Comparison of the molybdenum centres of native and desulpho xanthine oxidase. The nature of the cyanide-labile sulphur atom and the nature of the proton-accepting group. The non-functional form of xanthine oxidase known as the desulpho enzyme was compared with the functional enzyme in various ways, to obtain information on the structure of the molybdenum centre and the mechanism of the catalytic reaction. The desulpho enzyme, like the functional one, possesses a site for the binding of anions, presumably as ligands of molybdenum. Evidence is presented that in the Mo(V) e.p.r. signal from the desulpho-enzyme, as in that from the functional enzyme, a weakly coupled proton, in addition to a strongly coupled proton, interacts with the metal. Measurements were carried out by e.p.r. on the rate at which the proton strongly coupled to molybdenum exchanged, on diluting enzyme samples with 2H2O. For the desulpho enzyme the exchange rate constant was 0.40s-1, at pH 8.2 and 12 degrees C, and for the functional enzyme it was 85 s-1. It is shown that the great majority of reported differences between the enzyme forms are consistent with functional enzyme containing an (Enzyme)-Mo=S grouping, replaced in the desulpho form by (Enzyme)-Mo=O. Protonation of these groups, with pK values of about 8 and 10 respectively, would give (Enzyme)-Mo-SH and (Enzyme)-Mo-OH, these being the forms observed by e.p.r. The accepting group in the functional enzyme, for the proton transferred from the substrate while molybdenum is reduced in the catalytic reaction [Gutteridge, Tanner & Bray (1978) Biochem J. 175 869-878], is thus taken to be Mo=S.


Kinetic properties of aldehyde dehydrogenase from sheep liver mitochondria. The kinetics of the NAD+-dependent oxidation of aldehydes, catalysed by aldehyde dehydrogenase purified from sheep liver mitochondria, were studied in detail. Lag phases were observed in the assays, the length of which were dependent on the enzyme concentration. The measured rates after the lag phase was over were directly proportional to the enzyme concentration. If enzyme was preincubated with NAD+, the lag phase was eliminated. Double-reciprocal plots with aldehyde as the variable substrate were non-linear, showing marked substrate activation. With NAD+ as the variable substrate, double-reciprocal plots were linear, and apparently parallel. Double-reciprocal plots with enzyme modified with disulfiram (tetraethylthiuram disulphide) or iodoacetamide, such that at pH 8.0 the activity was decreased to 50% of the control value, showed no substrate activation, and the plots were linear. At pH 7.0, the kinetic parameters Vmax. and Km NAD+- for the oxidation of acetaldehyde and butyraldehyde by the native enzyme are almost identical. Formaldehyde and propionaldehyde show the same apparent maximum rate. Aldehyde dehydrogenase is able to catalyse the hydrolysis of p-nitrophenyl esters. This esterase activity was stimulated by both NAD+ and NADH, the maximum rate for the NAD+ stimulated esterase reaction being roughly equal to the maximum rate for the oxidation of aldehydes. The mechanistic implications of the above behaviour are discussed.


Preparative-scale enzymic synthesis of D-[14C]ribulose 1,5-bisphosphate. A procedure is described to prepare uniformly labelled D-[14C]ribulose 1,5-bisphosphate enzymically from uniformly labelled D-[14C]glucose through the coupled reactions catalysed by hexokinase (EC 2.7.1.1), glucose 6-phosphate dehydrogenase (EC 1.1.1.49), 6-phosphogluconate dehydrogenase (EC 1.1.1.44) and 5-phosphoribulokinase (EC 2.7.1.19). All reagents utilized in the method are commercially available. The procedure is a reliable preparative-scale method for synthesizing the dibarium salt of D-[14C]ribulose 1,5-biphosphate with a specific radioactivity up to 7 mCi/mmol and a purity near 90%. The final product was free of other 14C-labelled sugars, sugar phosphate esters, Pi and nucleotides.


Cartilage proteoglycan aggregates. Electron-microscopic studies of native and fragmented molecules. 1. Proteoglycan aggregates from bovine nasal cartilage were studied by using electron microscopy of proteoglycan/cytochrome c monolayers. 2. The aggregates contained a variably long central filament of hyaluronic acid with an average length of 1037nm. The proteoglycan monomers attached to the hyaluronic acid appeared as side chain filaments varying in length (averaging 249nm). They were distributed along the central filament at an average distance of about 36nm. 3. Chondroitin sulphate side chains were removed from the proteoglycan monomers of the aggregates by partial chondroitinase digestion. The molecules obtained had the same general appearance as intact aggregates. 4. Proteoglycan aggregates were treated with trypsin and the largest fragment, which contains the hyaluronic acid, link protein and hyaluronic acid-binding region, was recovered and studied with electron microscopy. Filaments that lacked the side chain extensions and had the same length as the central filament in the intact aggregate were observed. 5. Hyaluronic acid isolated after papain digestion of cartilage extracts gave filaments with similar length and size distribution as observed for the central filament both in the intact aggregate and in the trypsin digests. 6. Umbilical-cord hyaluronic acid was also studied and gave electron micrographs similar to those described for hyaluronic acid from cartilage. However, the length of the filament was somewhat shorter. 7. The electron micrographs of both intact and selectively degraded proteoglycans corroborate the current model of cartilage proteoglycan structure.


Isolation, characterization and distribution of adenosine 3':5'-cyclic monophosphate from Pinus radiata. Cyclic AMP was extracted in 0.1 M-HCl from tissues of Pinus radiata and purified by gel filtration on Sephadex G-10, and chromatography on Dowex AG1 (X2) and polyethyleneimine-cellulose in two separate solvent systems. Presumptive cyclic AMP from 10kg batches of pine needles was characterized by countercurrent distribution in the presence of cyclic [8-3H]AMP. Statistical analysis of the curves for radioactivity and mass (determined by the Gilman competitive-binding assay) showed that the fit of the curves was highly significant for seven degrees of freedom. The distribution of cyclic AMP within P. radiata and various other plant tissues was determined by the Gilman procedure. The results suggest that there is no relationship between variations in cyclic AMP concentrations and the known function of the tissue in which it was measured.


Hepatic storage of oligosaccharides and glycolipids in a cat affected with GM1 gangliosidosis. 1. Glycopeptides and glycolipids were isolated from normal cat liver and liver from a cat affected with GM1 gangliosidosis. 2. Bio-Gel P-6 chromatography of the crude glycopeptide fractions demonstrated three major peaks of hexose-containing compounds that were greatly increased in the mutant liver sample; these peaks contained oligosaccharides that comprised over 2% of the liver wet weight. 3. Two of the major pathological oligosaccharides, GP5 and GP6, were purified by chromatography on charcoal/Celite and Sephadex G-25. Oligosaccharides GP5 and GP6 had apparent mol.wts. of 1800 +/- 200 and 1350+/-200 respectively, and contained galactose, mannose and N-acetylglucosamine in molar proportions of 2.0:3.1:4.1 (GP5) and 1.0:2.2:2.7 (GP6). Periodate oxidation studies demonstrated the presence of galactose in a non-reducing terminal position. 4. The neutral glycolipid fraction from the mutant cat liver has a 1.3-fold increase in hexose content accompanied by an increased concentration of asialo-(ganglioside GM1). 5. There was a 2-fold increase of gangliosides in the mutant cat liver compared with normal cats. Ganglioside GM1 and a compound tentatively identified as N-glycolloyl-(ganglioside GM1) were the major glycolipids accumulated.


Simulation of the electron-paramagnetic-resonance spectrum of the iron-protein of nitrogenase. A prediction of the existence of a second paramagnetic centre. The e.p.r. spectra of the Fe-proteins of nitrogenase from all sources studied have unusual features in that they have very anisotropic linewidths and low integrated intensities. These characteristics can be explained by assuming that one of the two electrons accepted by these proteins is located at a rapidly relaxing paramagnetic centre that is unobservable by e.p.r., but causes anisotropic broadening of the e.p.r. signal of the other electron. Complex-formation between Fe-proteins and MgATP is described in terms of a 50-60 degrees rotation of the e.p.r.-observable centre.


Malate dehydrogenase of the cytosol. A kinetic investigation of the reaction mechanism and a comparison with lactate dehydrogenase. 1. The mechanisms of the reduction of oxaloacetate and of 3-fluoro-oxaloacetate by NADH catalysed by cytoplasmic pig heart malate dehydrogenase (MDH) were investigated. 2. One mol of dimeric enzyme produces 1.7+/-0.4 mol of enzyme-bound NADH when mixed with saturating NAD+ and L-malate at a rate much higher than the subsequent turnover at pH 7.5. 3. Transient measurements of protein and nucleotide fluorescence show that the steady-state complex in the forward direction is MDH-NADH and in the reverse direction MDH-NADH-oxaloacetate. 4. The rate of dissociation of MDH-NADH was measured and is the same as Vmax. in the forward direction at pH 7.5. Both NADH-binding sites are kinetically equivalent. The rate of dissociation varies with pH, as does the equilibrium binding constant for NADH. 5. 3-Fluoro-oxaloacetate is composed of three forms (F1, F2 and S) of which F1 and F2 are immediately substrates for the enzyme. The third form, S, is not a substrate, but when the F forms are used up form S slowly and non-enzymically equilibrates to yield the active substrate forms. S is 2,2-dihydroxy-3-fluorosuccinate. 6. The steady-state compound during the reduction of form F1 is an enzyme form that does not contain NADH, probably MDH-NAD+-fluoromalate. The steady-state compound for form F2 is an enzyme form containing NADH, probably MDH-NADH-fluoro-oxaloacetate. 7. The rate-limiting reaction in the reduction of form F2 shows a deuterium isotope rate ratio of 4 when NADH is replaced by its deuterium analogue, and the rate-limiting reaction is concluded to be hydride transfer. 8. A novel titration was used to show that dimeric cytoplasmic malate dehydrogenase contains two sites that can rapidly reduce the F1 form of 3-fluoro-oxaloacetate. The enzyme shows 'all-of-the-sites' behaviour. 9. Partial mechanisms are proposed to explain the enzyme-catalysed transformations of the natural and the fluoro substrates. These mechanisms are similar to the mechanism of pig heart lactate dehydrogenase and this, and the structural results of others, can be explained if the two enzymes are a product of divergent evolution.


