 of which are potassium-sparing diuretics, triamterene (2, 4, 7-triamino-6-phenylpteridine) and [2-phenyl-4, 7 diaminopteridine-6-(N-diethylaminoethyl) carboxamide] and 2 of which have no diuretic effects [2-phenyl-4, 7-diaminopteridine-6-(N-2-hydroxyethyl) carboxamide], on ouabain-induced ventricular tachycardia in intact pentobarbital-anesthetized dogs were investigated. Ouabain was given as a continuous infusion 2 mug/kg/min intravenously until 5 min after the onset of a sustained ventricular tachycardia. It was found that both 6-(N-dimethylaminopropyl) and 6-(N-diethylaminoethyl) carboxamide derivates of the pteridine had a significant protective effect against ouabain-induced ventricular tachycardia in dogs that had been pretreated with a dose of 5 mg/kg intravenously. At this dose the 2 pteridine compounds with diuretic activity exhibited a transient antiarrhythmic effect in abolishing the ouabain-induced ventricular tachycardia while those without diuretic properties failed to suppress the ventricular tachycardia.


Furosemide binding to human albumin and plasma of nephrotic children.

The extent and nature of furosemide (F) binding to human albumin (HA) and to the plasma of 6 children with nephrotic syndrome were studied by equilibrium dialysis at 37 degrees C and pH 7.4 with 14C-F. At a total concentration of 3.4 mug/ml (therapeutic range), the unbound fraction of F to 4 gm per 100 ml HA was 2.79 plus or minus 0.35. The degree of binding was relatively constant from 1.8 to 36 mug/ml of F concentration. The percentage of unbound F doubled when total concentration of the drug was increased more than 130 times (1.8 to 245 mug/ml). F has two classes of binding sites (n1 = 1.42, k1 = 5.07 times 10-4 M-minus 1; n2 = 3.4, k2 = 1.58 times 10-4 M-minus 1); interaction with HA involves hydrophobic, ionic, and hydrogen forces. Acetylsalicylic acid (ASA), acetazolamide, diazoxide, phenylbutazone, sulfisoxazole (S), and tolbutamide (T) decreased F binding. Combinations of ASA, S, and T exerted a strong additive displacing effect. The binding of the F metabolite (4-chloro-5-sulfamoylanthranilic acid, CSA) was studied at 1.3 and 2.6 mug/ml. The unbound fraction was 5 times that of F. CSA did not influence F binding. Studies with plasma of 7 healthy adults showed that albumin is the only plasma protein responsible for F binding. The plasma albumin concentration range of the children with nephrotic syndrome was 0.6 to 2.1 gm per 100 ml. There was some correlation between albumin concentration and binding of F (2.8 to 9.6% unbound); this corresponded with findings with HA. Albumin concentrations lower than 2 gm per 100 ml seemed to influence the extent of the unbound fraction of F considerably.


Plasma levels and effects of metoprolol on blood pressure and heart rate in hypertensive patients after an acute dose and between two doses during long-term treatment.

Plasma levels and the effect of orally administered metoprolol on the resting arterial blood pressure and heart rate have been studied during acute and steady-state conditions in patients with mild hypertension. The patients receiving an 80-mg dose had a mean maximum plasma level of about 100 ng/ml plasma in single-dose studies and about 140 ng/ml plasma during steady-state conditions. The corresponding values for the patients on the 50-mg dose were about 60 and 100 ng/ml plasma, respectively. The maximum concentrations were reached 1 hr after administration. After the single dose the elimination half-life of metoprolol in plasma was 4.3 plus or minus 0.7 hr in the patients receiving the 80-mg dose and 3.8 plus or minus 0.3 hr in the other group. The difference was not statistically significant. The elimination half-life in the plasma was about the same in the single-dose study and during steady state in both groups. The morning dose induced a decrease of the systolic blood pressure whereas the diastolic blood pressure was not significantly different from that recorded immediately before administration of metoprolol. For the 80-mg dose the systolic pressure dropped from 167 plus or minus 4 to 146 plus or minus 4 mm Hg in the single dose study and from 160 plus or minus 8 to 140 plus or minus 4 mm Hg at steady state. The corresponding values for the 50-mg dose were 150 plus or minus 3 to 135 plus or minus 3 mm Hg and 144 plus or minus 3 to 138 plus or minus 3 mm Hg, respectively. In experiments with placebo the systolic blood pressure was not significantly changed. There was no correlation between the plasma levels and the effect on the systolic blood pressure. Both doses of metoprolol markedly reduced the heart rate after the single dose as well as at steady state. The effect was linearly related to the logarithm of the plasma concentration, and the relationship was virtually the same as obtained previously for the effect on exercise heart rate in healthy volunteers.