Studies on the fate of pulmonary surfactant in the lung. 1. Radioactively labelled pulmonary surfactant was prepared in an isolated perfused lung system provided with [14C]hexadecanoate. 2. After intratracheal administration of pulmonary surfactant radioactively labelled components were rapidly distributed into different lung fractions, including macrophages (free cells), but most of the radioactive label was accumulated by the lung tissue. 3. Alveolar macrophages, maintained in a variety of culture media in the presence and absence of mineral particles, incorporated a low percentage (11%) of radioactively labelled components when incubated with the surfactant, although evolution of labelled CO2 (6% of the original total activity) suggested that some breakdown of the components had taken place. 4. In similar cultures little intracellular accumulation or extracellular release of non-esterified fatty acids was demonstrated, indicating minimal catabolism of the high-molecular-weight lipid components of surfactant (particularly phosphatidylcholine). 5. However, experiments in vitro designed to simulate the lysosomal degradation of endocytosed surfactant indicated that the macrophage had enzymes capable of releasing non-esterified fatty acids, particularly hexadecanoate, from the lipoprotein complex. 6. It is argued that lung cells, other than alveolar macrophages, may also have a role in surfactant turnover.


Effects of cholera toxin and guanosine 5'-[betagamma-imido]triphosphate on beta-adrenergic-receptor affinity. A comparison was made of the effects of cholera toxin and p[NH]ppG on the binding affinity of beta-adrenergic receptors in toad erythrocyte membranes. This was determined by studying the ability of isoproterenol and propranolol to compete for the receptor with (-)-[3H]dihydroalprenolol. p[NH]ppG decreased the receptor affinity for the agonist isoproterenol (i.e. a 'right' shift in the displacement-concentration curve), but was without effect on the affinity for the antagonist propranolol. Toad erythrocyte membranes after treatment with cholera toxin exhibited increased receptor affinity for isoproterenol (i.e. a 'left' shift in the displacement curve), but did not affect the affinity for propranolol. p[NH[ppG was able to exert its right shift even in cholera-toxin treated membranes. The ability of cholera toxin to alter beta-adrenergic-receptor affinity is interpreted as further evidence that the toxin affects the nucleotide-regulatory component of adenylate cyclase. The regulatory component affected may be the catecholamine-sensitive guanosine triphosphatase.


Calcium-activated hydrolysis of phosphatidyl-myo-inositol 4-phosphate and phosphatidyl-myo-inositol 4,5-bisphosphate in guinea-pig synaptosomes. 1. Addition of the bivalent ionophore A23187 to synaptosomes isolated from guinea-pig brain cortex and labelled with [(32)P]phosphate in vitro or in vivo caused a marked loss of radioactivity from phosphatidyl-myo-inositol 4-phosphate (diphosphoinositide) and phosphatidyl-myo-inositol 4,5-bisphosphate (triphosphoinositide) and stimulated labelling of phosphatidate. No change occurred in the labelling of other phospholipids. 2. In conditions that minimized changes in internal Mg(2+) concentrations, the effect of ionophore A23187 on labelling of synaptosomal di- and tri-phosphoinositide was dependent on Ca(2+) and was apparent at Ca(2+) concentrations in the medium as low as 10(-5)m. 3. An increase in internal Mg(2+) concentration stimulated incorporation of [(32)P]phosphate into di- and tri-phosphoinositide, whereas lowering internal Mg(2+) decreased labelling. 4. Increased labelling of phosphatidate was independent of medium Mg(2+) concentration and apparently only partly dependent on medium Ca(2+) concentration. 5. The loss of label from di- and tri-phosphoinositide caused by ionophore A23187 was accompanied by losses in the amounts of both lipids. 6. Addition of excess of EGTA to synaptosomes treated with ionophore A23187 in the presence of Ca(2+) caused a rapid resynthesis of di- and tri-phosphoinositide and a further stimulation of phosphatidate labelling. 7. Addition of ionophore A23187 to synaptosomes labelled in vivo with [(3)H]inositol caused a significant loss of label from di- and tri-phosphoinositide, but not from phosphatidylinositol. There was a considerable rise in labelling of inositol diphosphate, a small increase in that of inositol phosphate, but no significant production of inositol triphosphate. 8. (32)P-labelled di- and tri-phosphoinositides appeared to be located in the synaptosomal plasma membrane. 9. The results indicate that increased Ca(2+) influx into synaptosomes markedly activates triphosphoinositide phosphatase and diphosphoinositide phosphodiesterase, but has little or no effect on phosphatidylinositol phosphodiesterase.


A method for measuring relative changes in guanosine 3':5'-cyclic monophosphate in mouse neuroblastoma cells on muscarinic cholinergic stimulation. A convenient, inexpensive assay was developed for measuring relative changes in cyclic GMP in whole mouse neuroblastoma cells (clone NIE 115) based on labelling the cellular GTP pool with [8(-3)H]guanine. The time course of cell labelling and the distribution of radioactivity among possible products were studied; GTP is the only major labelled species. Radioactive cyclic GMP produced from the radioactive GTP on cell stimulation is isolated by column chromatography nad its identity has been rigorously established by paper chromatography and ion-exchange chromatography. The assay was used to study the time course of the cyclic GMP changes that occur after stimulation of neuroblastoma cells with carbamoylcholine and the dependence of the cyclic GMP changes on the carbamoylcholine concentration. The assay gives results comparable with those obtained by using a radioimmunoassay for cyclic GMP and should be applicable to other whole-cell and tissue-slice systems.


Recognition of the beta-subunit of human chorionic gonadotropin and sub-determinants by target tissue receptors. The beta-subunit of human chorionic gonadotropin, purified immunochemically to eliminate undissociated human chorionic gonadotropin, induced testosterone production by mouse Leydig cells at concentrations 400-fold higher than human chorionic gonadotropin. Steroidogenesis was also stimulated by a synthetic fragment of the beta-subunit of human chorionic gonadotropin conforming to the peptide sequence residues 39--71, whereas peptide sequence residues 39--56 and three C-terminal fragments (residues 115--145, 111--145 and 101--145) failed to cause steroidogenesis. These studies suggest the presence in the beta-subunit of human chorionic gonadotropin of determinants recognized by the tissue receptors, a part of these determinants residing between amino acid residues 57--71.


Rat adipose-tissue glycerol phosphate acyltransferase can be inactivated by cyclic AMP-dependent protein kinase. Rat adipose-tissue glycerol phosphate acyltransferase can be inactivated in a phosphorylation reaction catalysed by cyclic AMP-dependent protein kinase and reactivated by treatment with alkaline phosphatase. These results suggest that phosphorylation of glycerol phosphate acyltransferase may be involved in the hormonal control of esterification.


The role of phosphoenolpyruvate carboxykinase in amino acid metabolism in muscle. Starvation or feeding rats on a high-protein diet, valine or isoleucine, but not leucine, increases the activity of muscle phosphoenolpyruvate carboxykinase, but has no effect on NADP+-linked malate dehydrogenase. This suggests that muscle phosphoenolpyruvate carboxykinase is involved in oxidation or conversion of some amino acids to alanine.


Control of phosphatidylcholine synthesis and the regulatory role of choline kinase in rat liver. Evidence from essential-fatty acid-deficient rats. Choline kinase and phosphocholine cytidylytransferase catalyse the rate-limiting steps of the cytidine pathway for the synthesis of phosphatidylcholine [Infante (1977) Biochem. J. 167, 847--849]. Essential-fatty acid deficiency induces a 3.5-fold increase in the specific activity of choline kinase, whereas the specific activity of the cytidylytransferase remains unchanged in rat liver. This change in specific activity accounts for the calculated increase in flux through the cytidine pathway produced in vivo by the same dietary state [Trewhella & Collins (1973 Biochim. Biophys. Acta 296, 34--50], thus confirming the fact that choline kinase has a regulatory role in the cytidine pathway for the synthesis of phosphatidylcholine.


Inhibition of collagen gelation by action of the superoxide radical. Superoxide anion, a highly reactive free radical, was generated in vitro using enriched purified xanthine oxidase. Collagen solutions exposed to superoxide radical failed to gel normally when heated to 37 degrees C. The magnitude of the inhibition of gelation was propotional to duration of exposure and to flux of superoxide. Since inhibition of collagen gelation reflects alteration of collagen biochemistry, and/or collagen degradation, it is suggested that the action of free radicals produced in vivo by leukocytes may adversely affect the structural or functional integrity of cartilage and adjacent joint structures.


[Radiotherapy of carcinoma of the nasopharynz: clinical results (author's transl)]. A series of 64 patients with malignant tumours of the nasopharynx treated by cobalt irradiation is analyzed. The crude 5-year survival rate for the entire epithelial group was 32%; 55% for N- group, 25% for N+. Treatment methods avoiding complications are discussed.


Specificity of the inhibition of DNA synthesis by extracts from cloned normal, sarcoma-virus-transformed and revertant 3T3 cells. Extracts containing tissue-specific DNA-inhibitory activity were prepared from normal FL (Swiss) and BALBc3T3 cells, from these cells transformed with sarcoma virus and from revertants cloned from the transformed cell lines. By testing all extracts on all cell lines we found that (1) production of and susceptibility to the inhibitors were decreased in transformed BALB/c cells (2) specificity varied with expression of the transforming genome, as an extract from a given cell line inhibited the growth of its cell of origin, e.g. revertant, more than normal or transformed cells, and (3) there was also a DNA-synthesis stimulator.


Effects of Corynebacterium parvum on murine myeloid leukaemia. The effects of C. parvum on RFM/UN myeloid leukaemia were studied. Mice inoculated with 7.0 mg but not 0.7 mg C. parvum i.p. survived significantly longer than untreated leukaemic mice (P less than 0.001). Administration of silica abrogated the effects of C. parvum, whilst polyvinyl pyridine-N-oxide prevented the inhibitory effects of silica. These studies demonstrate that a single large dose of C. parvum, either before or after leukaemic-cell passage, can significantly prolong the survival of RFM mice bearing myeloid leukaemia. The effects of silica and PVNO on C. parvum suggest a critical role for macrophages in C. parvum effects on myeloid leukaemia.