Iodine absorption in burn patients treated topically with povidone-iodine.

Providone-iodine is used as a topical antimicrobial in burn patients. Although absorption of iodine has been thought to be negligible, several patients have recently been noted with substantial elevations of serum free iodide. Unexplained abnormalities occurred in several of these patients, renal failure, metabolic acidosis, and elevation of serum glutamic oxaloacetic transaminase. It is conceivable that the large iodide loads noted were at least in part responsible for these abnormalities.


Correlation of psychophysiologic variables with vocational rehabilitation outcome in patients with chronic obstructive pulmonary disease.

Despite the magnitude of the public health problem presented by respiratory diseases, there have been few studies concerned with vocational rehabilitation (VR) potential of patients with chronic obstructive pulmonary disease (COPD). Certain physiologic variables which show a high degree of relationship to VR success are identified. The three independent variables which most highly correlate with the VR potential of patients with COPD are the percentages predicted for the first-second forced expiratory volume (FEV(1.0)), forced expiratory flow between 25 and 75 percent of the forced vital capacity (FEF(25-75 percent)), and maximum voluntary ventilation (MVV). The mean "cutting" percentages for inclusion in VR programs were 50, 27, and 40, respectively. The emotional variables studied do not differentiate potential VR success or failure as clearly as the physiologic factors. The criteria set forth not only can be used by rehabilitation workers but could serve as a basis for future demonstration studies.


[Optimal time for treating cryptorchism (author's transl)].

In a ten-year span (1950-1960) a total of 133 patients were operated upon for maldescent of testes. A follow-up enquiry traced 102, of whom 79 were re-examined. Maldescent had been unilateral in 71, bilateral in eight. All of the latter were infertile. In the others the fertility rate was the greater the younger the patient at the time of operation, best results being noted in those operated upon during the first or second year of life. Acquired testicular disorders seemed to play a minor role, while the normally descended contralateral testis was apparently damaged in an as yet undetermined manner (autoantigen-antibody reaction?) It is concluded that the optimal moment for operating on testicular maldescent is (at the latest) the end of the second year of life.


[Endocrine and morphological investigation in undescended testis (author's transl)].

Determination of the basal and LH-RH-stimulated luteinizing hormone (LH) levels as well as testicular morphology in patients with surgically treated (n equals 112) and non-treated (n equal 96) undescended testes gave the following results: Unilateral of bilateral undescended testes are probably a form of primary, secondary or tertiary hypogonadism. In a high percentage abnormal basal and stimulated serum-LH values can be demonstrated. The testosterone values are within normal limits. The gonadal tissue damage in the abnormally situated testes of prepubertal boys seem to be congenital. With increasing duration of the malposition, particularly after the onset of puberty, a secondary tissue degeneration in the dystopic testis occurs. The damaging influence on the testicular tissue caused by the abnormal position seems to take place mostly at the end of prepuberty. A decrease in the number of spermatogonia in the tubuli of dystopic testes during the first 2 years of life is physiological. A surgical repositioning of the dystopic testis into the scrotum within the first 2 years of life is not indicated. Damage of gonadal tissue in the scrotal testis caused by the dystopic testis in unilateral maldescent could not be demonstrated. The optimal space for orchidopexy is between the third and fifth year of life.


Participation of NADPH-cytochrome C reductase in thyroid hormone biosynthesis.

Purified rat liver NADPH-cytochrome c reductase supports iodination of tyrosine in a system including NADPH, cytochrome c and thyroid perioxidase. Catalase inhibits the iodination of tyrosine, while superoxide dismutase has no effect. Antibody developed in the rabbit against purified rat liver NADPH-cytochrome c reductase inhibits both reduction of cytochrome c and tyrosine iodination supported by the enzyme. The antibody forms a single precipitation line with thyroid extract, and inhibits NADPH cytochrome c reductase activity of the thyroid. The antibody partially inhibits iodination in a thyroid mitochondrial-microsomal fraction, but does not inhibit NADH-dependent iodination. The immunochemical studies indicate the participation of NADPH-cytochrome c reductase in thyroidal H2O generation, and the independent existence of NADPH-dependent and NADH-dependent H2O2 generation mechanisms in the thyroid.


Release of vasopressin by angiotensin II.

To test the hypothesis that angiotensin II releases antidiuretic hormone (ADH) after injection into ventricular cerebrospinal fluid, conscious adult male Sprague-Dawley rats with a lateral cerebroventricular cannula received an intraventricular injection of 0, 10, 50, or 100 ng angiotensin II. Trunk blood was collected 90 seconds later for radioimmunoassay of ADH. Plasma ADH, pg/ml (mean plus or minus S. E.), for the four dose levels were 2.8 plus or minus 0.7, 9.6 plus or minus 2.5, 22.6 plus or minus 5.6 and 25.0 plus or minus 5.0, respectively. The increases produced by angiotensin were statistically significant (p smaller than 0.05). Plasma ADH of the 10 ng group was intermediate between control and the two highest angiotensin doses (p smaller than 0.05), suggesting a dose-response relationship. These data provide direct evidence that angiotensin releases ADH by central mechanisms.


Amino terminal residue heterogeneity of bovine and ovine growth hormones as revealed by polyacrylamide gel electrophoresis.

Prolonged electrophoresis (4 to 16 h) of bovine and ovine growth hormones on analytical polyacrylamide gels in Tris-EDTA-borate buffers of pH 7.6 to 8.1 resulted in the separation of alanyl, phenylalanyl, and methionyl terminal polypeptide chains of these hormones similar to that obtained by isoelectric focusing in Ampholine pH gradients. Their relative anodic mobilities were methionyl greater than phenylalanyl greater than alanyl. The mobilities of the respective monomers were inversely related to their isoionic points as determined by isoelectric focusing in Ampholine pH gradients. The resolution of the different monomers is attributed to differences in the dissociation constants of the amino groups of the alanyl, phenylalanyl and methionyl terminal residues. Rat growth hormone, which consists of monomers with only a phenylalanyl amino terminus, did not display heterogeneity on gel electrophoresis. The applicability of employing polyacrylamide gel electrophoresis for the assessing of amino terminal residue heterogeneity is noted.


Study of the mechanism of thyroid hormone secretion in an in vitro model system: indirect evidence for fusion of lysosomes with thyroglobulin liposomes.

The interaction between [131I]-thyroglobulin liposomes and thyroidal lysosomes was used as an in vitro model system for analyzing the relation of colloid droplets to lysosomes in follicular cells. The rates of hydrolysis of [131I]-thyroglobulin in a liposome-lysosome system (Lipo-Lyso system) and a thyroglobulin-lysosome system (TG-Lyso system) were compared. Hydrolysis of thyroglobulin in the Lipo-Lyso system increased hyperbolically and was greater than that in the TG-Lyso system for about 10 h. Liposomal thyroglobulin was not degraded by the 20,000 X g supernatant obtained on disruption of the lysosomal fraction. On varying the pH of the incubation medium, the highest activity was observed under acidic conditions in both systems. Under neutral or weakly alkaline conditions, the Lipo-Lyso system still showed 50% of the maximal hydrolytic activity, while the TG-Lyso system showed no activity. ATP and anaerobic conditions had no effect on either system. Cysteine (5 X 10-2M) and p-chloromercuribenzene sulfonic acid (PCMBS, 10-3 M) had not influence on hydrolysis in the Lipo-Lyso system, but in the TG-Lyso system cysteine greatly increased, and PCMBS significantly reduced the rate of hydrolysis. Dibutyryl cyclic AMP had no effect. Chlorpromazine (Cpz) decreased liposomal thyroglobulin hydrolysis in a concentration-dependent manner. In the TG-Lyso system, concentrations of 10-4M Cpz had no effect. These results strongly suggest that liposomes rapidly fused with lysosomes, providing optimal conditions for hydrolysis of thyroglobulin.


Triiodothyronine binding to liver nuclear solubilized proteins in vitro.

Nuclear proteins extracted from purified nuclei with 0.4M KCl at pH 7.4 OR 8.5 are able to bind L-triiodothyronine (T3) giving rise to nuclear thyroid hormone binding protein-T3 (NTBP-T3) complexes. Binding is maximum in 3 h at 20 C. It is thermolabile even at 36 C, inhibited by p-hydroxymercuribenzoate and markedly enhanced by dithiothreitol. Optimum pH is between 7.8 and 8.5. Divalent cations are not necessary. The NTBP-T3 complex exhibits similar anodal electrophoretic migration in polyacrylamide gel at pH 8.5, whether formed in vivo or in vitro. Scatchard plots obtained with various amounts of T3 from 0.15 nM TO 0.15 MUM and either unlabeled nuclear proteins or in vivo formed NTBP-[125I]-T3 complexes, give apparent association constants K-a of 0.2 X 10-10 M minus at pH 7.4 and 0.8 X 10-10 M minus 1 at pH 8.5. Capacity is about 0.5 pmol T3 per mg protein or 800 pg/g liver. The presence of dithiothreitol markedly enhances the Ka. The nuclear binding sites are not highly specific for L-T3 since they are able to bind D-T3 with almost equal affinity and triiodothyroacetic acid with a higher affinity. L-thyroxine (T4) can also displace L-T3 but with about 10-fold lesser effectiveness. Nuclear binding proteins of low capacity and high affinity have been demonstrated in vitro. The NTBP-T3 complexes formed in vivo, with whole nuclei, or in vitro are indistinguishable.