Effect of prostaglandins and hormones on cyclic AMP formation in rat hepatomas and liver tissue. The formation of cyclic AMP was studied in normal liver, subcutaneous hepatomas derived from MH1C1 cells, and premalignant liver and primary hepatomas induced by the carcinogens 2-acetylaminofluorene (AAF) and 4-dimethylamino-azobenzene (DAB). While only very slight effects of prostaglandins (PG) were seen in slices of normal liver, all the hepatomas responded strongly to PGE1 and PGE2. The hepatomas also had increase PGE1-sensitive adenylate-cyclase activity. PGF1alpha and PGF2alpha did not increase the cAMP level significantly either in the liver or in the hepatomas. During AAF carcinogenesis the response to PGE1 increased slightly during the carcinogen feeding, and was greatly elevated only in the fully developed hepatomas. This is in contrast to the increase in adrenalin response seen during carcinogenesis, which starts much earlier, and reaches a peak value within 8--10 weeks. It is concluded that various hepatomas have elevated responsiveness to PGE1 and PGE2 as well as to adrenalin, but the course of change in the tissues' ability to respond to these agents during carcinogenesis is very different.


Inherited aplastic anaemia with increased endoreduplications: a new syndrome of Fanconi's anaemia variant? Two sisters with aplastic anaemia without other congenital anomalies are described. Peripheral blood cytogenetic studies revealed an increase in endoreduplications in the absence of other unstable chromosome anomalies. Increased expression of T-antigen following SV40 virus infection in vitro was demonstrated in both sisters, as well as other normal family members. We feel that these patients represent a variant of Fanconi's anaemia. The importance of performing chromosome studies in idiopathic aplastic anaemia is emphasized.


Structure of the dimyristoylphosphatidylcholine vesicle and the complex formed by its interaction with apolipoprotein C-III: X-ray small-angle scattering studies. Single bilayer vesicles of dimyristoylphosphatidylcholine have been investigated by small-angle X-ray scattering at 28 degrees C. The results indicate that these vesicles are hollow spherical shell structures with an outer radius of approximately 12 nm and a molecular weight of (3.2 +/- 0.5) X 10(6). The shell was found to be 4.4 +/- 0.2 nm thick with a cross-sectional electron-density profile characteristic for a single phospholipid bilayer. Upon interaction of these vesicles with apolipoprotein C-III from human very low density lipoproteins at a protein/lipid ratio greater than 0.08 (g/g), a complex containing 0.25 g of protein/g of lipid, with molecular weight of (3.9 +/- 0.4) X 10(5), is formed. The shape analysis indicates a highly asymmetric particle with an internal partition of low and high electron density resembling that produced by a bilayer structure. Model calculations and curve-fitting procedures show good agreement between the experimental scattering curve and that computed for an oblate ellipsoidal structure with dimensions of 17 X 17 X 5 nm and a 1 nm thick shell of high electron density surrounding the core of low electron density.


Cation-induced regulatory mechanism of GTPase activity dependent on polypeptide initiation factor 2. Initiation factor IF-2 ribosome dependent GTP hydrolysis (uncoupled GTPase) presents a bell-shaped pH profile which is shifted by changes in ionic strength. At low ionic strength (I = 25 mM) the maximal hydrolytic activity occurs at pH 7.5; when the ionic strength is increased the pH optimum of the reaction is shifted toward more acidic values. Such behavior can be satisfactorily explained as the effect of an electrostatic potential developed by a neighboring polyanion, presumably RNA, on the catalytic site. The addition of fMet-tRNAfMet or AcPhe-tRNAPhe and messenger RNA (coupled GTPase) changes the ionic strength--pH characteristics of the reaction. Thus there is an effect, direct or indirect, of components located at the ribosomal P site. Investigation of the effect of neighboring polyanions on the catalytic activity of the factor-dependent ribosomal GTPases can be seen to provide information about their functional significance that is complementary to that gained from direct structural studies.


Cyclic nucleotide dependent protein kinase and the phosphorylation of endogenous proteins of retinal rod outer segments. Cyclic nucleotide dependent protein kinase has been extracted wiht Tris or Lubrol PX from purified rod outer segments (ROS) of bovine retina. The activity of the enzyme is unaffected by light but is stimulated by either cyclic guanosine 3',5'-monophosphate (cGMP) or cyclic adenosine 3',5'-monophosphate (cAMP). Most of the solubilized enzyme elutes from DEAE-cellulose with about 0.18 M NaCl (type II protein kinase). An endogenous 30,000 molecular weight protein of the soluble fraction of ROS as well as exogenous histone are phosphorylated by the protein kinase in a cyclic nucleotide dependent manner. The Tris-extracted enzyme can be reassociated in the presence of Mg2+ with ROS membranes that are depleted of protein kinase activity. The reassociated protein kinase is insensitive to exogenous cyclic nucleotides, and it catalyzes the phosphorylation of the membrane protein, bleached rhodopsin. While the soluble and membrane-associated protein kinases may be interchangeable, they appear to be modulated by different biological signals; soluble protein kinase activity is increased by cyclic nucleotides whereas membrane-bound activity is enhanced when rhodopsin is bleached by light.


Spin-label techniques for monitoring macromolecular rotational motion: empirical calibration under nonideal conditions. Practical techniques are demonstrated for determining rotational correlation times of macromolecules from the first harmonic absorption electron spin reasonance spectra of tightly bound spin labels. The techniques are developed to compensate for such nonideal conditions as residual label motion, temperature dependence of rigid limit spectral parameters, and the presence of inhomogeneous line broadening. These effects are all shown to be of importance in monitoring the rotational motion of carbonmonoxyhemoglobin which is spin labeled with the tightly bound nitroxide label, 4-maleimido-2,2,6,6-tetramethylpiperidinyl-1-oxy. Spin-label interactions with other paramagnetic agents are also shown to produce spectral changes which are qualitatively similar to, but quantitatively different from, those resulting from increases in the rate of rotational motion.


Lifetimes and NADH quenching of tryptophan fluorescence in pig heart lactate dehydrogenase. The decay of tryptophan emission from pig heart lactate dehydrogenase following pulsed excitation has been recorded in Tris buffer solution at pH 7.4. All tryptophan residues emit. A good least-squares two-component fit is obtained with I(t)-0.53e-t/1.2 + 0.47e-t/68. A longer lived emitter (r=7.4--8.1 ns) is also observed. Bound NADH strongly quenches most of the 6.8-ns emission, but the 1.2-ns component is relatively unaffected. The fluorescence is moderately quenched by acrylamide and only slighty quenched by I- and Cs+. The pulsed and steady-state fluorescence is discussed in terms of a model with three lifetime classes of tryptophan, viz., 1, 4, and 8 ns. The three-dimensional structure of the enzyme--NADH complex is used to develop a description of the individual residues in terms of their lifetimes and sensitivity to NADH and I- quenching. The nonlinear NADH quenching is due to intersubunit energy transfer from Trp-248 to NADH.


Complete exchange of viral cholesterol.

Complete exchange of viral cholesterol. The exchange of the cholesterol in the membranes of two enveloped viruses, Sindbis virus and vesicular stomatitis virus, with cholesterol present in lipid vesicles and in serum was measured. Biosynthetically labeled viral cholesterol underwent spontaneous and complete transfer to both lipid vesicles and to serum. The rate with which and the extent to which this process occurred were very similar for these two viruses. During incubation with lipid vesicles in excess, half of the viral cholesterol underwent transfer in approximately 4 h and more than 90% underwent transfer in 24h at 37 degrees C. Similar rates and extents of movement of viral cholesterol were observed when incubations were carried out with vesicles which contained cholesterol and phospholipid in the same molar ratio as in the virus or with egg lecithin vesicles which contained no cholesterol. When labeled cholesterol was present initially in the lipid vesicles, movement of cholesterol from the vesicles to the virus was observed. One implication of the fact that viral cholesterol undergoes extensive exchange with serum cholesterol is that cellular cholesterol is in equilibrium with that in the extracellular fluid.


Diastereomers of 5'-O-adenosyl 3'-O-uridyl phosphorothioate: chemical synthesis and enzymatic properties. A procedure is described for the synthesis of the title compounds via phosphotriester intermediates. The 2-cyanoethyl group is used to protect the P-SH function during the course of the synthesis. Resolution of the phosphorus diastereomers is accomplished at the phosphotriester stage. Removal of the 2-cyanoethyl group without racemization, followed by removal of the other protective groups, affords the optically pure diastereomers of 5'-O-adenosyl 3'-O-uridyl phosphorothioate. Their designation as Rp and Sp follows from the stereospecificity in the hydrolysis catalyzed by RNase A. These diastereomers are useful for the investigation of the stereospecificity as well as of the stereochemical course of action of nucleases. Snake venom exonuclease hydrolyses only the Rp diastereomer, whereas both diastereomers are substrates for RNases A and T2. The results with the latter indicate that RNase T2 also operates by an in-line mechanism.


Isolation and characterization of baby hamster kidney (BHK-21) cell modulator protein. A Ca2+-dependent modulator protein has been isolated from BHK-21 cells. The purification requires heat treatment, ion-exchange chromatography, and gel filtration. The protein appears homogenous on sodium dodecyl sulfate--polyacrylamide and isoelectric focusing gels. The protein comigrates with purified smooth muscle and brain modulators. BHK-21 modulator is characterized by a high content of aspartic and glutamic acids and by a high phenylalanine/tyrosine ratio. It lacks both cysteine and tryptophan. The protein is effective in activating brain-modulator-deficient phosphodiesterase. It can also be used in assay systems to generate Ca2+-sensitive actin activation of both BHK-21 and smooth muscle myosins. Therefore, it is proposed that the BHK-21 modulator protein is a component of the Ca2+-dependent  mechanism involved in the regulation of actin--myosin interactions in BHK-21 cells.