Properties of a DNA-adenylate complex formed in the reaction between mammalian DNA ligase I and DNA containing single-strand breaks.

The major DNA ligase from calf thymus (mammalian DNA ligase I) forms a covalent enzyme-AMP complex on incubation with ATP [Söderhäll & Lindahl, J. Biol. Chem. 248, 672-675, (1973)]. The reaction of this complex with DNA has now been studied. When the ligase-adenylate complex is incubated at 0 degrees C for short time periods with DNA containing single-strand breaks, a DNA-AMP complex can be isolated from the reaction mixture by isopycnic centrifugation in CsCl. Incubation at pH 6.5 increased the amount of DNA-AMP complex that could be isolated 10-20-fold relative to that obtained at pH 7.4. Under the same conditions, incubation of the ligase-AMP complex with DNA free from single-strand breaks did not lead to detectable DNA-AMP formation. The DNA-AMP complex was resistant to treatment with dilute acid and alkali indicating the presence of a covalent linkage. Further, this complex was sensitive to DNase but resistant to pronase and RNase. Free AMP was released on further incubation of the isolated DNA-AMP complex with thymus DNA ligase I and Mg2+, suggesting that the complex is a reaction intermediate. Degradation of the DNA-AMP complex with several reagent enzymes indicated that the AMP residues were bound at the 5' ends of the single-strand breaks in DNA by pyrophosphate bonds.


18-Hydroxylation of deoxycorticosterone by reconstituted systems from rat and bovine adrenals.

18-Hydroxylation of deoxycorticosterone was studies with rat or bovine adrenal mitochondria or with reconstituted systems obtained from these fractions. The reconstituted systems consisted of a partially purified preparation of cytochrome P-450 from rat adrenals and a partially purified NADPH-cytochrome P450 reductase preparation from bovine adrenals. In some experimenta a soluble cytochrome P-450 fraction from bovine adrenals was used. Adrenodoxine and adrenodoxine reductase were shown to be the active components of the NADPH-cytochrome P-450 reductase preparation. Optimal assay conditions were determined for 18-hydroxylation by the crude mitochondrial fraction as well as by the reconstituted systems. In the presence of excess NADPH-cytochrome P-450 reductase fraction, the rate of 18-hydroxylation was linear with time and with the amount of cytochrome P-450. In incubations with intact rat adrenal mitochondria to which Ca2+ and an excess NADPH had been added, NADPH-cytochrome P-450 reductase increased the rate of 18-hydroxylation about 100%, indicating that NADPH-cytochrome P-45o reductase was to some extent rate-limiting. The rate of 18-hydroxylation of deoxycorticosterone by the reconstituted system as well as by intact mitochondrial fraction was much higher than the rat of 18-hydroxylation of corticosterone and progesterone. When the cytochrome P-450 preparation from rat adrenals in the reconstituted system was substituted for cytochrome P-450 from bovine adrenals, the rate of 18-hydroxylation decreased considerably. Under all experimental conditions, the 18-hydroxylation of deoxycorticosterone occurred with a concomitant and efficient 11beta-hydroxylation. Provided the source of cytochrome P-450 was the same, the ratio between 11beta- and 18hydroxylation was constant under all conditions and was not significantly different in the presence of metopirone, carbon monoxide, cytochrome c or different steroids. It is suggested that identical or at least very similar types of cytochrome P-450 are involved in 11beta- and 18-hydroxylation of deoxycorticosterone.


Extracellular enzyme system utilized by the fungus Sporotrichum pulverulentum (Chrysosporium lignorum) for the breakdown of cellulose. 1. Separation, purification and physico-chemical characterization of five endo-1,4-beta-glucanases.