H3.H4 tetramer directs DNA and core histone octamer assembly in the nucleosome core particle. The way in which histones interact with DNA during in vitro assembly of nucleohistone has been examined. Chicken erythrocyte core histones H2A, H2B, H3, and H4 and lambdaDNA in 2 M NaCl were allowed to interact by stepwise decrease in the salt concentration. Binding, although weak, was first observed at 1.4 M NaCl and was essentially completed at 0.6 M NaCl. Analysis of the DNA-bound histones revealed that each of the histones in the pairs H2A,H2B and H3,H4 was always present in equimolar amounts and that the relative proportion of each pair was constant between 1.4 and 0.8 M NaCl. Evidence is presented suggesting that binding occurred via complexes of the four histones, the nature of which is likely to reflect the equilibrium among the octamer and its products of dissociation (Ruiz-Carrillo, A., & Jorcano, J.L. (1979) Biochemistry (preceding paper in this issue)). The presence of complexes of the four core histones is, however not required for the correct assembly of the nucleosome core particle. Nucleohistones obtained by adding at progressively lower ionic strengths the dimer H2A.H2B to the H3.H4-DNA complex (split reconstitutions) had the same characteristics as those assembled with the core histone complexes.


Molecular weight of (Na+,K+)ATPase from shark rectal gland. The (Na+,k+)ATPase from the rectal gland of Carcharhinus obscurus has been solubilized in Lubrol WX as an active complex containing 379,900 g of protein and 61 mol of phospholipid. This detergent-lipid-protein complex contains two catalytic subunits of molecular weight 106,400 and four glycopeptide subunits of protein molecular weight 36,600. The latter subunit has a total molecular weight of 51,700 when the carbohydrate is included. Attempts to dissociate this active enzyme complex to smaller size by increasing the detergent concentration led to inactivation. Thus, the smallest active particle in the presence of Lubrol WX contains the two polypeptide subunits in a mole ratio of 2:4 under conditions where the micellar form of detergent is present at a 70:1 molar ratio. This large excess of Lubrol WX eliminates any possibility of artificial togetherness as the result of statistical considerations.


Cyclic adenosine monophosphate dependent and independent phosphorylation of sarcolemma membrane proteins in perfused rat heart. This study was initiated in order to elaborate further on the mechanism by which epinephrine modulates cardiac function via protein phosphorylation. A membrane fraction has been isolated from freeze-clamped perfused rat heart that contains two phosphoproteins. These proteins have molecular weights of 36,000 (A protein) and 27,000 (B protein). The phosphorylation of the A protein occurs during the equilibration of the heart with inorganic [32P]phosphate. The phosphorylation of the B protein occurs in response to epinephrine. The A and B proteins are apparently identical with two phosphoproteins in enriched preparations of sarcolemma. The protein of the sarcolemma preparation equivalent to the A protein is phosphorylated in vitro by both cAMP-independent and cAMP-dependent protein kinases. The phosphorylation of the protein of the sarcolemma preparation equivalent to the B protein is catalyzed by the cAMP-dependent protein kinase. Thus the patterns of phosphorylation of these proteins in vivo and in vitro are compatible. The phosphorylation of the B protein has been documented in vitro to modulate calcium transport (Will, H., et al. (1973) Acta Biol. Med. Ger. 31, 45-52), but the response to epinephrine in the perfused heart is not apparently coordinated with the catecholamine-induced inotropic effect.


Azethoxyl nitroxide spin labels. ESR studies involving thiourea crystals, model membrane systems and chromatophores, and chemical reduction with ascorbate and dithiothreitol. Trans- and cis-azethoxyl nitroxides 1, 2, 3 and 4 can be trapped in the cavities of thiourea crystals. The presence of a single gauche conformation on either side of the pyrrolidine ring within the crystals was indicated by the ESR spectra. Rotation about the long molecular axis then corresponds approximately to y-axis motion of the nitroxide moiety. Proxyl nitroxides in which the nitroxide group is located on the penultimate carbon of long chain lipids can also be trapped and were shown to adopt the azethoxyl conformation in the thiourea crystals. The measured deltaA values (A parallel to - A perpendicular) of oriented egg lecithin multilayers containing trans- and cis-azethoxyl nitroxides 1 and 2 were quite small, consistent with the unique orientation of the nitroxide principal axes with respect to the long axis of the molecule. The deltaA values for a series of lipids bearing a label near the terminus of the chain were very similar to that of 1, showing that the azethoxyl conformation is likely the predominant one in these labels in orienting systems. Computer simulations of the ESR spectra of 1 and 2 in egg lecithin vesicles provided values for molecular orientation and motion parameters consistent with those expected from a consideration of molecular models in the extended (all trans) conformation. Azethoxyl nitroxides have also proven useful in the investigation of motion restricted (boundary) lipid in a lipid-protein system. Thus, the values (69 +/- 10%) for the amount of boundary lipid in the chromatophore membranes from Rhodopseudomonas sphaeroides as determined using trans- 2 and cis- 2 are in good agreement with values using 16-doxylstearic acid (64 +/- 3%). The fact that all three labels show about the same fraction of boundary lipid in this system indicates that the lipid binding sites are relatively insensitive to the geometry of the lipid chain. Also, both 1 and 2 appear to be able to detect a third lipid environment not seen with the doxyl fatty acid. The apparent fluidity of this component lies between that of boundary and bilayer lipid. The unique orientation of the nitroxide principal axes with respect to the long molecular axis in azethoxyl nitroxides 1 and 2 allows detection of hindrance to rotation about the long molecular axis, in contrast to the analogous doxyl and proxyl fatty acids. Comparative reduction studies using ascorbate and dithiothreitol indicate that azethoxyl nitroxides are slightly more resistant toward reduction than proxyl nitroxides and much more resistant than doxyl nitroxides.


Interaction of plasma lipoproteins with erythrocytes. I. Alteration of erythrocyte morphology. Intact erythrocytes incubated in the presence of low density lipoproteins (LDL) undergo a time-dependent morphologic transformation from biconcave discs to spherocytes within 4 h. No shape change is observed when erythrocytes are incubated with high density lipoproteins (HDL). The LDL-induced change in erythrocyte morphology occurs without concomitant leakage of hemoglobin from the cell or depletion of intracellular ATP; no change in the distribution of the major lipids of the erythrocyte membranes was detected. The alteration of morphology does require attachment of LDL to the erythrocyte surface. The LDL-induced morphologic alteration is inhibited by HDL, but not by serum albumin. HDL prevent the attachment of LDL to the cell membrane; however, the HDL subfractions, HDL2 and HDL3, are only partially effective. These data suggest that normal erythrocyte morphology and cell function may depend on the concentration and composition of the circulating lipoproteins.


Interaction of plasma lipoproteins with erythrocytes. II. Modulation of membrane-associated enzymes. When incubated with intact erythrocytes, low density lipoproteins (LDL) decrease the phosphate content of erythrocyte spectrin allowing the cells to undergo morphological transformation. The phosphate content of spectrin depends on the balance between the activity of membrane-associated cyclic AMP-independent protein kinases and phosphoprotein phosphates. LDL do not influence the activity of membrane-associated cyclic AMP-independent protein kinases; these lipoproteins activate by 2-fold and greater membrane-associated phosphatases as determined by hydrolysis of p-nitrophenyl phosphate and by phosphate hydrolysis of phosphorylated erythrocyte membrane proteins. We conclude that LDL interact at the exterior surface of the erythrocyte to stimulate dephosphorylation of spectrin. The significance of this conclusion is augmented by the fact that spectrin, the target for LDL-induced dephosphorylation, specifies cell morphology and modulates the distribution of cell-surface receptors. LDL also render erythrocyte acetylcholinesterase less susceptible to inhition by F-. Lipoproteins in the high density class (HDL) do not stimulate dephosphorylation of spectrin, and they are consequently unable to alter erythrocyte morphology. HDL do prevent the LDL-induced activation of membrane phosphatase. The inhibitory capacity of HDL is observed over the range of LDL:HDL (w/w) which exists in the plasma of normolipemic humans.


Mathematical modelling of ATP, K+ and Na+ interactions with (Na+ + K+)-ATPase occurring under equilibrium conditions. The controlling effect of ATP, K+ and Na+ on the rate of (Na+ + K+)-ATPase inactivation by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-C1) is used for the mathematical modelling of the interaction of the effectors with the enzyme under equilibrium conditions. 1. Of a series of conceivable interaction models, designed without conceptual restrictions to describe the effector control of inactivation kinetics, only one fits the experimental data described in a preceding paper. 2. The model is characterized by the coexistence of two binding sites for ATP and the coexistence of two separate binding sites for K+ and Na+ on the enzyme-ATP complex. On the basis of this model, the effector parameters fitting the experimental data most closely are estimated by means of nonlinear least-squares fits. 3. The apparent dissociation constants for ATP fo the enzyme-ATP complex or of the enzyme-(ATP)2 complex are computed to lie near 0.0024 mM and 0.34 mM, respectively, irrespective of whether K+ and Na+ were absent or K+ and K+ plus Na+, respectively, were present in the experiments. 4. The origin of the high and the low affinity site for binding of ATP to the (Na+ + K+)-ATPase molecule is traced back to the coexistence of two catalytic centres which, although primarily equivalent as to the reactivity of their thiol groups with NBD-C1, are induced into anticooperative communication by ATP binding and thus show an induced geometric asymmetry. 5. On the basis of the interaction model outlined under item 2 the apparent dissociation constant for K+ or Na+ in the (K+ + Na+)-liganded enzyme-ATP complex are computed to be 1.7 mM and 3.5 mM, respectively. 6. The conclusions concerning the coexistence of two primarily equivalent but anticooperatively interacting catalytic centres and the coexistence of two separate ionophoric centres for Na+ and K+ correspond to the appropriate basic postulates of the flip-flop concept of (Na+ + K+)-ATPase mechanism.