Five endo-1,4-beta-glucanases (EC 3.2.1.4) have been separated from culture solutions OF THE ROT FUNGUS Sporotrichum pulverulentum (formerly called Chrysosporium lignorum) grown on powder cellulose as the sole carbon source. They have been extensively purified and characterized with regard to some physicochemical properties. The purifications have been carried out on a quantitative basis, the purity of the enzymes being tested in several ways. After purification they all showed one single protein band in analytical polyacrylamide electrophoresis, on dodecyl-sulphate gels and in analytical isoelectric focusing on flat-bed polyacrylamide gels. One exo-1,4-beta-glucanase has also been identified in the culture solution and separated from the endo-1,4-beta-glucanases. From the data obtained during the quantitative purification it has been possible to calculate that the ratio of activity between the five endoglucanases T1, T2a, T2b, T3a, and T3b in the culture solutions is 4:1:1:1:1. It has also been calculated that the weight ratio endoglucanase protein to exoglucanase protein is approximately 1:1. Flat-bed isoelectric focusing has been used for the identification of the individual endoglucanases and a new zymogram technique, useful for studies of carbohydrases in general, has been developed. The molecular weights, determined by ultracentrifugation, and calculated on the basis of a knowledge of the amino acid composition and carbohydrate content vary between 28200 and 37500. Small but significant differences in the amino acid compositions of the different endoglucanases have been found. The carbohydrate content varies between 0 and 10.5%, all but one of the enzymes being glycoproteins. For two of these the exact carbohydrate composition has been determined. Enzyme T1 contains 2 glucose and 19 mannose units per enzyme molecule while enzyme T2b contains 5 mannose, 7 galactose, 1 glucose and 1 arabinose unit per molecule.


Extracellular enzyme system utilized by the fungus Sporotrichum pulverulentum (Chrysosporium lignorum) for the breakdown of cellulose. 3. Purification and physico-chemical characterization of an exo-1,4-beta-glucanase.

An exo-1,4-beta-glucanase from culture solution of the rot fungus Sporotrichum pulverulentum (formerly called Chrysosporium lignorum) grown on powder cellulose as the sole carbon source has been extensively purified and characterized with respect to some physico-chemical properties. The purification has been carried out in a five-step procedure comprising chromatography on DEAE-Sephadex, gel filtration on polyacrylamide P-150, activation on a Dowex 2-X8 anion exchanger, chromatography on Concanavalin A-Sepharose and chromatography on SP-Sephadex. The purified enzyme was found to be pure and homogeneous by analytical polyacrylamide electrophoresis, by electrophoresis on dodecylsulphate gels and by analytical polyacrylamide electrophoresis, by electrophoresis on dodecylsulphate gels and by analytical isoelectric focusing. A single symmetrical peak was obtained with the free zone electrophoresis method. The purification factor is about 15 and the yield of exo-1,4-beta-glucanase activity 7%. After purification, the enzyme showed no viscosity-decreasing activity towards carboxymethyl-cellulose solutions. The exo-1,4-beta-glucanase was isoelectric at pH 4.3 (4 degrees C). A molecular weight of 48600 was calculated on the basis of a knowledge of the partial specific volume, ultracentrifugation data and the amino acid composition. The enzyme contained no carbohydrate.


Purine nucleoside phosphorylase from Escherichia coli and Salmonella typhimurium. Purification and some properties.

The purine nucleoside phosphorylases from Escherichia coli and from Salmonella typhimurium have been purified to electrophoretic homogeneity and crystallized. Comparative studies revealed that the two enzymes are very much alike. They obey simple Michaelis-Menten kinetics for their substrates with the exception of phosphate for which they show negative cooperativity. Gel filtration on Sephadex G-200 of the native enzymes revealed a molecular weight for both enzymes of 138000 plus or minus 10%. By use of dodecylsulphate gel electrophoresis a subunit molecular weight of 23700 plus or minus 5% was determined, suggesting that both enzymes consist of six subunits of equal molecular weight. When the subunits were partially crosslinked with dimethyl suberimidate before dodecylsulphate electrophoresis six protein bands were observed in agreement with the proposed oligomeric state of the enzyme, consisting of six subunits of equal molecular weight. Analysis of the amino acid composition also indicates that the subunits are identical. 6M guanidinium chloride dissociates the enzymes; association experiments with native and succinylated enzymes suggested that only the hexameric form is active. Both enzymes could be dissociated into subunits by p-chloromercuribenzoate; this dissociation is prevented by the substrates: the nucleosides, the pentose 1-phosphates, and mixtures of phosphate and purine bases.