Viral and non-viral induced fusion of pronase-digested human erythrocyte ghosts. Human erythrocyte ghosts but was able to fuse only iso-human erythrocyte ghosts. Iso- and hypo-human erythrocyte ghosts were incubated with the proteolytic enzyme pronase under isotonic (iso-human erythrocyte ghosts) or hypotonic (hypo-human erythrocyte ghosts) conditions. Gel electrophoresis and electron microscope (freeze-etching) studies revealed that most of the erythrocyte membrane polypeptides were hydrolyzed by pronase under hypotonic conditions. Sendai virus readily agglutinated both pronase-digested iso-human erythrocyte ghosts and hypo-human erythrocyte ghosts were fused by the non-viral fusogenic agent glyceromonooleate. Freeze-etching studies revealed that during fusion the membranes of pronase-digested human erythrocyte ghosts are intermixed.


The effect of cyclic nucleotides and protein phosphorylation on calcium permeability and binding in the sarcoplasmic reticulum. In the absence of cyclic nucleotides heart microsomes have two classes of calcium binding sites with binding constants of 0.69 and 0.071 micron-1 and capacities of 2.2 and 9.7 nmol/mg protein, respectively. Neither cyclic AMP nor monobutyryl cyclic AMP affect binding but cyclic GMP and monobutyryl cyclic GMP cause the complete loss of the high affinity calcium binding sites, Cyclic GMP (but not monobutyryl cyclic GMP) also causes a decrease in the binding constant of the low affinity binding sites. AMP, GMP and Tris-butyrate do not affect calcium binding. The effects of the cyclic nucleotides are direct and are not mediated by protein phosphorylation. Phosphorylation of microsomal proteins increases the binding constant but not the capacity of the high affinity calcium binding sites. The capacity and also, perhaps, binding constant of the low affinity sites is also increased by phosphorylation. In additon to their effects on calcium binding the cyclic nucleotides also affect the movements of calcium into and out of the microsomes. The effects are again direct and not mediated by protein phosphorylation. Cyclic GMP decreases the rate of Ca2+ efflux from preloaded cardiac microsomes and also appears to decrease the rate of uptake of Ca2+ by cardiac microsomes though this effect is less clear cut than the action on efflux. The cyclic nucleotide has a half maximal effect at a concentration of 100 microns. By contrast cyclic AMP increases the rate of influx of Ca2+ into heart microsomes and the rate of efflux of Ca2+ from preloaded preparations. The effect is, however, rather slight. It is suggested that the most obvious interpretation of these results is that cyclic GMP decreases the Ca2+ permeability of the cardiac microsomal membrane while cyclic AMP increases the permeability. In contrast to the results found with membrane preparations from certain other tissues phosphorylation of cardiac microsomal proteins does not appear to alter Ca2+ efflux or influx out of, or into, cardiac microsomal preparations. It is thus concluded that phosphorylation of cardiac microsomal proteins does not affect the Ca2+ permeability of the microsomal membrane.


Electron spin resonance, hematological, and deformability studies of erythrocytes from patients with Huntington's disease. Electron spin resonance, hematologic, and deformability studies of erythrocytes from patients with Huntington's disease have been performed A decreased deformability of Huntington's disease erythrocytes compared to normal controls was demonstrated. No difference in erythrocyte hematologic indices, osmotic fragility, reticulocyte counts, or intracellular Na+ concentration was found. Huntington's disease serum had no demonstrable effect on electron spin resonance parameters of a protein-specific spin label attached to membrane proteins in control erythrocytes compared to the effect of control serum. This finding suggests that under the conditions employed no serum component or circulating factor is responsible for the changes in the physical state of membrane proteins in Huntington's disease erythrocytes (Butterfield, D.A., Oeswein, J.Q. and Markesbery, W.R. (1977) Nature 267, 453--455). No alteration in lipid fluidity of Huntington's disease erythrocyte membranes could be discerned suggesting that the underlying molecular defect in Huntington's disease involves a membrane protein. The results of the present studies on erythrocytes strongly support the concept that Huntington's disease is associated with a generalized membrane abnormality.


The purification of Q-containing tRNAs by periodate modification. Purification and nucleoside composition of two related Drosophila tyrosine tRNAs. A simple procedure for the purification of Drosophila tRNAs which contain the hypermodified nucleoside Q was developed. The cis-diol group of Q renders it susceptible to attack by sodium periodate, with the resultant formation of a hydrophobic pyrrol ring structure. The increase in hydrophobicity after periodate modification, of those tRNA species which contain the Q nucleoside, causes them to be selectively retarded on benzoylated DEAE-cellulose or RPC-5 columns, and allows their isolation in essentially pure form. Utilizing this property, the Q-containing tyrosine tRNA from Drosophila, tRNATyr1delta, was purified. The non-Q-containing tyrosine tRNA, tRNA, tRNATyr1gamma, was also purified, and the nucleoside compositions of the two were determined and compared. In addition to the Q nucleoside, these tRNAs appear to differ by the presence (tRNATyr1delta) or absence (tRNATyr1gamma) of 5-methylcytidine. In all other respects, these tRNAs appear to be identical, and are probably products of the same gene which differ in their levels of post-transcriptional modification.


Thiaisoleucine and protein synthesis.

Thiaisoleucine and protein synthesis. Thiaisoleucine is an isoleucine analogue having the gamma-methylene group of the valerianic carbon chain substituted by a sulphur atom. It has been demonstrated that thiaisoleucine is activated and transferred to tRNAIle by rat liver aminoacyl-tRNA synthetase and inhibits isoleucine incorporation into polypeptides in protein synthesizing systems from rat liver or rabbit reticulocytes, whereas it does not affect either leucine incorporation or ribosome run-off or polypeptide chain elongation rate. All tests were performed in comparison with O-methyl-threonine, an isoleucine analogue with the gamma-methylene group substituted by an oxygen atom. In all the reactions studied, both thiaisoleucine and O-methyl-threonine act as competitive inhibitors of isoleucine. With respect to O-methyl-threonine, thiaisoleucine shows higher activity as an isoleucine inhibitor.


Solubilization of membrane-bound adenylate cyclase of isolated rat adrenal cortex cells. The membrane-bound adenylase cyclase (ATP pyrosphosphate-lyase (cyclizing), EC 4.6.1.1) of isolated rat adrenal cortex cells can be rendered soluble using 0.02 M Lubrol 12A9. The solubilized enzyme can be filtered through Milipore filters with pores 0.22 micron in diameter. Using gel filtration, on Sephadex G-200, adenylate cyclase activity was eluted with a distribution coefficient of 0.139, whereas on Sephadex G-100 the activity was eluted in the excluded volume. Half-maximum activation of the postulated guanyl nucleotide regulator site of adenylate was achieved with 5'-guanylyl-imidodiphosphate at a concentration of 1 . 10(-6)M. In contrast, however, using intact isolated rat adrenal cortex cells the guanyl nucleotide regulator site could not be stimulated by 5'-guanylyl-imidodiphosphate.


Conversion of dihydroorotate to orotate in parasitic protozoa. The conversion of dihydroorotate to orotate, one of the key reactions in the de novo pyrimidine biosynthetic pathway, has been studied in a number of parasitic protozoa. Enzyme activities capable of carrying out this reaction were detected in six members of the Kinetoplastida (Trypanosoma brucei, Trypanosoma congolense, Trypanosoma vivax, Trypanosoma lewisi, Trypanosoma cruzi, Leishmania enriettii) and three members of the genus Plasmodium (P. knowlesi, P. berghei, P. gallinaceum). The mechanism of the reaction in the two groups of protozoa were quite distinct. In the Kinetoplastida, the enzyme is an hydroxylase which occurs in the soluble fraction of the cell and probably requires tetrahydrobiopterin for activity. In contrast, in Plasmodium, the enzyme is a dehydrogenase which is particulate, probably mitochondrial, and intimately connected to the electron transport chain to which it passes electrons directly, probably at the ubiquinone level. Neither activity is regulated by fully formed pyrimidines. The enzyme in Plasmodium is similar in mechanism to the isofunctional mammalian enzyme. However, since malarial ubiquinones are apparently different from those in the mammal and since menoctone, which is active in vivo in experimental malaria, is a good inhibitor of the malarial enzyme, it could represent a useful target for chemotherapeutic attack. The enzyme in the Kinetoplastida is quite distinct from that in the mammal so that it too apparently falls into this category, though none of the currently used antitrypanosomal drugs appears to block it activity at physiological concentrations.


Enzymic effects of beta-glucosidase destruction in mice. Changes in glucuronidase levels. 1. The injection into mice of a single dose of conduritol B epoxide, a covalent inhibitor of glucosidases, quickly produced changes in tissue levels of beta-D-glucuronidase (EC 3.2.1.31). The specific activity of the enzyme decreased in liver, spleen and kidney while brain showed little change. The inhibitor did not act on glucuronidase in vitro, so the effect of the inhibitor is complex, possibly a result of the loss of glucosidase activity. Since glucuronidase contains glucose, we suggest that the transport of the enzyme between subcellular regions and tissues involves loss of part of the glucose moieties. 2. Levels of glucocerebrosidase (D-glucosyl-N-acylsphingosine glucohydrolase, EC 3.2.1.45) dropped very rapidly after epoxide injection, reaching a minimum at 1 h in liver. There was a noticeable restoration of activity within the next 1--2 h. Aryl beta-glucosidase (EC 3.2.1.21) decrease somewhat less than cerebrosidase, reaching a minimum within 2 h. It too showed some recovery of activity within 3 h. 3. Acid phosphatase rose slightly in liver but not in brain. alpha-L-Fucosidase and angiotensin-converting enzyme were not affected by the epoxide injection. The latter two enzymes are known to contain glucose. 4. Injection of a hemolyzing agent, phenylhydrazine, produced an increased level of glucuronidase in liver and spleen within 6 days, but not in kidney. This enhancement was a little less in mice previously injected with the glucosidase inhibitor. 5. Mice injected with the epoxide once a day eight times showed a distinct rise in brain glucuronidase level, as well as a rise in brain weight. However, the other organs showed only the same decrease in glucuronidase specific activity noted with the single injection protocol. It is suggested that the difference is due to the blood-brain barrier, which could slow the loss of brain glucuronidase from the extracellular fluid.


Soluble cyclic AMP-dependent protein kinases from chick kidney. Effects of dilution and non-protein inhibitors. Cyclic AMP-dependent protein kinases prepared from crude cytosols of chick kidney, rat kidney and rat liver were found on dilution to exhibit complex kinetics. Dilution of the cytosols appears to increase the state of activation of the enzymes. This effect was due to the presence of inhibitory agents in the cytosol which had a greater inhibitory effect on the cyclic AMP-dependent than on the cyclic AMP-independent enzyme. Two types of inhibitory activity were found by column chromatography, one resistant to trichloroacetic acid precipitation and boiling but affected by trypsin digestion and the other resistant to boiling and trypsin digestion but precipitated by trichloroacetic acid. Inhibitory activity corresponding to the former characteristics has been described previously but the presence of additional soluble inhibitory agents in the cytosol has not been documented. The complete characterisation of this previously undescribed inhibitory activity requires further investigation. The relevance of such cytosolic inhibitory activity to the interpretation of states of activation of protein kinase enzymes is discussed.


Decreased number of beta-adrenergic receptors in hypertensive vessels. Responsiveness to inotropic agents is altered in hypertension and may contribute to its initiation and maintenance. A biochemical basis for this change was provided by the observation that the number of beta-adrenergic receptors, as reflected in specific [3H]dihydroalprenolol binding, was diminished in both arteries and veins of spontaneously hypertensive rats. There was no change in the affinity of dihydroalprenolol for the binding sites or in the capacity of isoproterenol to displace dihydroalprenolol. The decline in beta-adrenergic receptor numbers is not secondary to blood pressure elevation but may, instead, contribute to the pathogenesis of hypertension.


Pulse-radiolytic investigations of catechols and catecholamines. II. Reactions of Tiron with oxygen radical species. Transient spectra and kinetic data of Tiron (1,2-dihydroxybenzene-3,5-disulphonic acid) are reported, obtained after pulse-radiolytic oxidation by hydroxyl radicals (.OH), superoxide anions (O-2) or a combination of both oxygen radicals. The rate constant with .OH radicals was determined at 1.0.10(9) M-1.s-1. Contrary to a previous report (Greenstock, C.L. and Miller, R.W. (1975) Biochim. Biophys. Acta 396, 11--16), the rate constant with O-2 of 1.0.10(7) M-1.s-1 is lower by one order of magnitude; also the semiquinone absorbs at 300 nm rather than at 400 nm. The ratio of the rate constants with .OH and O-2 of 100 again demonstrates that any oxidation reaction by the latter radical is unspecific due to the more efficient reaction of .OH radicals, leading to the same products with catechol compounds.


Phosphodiesterase and GTPase in rod outer segments. Kinetics in vitro. The hydrolysis of cyclic guanosine monophosphate (cyclic GMP) and of guanosine triphosphate (GTP) by the broken rods of the frog retina after a flash of light have been studied in vitro with a constant perfusion method. The activation has an onset apparently instantaneous as observed with the existing possible time resolution of 3 s. The activation is followed by a partial inactivation that does not bring the activity back to the pre-flash level. GTP or the non-hydrolysable guanyl-5'-ylimidodiphosphate (GMP-PNP) is required for the normal light-activation of the phosphodiesterase and in its absence both the speed of activation and the sensitivity are greatly reduced. The activation speed, the sensitivity (threshold at approx. 0.00004% bleaching), and the kinetic constants do not exclude a direct role in the process of excitation for the phosphodiesterase and suggest a subsidiary but as yet undefined role for the GTPase.


beta-Adrenergic and muscarinic cholinergic receptors in rat submaxillary glands. Effects of thyroidectomy. Administration of triiodothyronine to thyroidectomized rats increased the density of beta-adrenergic receptors in rat submaxillary gland without significantly changing the density of muscarinic cholinergic receptors. Thus, thyroid hormone appears to regulate beta-adrenergic sensitivity in the rat salivary gland.


Glucose activation of liver glycogen synthase. Insulin-mediated restoration of glucose effect in diabetic rats is blocked by protein synthesis inhibitor. The loss of glucose regulation of glycogen synthase in perfused livers from diabetic rats was associated with a substantial reduction in synthase phosphatase activity. Treatment of diabetic rats with insulin alone resulted in total restoration of the glucose effect and synthase phosphatase activity, while simultaneous treatment with cycloheximide severely reduced the hormonal effect. Although treatment of normal rats with cycloheximide had no effect on glucose activation of synthase, it did result in severe depletion of liver glycogen, increased liver glycogen phosphorylase activity, and elevation of liver adenosine 3',5'-monophosphate (cyclic AMP), but without elevation of liver protein kinase activity. Simultaneous treatment of alloxan-diabetic rats with insulin and cycloheximide resulted in reduction of total liver glycogen, increased phosphorylase activity, a reduction in the ability of insulin to lower hepatic cyclic AMP, and a further reduction of protein kinase activity. In summary, the effect of insulin treatment of diabetic rats to restore glucose regulation of hepatic glycogen synthase probably involves synthesis of new protein, and the data remain consistent with the hypothesis that the defect may be due to a diabetes-related deficiency in a specific synthase phosphatase and/or alteration of the synthase molecule itself.


Production of cyclic AMP from extracellular ATP by intact LM cells. Intact LM cells, a line of cultured mouse fibroblasts, exhibited an adenylate cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) activity in the presence of exogenous [alpha-32P]ATP which was 20--30% of that observed with comparable preparations of lysed cells. The extent of NaF and prostaglandin E1 stimulation was comparable in intact cells and lysed cells. 96% of the added ATP and 92% of the cyclic AMP produced by intact cells could be isolated extracellularly in the incubation medium. Cellular integrity under assay conditions was monitored by trypan blue exclusion. These data suggest that LM cells contain an adenylate cyclase activity which is accessible to extracellular ATP.


The regulatory effects of growth rate and cyclic AMP levels on carbon catabolism and respiration in Escherichia coli K-12. Cyclic AMP levels in glucose and succinate-limited and ammonia-limited glucose-containing continuous cultures of Escherichia coli were measured at different bacterial growth rates. Intracellular cyclic AMP concentrations were fairly constant (about 5 micrometer) at all dilution rates used when glucose was limiting. In ammonia-limited glucose cultures the cyclic AMP content was much lower (about 0.3 micrometer). In succinate-limited cultures cyclic AMP levels fell from 2.7 to 0.8 micrometer as dilution rate increased from 0.05 to 0.4 h-1. The effects of cyclic AMP on respiratory and carbon catabolic enzyme levels were studied. There was no indication of a direct cyclic AMP involvement in the regulation of these cellular functions. It seems more likely that the variations in enzyme levels observed resulted from variation of the specific growth rate of cultures.


The heparan sulfates of Swiss mouse 3T3 cells. The effect of transformation. Three major pools of heparan sulfate have been isolated from cultures of Swiss mouse 3T3 and SV40-transformed 3T3 cells: cell-surface, medium, and intracellular heparan sulfates. The cell-surface heparan sulfate is a high molecular weight proteoglycan which is partially degraded by pronase. Before pronase treatment, it has a peak molecular weight (as estimated by gel filtration) of approx. 7.2 . 10(5) in contrast to only 2.4 . 10(5) after pronase treatment. The medium heparan sulfate appears to be similar in structure to the cell-surface heparan sulfate, since they coelute on Bio-Gel A-15m and DEAE-cellulose, and are both proteoglycans. In contrast, the intracellular heparan sulfate has a low molecular weight (6.0 . 10(3)) and has little if any attached protein. Both the medium and intracellular heparan sulfate exhibit the transformation-associated change in structure reported earlier for cell-surface heparan sulfate (Underhill, C.B. and Keller, J.M. )1975) Biochem. Biophys. Res. Commun. 63, 448--454). This transformation-associated change, detected by DEAE-cellulose chromatography is not the result of changes in either molecular weight or protein core. Cellulose acetate electrophoresis of the cell-surface heparan sulfate at pH 1 suggests that the transformation-associated change in structure is due to a difference in sulfate content. Both types of heparan sulfate are produced in mixed cultures of 3T3 and SV3T3 cells, indicating that neither serum factors in the culture medium nor secreted cell products are responsible for the transformation-associated change in heparan sulfate structure. The presented data are discussed with respect to the postulated role of heparan sulfate in cell social behavior.


[Aminoglycoside-3'-phosphotransferase I from aminoglycoside-polyresistant strain E. coli 182]. An aminoglycoside-3'-phosphotransferase I catalyzing phosphorylation of some aminoglycoside antibiotics with the 3'-hydroxyl group has been purified from the cells of aminoglycoside resistant strain E. coli 182 by competitive affinity chromatography on neomycin-Sepharose and gel-filtration on Sephadex G-100. The product of enzymatic phosphorylation of kanamycin A was isolated and identified as kanamycin-3'-phosphate by NMR, thin-layer chromatography and chemical characterization. The kinetic properties of the enzyme were studied. The pH-optimum was between 7,8--8,0; the [S]0.5 values for kanamycin, neomycin and paromomycin were 2.10(-5) M, the energy of activation was 15,9 kcal/mol. The bivalent cations were required for activity of the enzyme, Mg2+ was the most effecient. The relative aminoglycoside antibiotics containing no 3'-hydroxyl group were competitive inhibitors of the enzyme activity with Ki values close to [S]0.5.


[Substrate and inhibitory specificity of riboflavin kinase from Pichia guilliermondii yeast]. The interaction of purified riboflavin kinase (EC 2.7.1.26) from Pichia guilliermondii with 44 structural vitamin B2 analogues is studied. The presence of D-ribityl lateral chain in an analogue structure is found to be necessary for the substrate activity. The substitution of CH3 groups in the 7 and 8 positions of isoalloxazine ring in the riboflavin molecule for CF3, Cl, H, NH2 and N(CH3)2 resulted in the decrease of the analogue affinity to riboflavin kinase as compared with the natural substrate, vitamin B2. The most efficient enzyme inhibitors of analogues without substrate properties turned to be trifluoromethylisoalloxazines, containing 2'-hydroxyethyl group at N10. The elongation of D-ribityl lateral chain, the elimination of change of CH3-groups in the 7 and 8 positions for CF3- Cl-, COOH-substitutors resulted in the decrease of the inhibitory effect of flavines. Modifications in the structure of isoalloxazine ring, etherification of OH-groups in the lateral D-ribityl chain, and the introduction of volume substitutors (N-piperidyl, D-ribitylamine, hydroxyethylamine) prevented the interaction of the analogue with riboflavin kinase. Flavin nucleotides (FMN and FAD) did not affect the rate of vitamin B2 phosphorylation.


[Activities of enzymes from canine kidney plasma membranes under effects of polyene antibiotics in vivo and in vitro]. The effects of amphotericin B drug containing sodium deoxycholate (DOC) and those of DOC and nistatin on the activities of Na+, K+-ATPase and 5'-nucleotidase of canine kidney plasma membranes were studied. It was found that the activities of Na+, K+-ATPase and 5'-nucleotidase were markedly inhibited only after intravenous injection of amphotericin B, whereas the other agents tested caused no changes in the enzyme activities. Similar results were obtained in vitro. In the presence of amphotericin B the activity of Na+, K+-ATPase was noticeably inhibited already at the antibiotic concentration of 0,1 mkg per mg of membrane protein. It was found that the injection of amphotericin B, DOC and nistatin did not qualitatively or quantitatively affect the phospholipid composition of the plasma membranes. This is indicative of the lack of correlation between the enzyme activities and changes in the phospholipid composition of the plasma membranes under effects of amphotericin B. The pyrimidine derivative--amygluracyl--markedly removes the inhibiting effect of amphotericin B on the enzyme activity of plasma membranes.


[Mechanisms of rat liver thiamine pyrophosphokinase activation by magnesium ions]. The role of Mg2+ in the activation of thiamine pyrophosphokinase from rat liver was studied. The dependence of the thiamine pyrophosphokinase reaction rate on total and free ATP concentrations suggests that the role of the metal comes down to optimization of conditions of the active enzyme-substrate complex formation due to incorporation of the reactions of Mg2+ and ATP-4 consecutive acception, the affinity of the latter for the enzyme being higher than that of Mg-ATP-2. The kinetic parameters of the reaction were determined. In the absence of ATP-4 free Mg2+ ions were shown to compete with the Mg-ATP-2 complex (Ki = 18 . 10(-3) M). Thiamine pyrophosphokinase is also inhibited by free ATP-4 with Ki = 3 . 10(-3) M.


[Melting and reassociation of double-stranded RNA of the encephalomyocarditis virus]. The processes of melting and reassociation of double-stranded RNA in dimethylsulfoxide were studied. The addition of a small amount of LiCl results in great results in great reduction of Tm (temperature of melting), whereas the NaCl produces the opposite effect. It is suggested, that LiCl coordinates the molecules of H2O, reducing their activity, and consequently destabilises dsRNA. Mild conditions for melting and reassociation of RNA can be created. It was found that under optimal conditions for dsRNA melting, the degree of strand separation depends on the overall concentration of RNA, irrespective of the type of RNA added to the dsRNA preparation. Reassociation of dsRNA of EMC virus proceeds much faster than that of dsRNA of a related poliovirus. Addition of poly(C) to an annealing mixture slows down the rate of reassociation of EMC dsRNA, producing no effect on the poliovirus dsRNA reassociation. It is suggested that the presence of large poly(C) and poly(G) tracts in the complementary strands of the RNA determines its anomalous fast reassociation. Upon incubation of completely separated strands of EMC dsRNA in a water solution with high ionic strength partially double-stranded aggregates are formed. The formation of aggregates is prevented by addition of poly(A), which indicates that they are produced by "zippening" of a molecule starting with poly(A):poly(U) region. The significance of homopolymeric regions for stability of dsRNA of the EMC virus as well as their role in viral multiplication are discussed.


Hormonal regulation of three urea cycle enzymes in rat fetal liver. The purpose of such studies is to identify the hormones that act on the developmental formation of individual urea cycle enzymes, namely argininosuccinate synthetase, argininosuccinate lyase and arginase in the rat fetal liver. The development of argininosuccinate synthetase activity which is completely blocked by the suppression of glucocorticoids in the fetal liver is affected by neither glucagon nor thyroxine. The activity of argininosuccinate lyase which is never changed by the suppression or addition of glucocorticoids, is under the influences of glucagon and thyroxine at a late fetal period and the enzyme activity can be precociously induced in utero by injection of dibutyryl cyclic AMP to fetuses. Cortisol has been shown to precociously stimulate fetal liver arginase activity after an intraperitoneal injection, but the rise in activity at the late fetal period is not completely prevented by suppression of glucocorticoid and it seems that glucagon and thyroxine may also promote its developmental formation just before birth.


Effect of certain cerebral hemispheric diseases on dreaming. Dreaming may be altered by cerebral hemispheric disease. A woman who sustained a probable left posterior cerebral artery thrombosis, with right homonymous hemianopsia and alexia, had virtual cessation of dreaming for at least 9 months. Four individuals with temporal lobe epilepsy experienced recurrent painful (frightening) dreams, which in two patients showed features identical to seizures. Sleep recordings showed abnormalities in all four, including rhythmic temporal epileptiform activity during REM sleep. Lesions in parieto-occipital loci may interfere with production of the visual imagery required for dreaming (negative symptom in the Jacksonian sense) while epileptic activity in temporal loci may produce painful repetitive dream imagery (positive symptom).


Electrographic analysis of the sleep cycle in young depressed patients. Electrographic (EEG) patterns of nocturnal sleep were investigated in young psychiatric patients during unipolar depressive episodes. EEG-sleep data was recorded in 20 non-psychotic depressed patients all under 26 years old individually matched with a normal control group. All 20 subjects slept in the laboratory for 1-3 consecutive baseline nights from 12-8:00 a.m. During a subsequent extended condition 14 in each group were allowed to sleep ad-lib. Although the mean total time asleep on baseline nights was about the same between groups (greater than 7.1 hr), the depressives had a statistically significant reduction in REM time, increased transitions into stage 1, but most especially averaged: (a) less stage 4; and (b) more stage 1. Compared with the prior eight-hour night 27/28 subjects among both groups exhibited elevated time asleep during the extended condition, but the patients' mean total 10.3 hr sleep was significantly greater by 1.5 hr than the controls (X = 8.8 hr). Sleep exceeding 9 hr on the ad-lib night was a consistent phenomenon which occurred in significantly more (11/14) young depressed patients contrasted to 4/14 control subjects. These findings indicate that young persons with primary affective disorders do not exhibit nocturnal EEG disturbances of comparable severity to most older depressed patients such as reduced time asleep, increased wakefulness or lowered slow-wave (stages 3 and 4) sleep. Although no direct evidence of symptomatic 'hypersomnia' in these patients was provided, the present results demonstrated that some young persons with clinical depression have the capacity to sleep for sustained periods.


Recent data and significance of intracisternal tubular inclusions in human and animal cells. In 24 out of 60 patients with nasopharyngeal carcinoma (NPC) the cells were shown to contain a tubulo-reticular network in the dilated cisternae of the ergastoplasm which corresponds to the entity described previously as "intracisternal-tubular inclusion" (ITI). The significance of such inclusions is discussed in different pathological conditions and the literature is reviewed. This is the first report of such inclusions in nasopharyngeal carcinoma.


Investigation of the significance of a carbon and redox balance to the measurement of gaseous metabolism of Saccharomyces cerevisiae. A complete carbon and redox balance for Saccharomyces cerevisiae grown in batch culture with ethanol as the limiting carbon and energy source is reported. A novel method, which allowed the determination of carbon dioxide contained in the culture medium and biomass, is described and revealed amounts considerably in excess of what was expected from equilibrium data. Furthermore, elemental composition of the biomass was used to calculate the amount of oxygen required for biosynthetic reactions. When these corrections are applied to experimentally measured gas metabolism data, apparently anomalous results are shown to be consistent with the overall metabolism of bakers' yeast. These findings have wide implications to the quantitative study of the metabolism and energetics of facultative aerobes.


[State of vascular tone and several blood biochemical indices in experimental atherosclerosis and sensitization]. A study of the vascular tone of the posterior extremities in rabbits, of some blood lipid metabolism indices under conditions of experimental atherosclerosis and sensitization showed an increase in the vascular resistance in the areas investigated, and disturbance of lipid metabolism in two experimental groups of animals. The data obtained support the literature evidence that allergic component serves as one of the factors complicating the atherosclerosis course.


[Electron microscopic study of isolated proteoglycans]. Chemically isolated separate preparations of the non-aggregating protein-chondroitin-keratin sulphate (PCKS) fraction from the hyaline cartilage and hyaluronic acid (HUA) of the vitreous body and of the umbilicus were investigated by electron microscopy. PCKS and HUA in films without cytochrome c were present in the form of granules and differed by structural organization. The proteoglycans of the cytochrome c films were seen as finely-filamentous-cellular network, and were distinctly differentiated by their macromolecular organization. A mixture of both proteoglycans formed complexes as a result of a noncovalent interaction. Uranyl acetate ensured a good contrasting of proteoglycans, especially of PCKS, without cytochrome c.


[Effect of taurine on cyclic AMP and GMP levels in the hearts of rats exposed to stress]. Taurine produced no effect on the cyclic nucleotides level in the heart of intact rats but sharply inhibited the cAMP and cGMP level elevation in the rat heart occuring in stress. After atropine pretreatment of the animals no effect of taurine on the heart cGMP level was observed; its effect on the cAMP level was significantly inhibited against the background of partial beta-adrenoreceptors block. It is suggested that taurine is a nonspecific regulator of the myocardial cells sensitivity to the biologically active drugs.


[Reproduction of murine encephalomyocarditis virus in enucleated Krebs 2 ascitic carcinoma cells]. Enucleated cells of ascites carcinoma Krebs 2 (cytoplasts) were prepared. The cells were centrifugated for this purpose in a discontinuous Ficoll density gradient containing cytochalasine B. The cytoplasts formed turbid bands in Phycol solution at the density of 1.037--1.053 g/ml. The cytoplasts failed to synthesize RNA, but proteins were synthesized for several hours. Encephalomyocarditis virus replicated in the ascites carcinoma cytoplasts, but its yield was 10 to 100-fold lower as compared to that in the intact cells. Apparently the reduction of the virus yield was not due to the low efficiency of early stages of the virus-cytoplast interaction. Viral infection resulted in inhibition of the synthesis of cellular proteins in the cytoplasts as well as in the intact cells.


[Non-infiltrating intraductal carcinoma of the breast (author's transl)]. Non-infiltrating intraductal carcinoma may be considered a type of "carcinoma in situ" of the breast. In a review of 47 cases diagnosed and treated at Gustave-Roussy Institute between 1956--1972, it appears that the early symptoms of this rare type of breast carcinoma (it occurs only in 2.4% of all breast cancers) were a bloody discharge (38%) or Paget's disease of the nipple (11%). The histological examination was of the utmost importance in these cases due to the diagnostic uncertainties between benign hyperplastic lesions and authentic carcinomas as well as between infiltrating carcinomas and strictly intraductal carcinomas. Frozen section was only accurate in 30% of cases. The high frequency of multicentric foci (76%) contrasted with the absence of lymph node involvment (none of the 23 cases in which at least one node was excised, showed lymph node metastases). Treatment was only of ablation of the whole mammary gland, except in 6 patients who had a tumorectomy, two of whom also received radiotherapy. Local recurrence occurred in 4 patients, 3 of whom had only tumorectomy. The contralateral breast was affected in 2 cases. No patient under follow-up died of cancer within 5 years. The peculiar and highly favorable course of non-infiltrating intraductal carcinoma calls for an adequate therapy which could later be followed by a plastic reconstructive surgery should the patient wish to have this procedure.


[Differences in the fixation of antibodies in presence or absence of complement on SV40 transformed cells (author's transl)]. The fixation of antibodies directed against viral induced antigens was observed on SV40 transformed hamster fibroblasts. These cells were incubated for various periods of time with sera of tumor bearing hamster in the presence or absence of fresh guinea pig complement. The fixation of antibodies was revealed by an anti hamster immunoglobulins immune serum labeled with 125 iodine. Autoradiographic observations were made by optic and electron microscopy. The fixation of antibodies is quite different in the presence or in the absence of complement. Cells incubated with antibodies alone show a regular label on the plasma membrane with no decrease in the total cell population as compared to the controls. When the complement is added, one fraction of the cells is heavily labeled detached and progressively destroyed. Some of the cells (2/10) still attached on the flask are heavily labeled on the plasma membrane, the major part being nearly negative.


Visual receptive fields of units in the pulvinar of cebus monkey. Visually driven units, isolated in the ventrolateral group -- Pv1g (109) and in subnucleus Pmu (33) of the pulvinar of the cebus monkey, were studied in acute and chronic preparations under nitrous oxide N2O/O2 anesthesia during periods of EEG arousal. Taking into consideration the response properties to static or moving stimuli as well as the organization of the receptive fields, units isolated in the pulvinar were subdivided into 8 groups. Units displaying dynamic properties predominate over static ones. Static units were classified in 3 groups; of these, one showed uniform receptive fields; the remaining two groups, with non-uniform RFs, were further subdivided in terms of orientation selectivity. By testing for directional sensitivity, organization of the RFs and orientation selectivity, the dynamic units were divided in 5 groups. Among these there was a predominance of directional units, displaying uniform RFs and showing orientation selectivity. Although the receptive fields would extend into the ipsilateral hemifield (up to 10 degrees), their centers were always located in the contralateral visual hemifield. Binocularly driven units predominate in both static and dynamic categories.


Effects of intraventricular injection of 6-hydroxydopamine in the developing kitten. 1. On the sleepwaking cycles. Intraventricular 6-OHDA was injected in kittens at different stages of development, and the subsequent sleep polygram was analyzed, in order to determine the role of the catecholaminergic system in the ontogenesis of sleep regulations during the first and the second postnatal months. 6-OHDA, used with or without previous chlorimipramine treatment, led within a 10-day period to drastic reductions of the endogenous monoamines in the forebrain of all age groups. Although the neurotoxicity of 6-OHDA was almost constant among the different age groups, the effects on sleep depended on the age of the animals at the time of the injection. In the 5-week-old injected kittens, 6-OHDA affected PS according to an adult-like pattern. In the 3-week-old kittens, 6-OHDA alone (leading to both catecholamines and serotonin decreases) induced the same PS deficit as in the adult cat. In the 1- and 2-week-old kittens, neither 6-OHDA alone, nor 6-OHDA with previous chlorimipramine treatment, distrubed the sleep regulations. These data are compared to similar experiments performed in the adult cat. They are discussed in terms of sleep control ontogenesis. It is concluded that the catecholaminergic system plays no important role in the mechanisms of sleep regulation in the early postnatal period in the kitten, whereas its regulatory influence on PS is confirmed in the juvenile animal. The functional maturation of the catecholaminergic system in terms of sleep regulation is achieved between the third and the fifth week of postnatal life.


Pharmacological distinction between the excitatory junctional potential and the glutamate potential revealed by concanavalin A at the crayfish neuromuscular junction. The effect of concanavalin A(Con A) on desensitization of the glutamate receptor was investigated in the crayfish opener muscle. The depolarization of the crayfish muscle fiber caused by bath-applied L-glutamate was greatly augmented by Con A. The time course of the appearance of the augmentation was slow. Con A completely prevented the development of desensitization of the glutamate receptor. When L-glutamate was applied iontophoretically with a constant current pulse, a decline of the depolarization was seen during the course of the drug application which was presumably due to desensitization of the glutamate receptor. The glutamate potential was slightly increased by Con A, though the increase was transient. On the other hand, the amplitude of excitatory junctional potentials (EJPs) was not increased but decreased by addition of Con A. In normal saline, the amplitudes of both glutamate potentials and EJPs remarkably decreased because of desensitization of the glutamate receptor, but the decrease in amplitude of the glutamate potential was completely prevented by previous application of Con A. On the other hand, Con A had no influence upon the decrease in amplitude of EJPs. These results show that there is a pharmacological difference between the glutamate potential and EJPs.


Effects of age on brain oxidative metabolism in vivo. Non-invasive optical techniques were used to monitor the effects of increasing cerebral energy demand on metabolic capabilities and vascular reactivity in young and aged brain. Low level of electrical stimulation of the cortex, in both young (4--7 months) and aged (24--28 months) rat brain, were accompanied by transient oxidations of NADH and cytochrome oxidase (a,a3) as measured by microfluorometry and reflection spectrophotometry respectively. Stimulation sufficient to produce spreading cortical depression was accompanied by an oxidation of both NADH and cytochrome a,a3 in young brain together with an increase in local blood volume. There was either no change or a slight disoxygenation of hemoglobin. In aged brain, however, spreading depression was associated with an oxidation of NADH and a reduction of cytochrome a,a3 together with an increase in local blood volume and an oxygenation of hemoglobin. The present results indicate that the relationship between microcirculation and the terminal oxidase step of the respiratory chain is altered in aged brain when energy demand is high.


Developmental alteration of rat brain synaptic membranes. Reaction of glycoproteins with plant lectins. Synaptic membranes were isolated from the forebrains of rats of increasing postnatal ages. Developmentally related changes in the structure and concnetration of synaptic membrane glycoproteins were indicated by: (1) a 2--3 fold increase in glycoprotein sialic acid between 5 and 60 days; (2) a similar increase in the number of membrane receptors for the lectins concanavalin A and wheat germ agglutinin; (3) transient increases between 10 and 17 days in the receptors for lentil and castor bean lectins and (4) an age dependent stimulation of castor bean lectin binding by neuraminidase. Labelling of SDS polysacrylamide gels of synaptic membranes with [125I]concanavalin A or wheat germ agglutinin revealed specific, age dependent changes in the lectin binding properties of individual molecular weight classes of glycoproteins. Differences in the glycoproteins composition of synaptic junctional complexes isolated from 10 and 28-day-old brains were also revealed by lectin binding studies. The results indicate that the number and structure of oligosaccharides associated with synaptic membrane glycoproteins are under developmental regulation.


Pharmacological and immunological characterization of the Leu5 analogue of human beta-endorphin. The potencies of beta h-endorphin, Met (O)5-beta h-endorphin, and synthetic Leu5-beta h-endorphin have been compared in three bioassays of opioid activity, and in two radioimmunoassays. In all assays, a peptide isolated from hemodialysates from a psychotic patient behaved like Leu5-beta h-endorphin; it has been distinguished unambiguously from beta h-endorphin and Met(O)5-beta h-endorphin. Leu5-beta h-endorphin was one-fifth as potent as beta h-endorphin in guinea pig ileum myenteric plexus, but was only slightly less active in mouse vas deferens and in guinea pig brain opiate receptor binding assay. The low cross-reactivity of Leu5-beta h-endorphin relative to beta h-endorphin with an antiserum raised to beta-endorphin suggests that the preferred solution conformations of these peptides are different. In all bioassays beta h-endorphin was 2- to 3-fold less potent than beta c-endorphin.


Effect of cyclic nucleotides on high affinity uptake of L-glutamate and taurine in glial and neuroblastoma cells. Cultured glial cells (clones NN and I6) and neuroblastoma cells (clones M1 and M1NN) were used to study the effect of cyclic nucleotides on high affinity uptake of L-glutamate and taurine. Treatment of NN cells with a mixture of dBcAMP and BrcGMP for one or two days resulted in a more than 3-fold increase in the Vmax of L-glutamate high affinity uptake. Similar but smaller change was observed in a related glial clone (I6). Effects of cyclic nucleotide derivatives on high affinity uptake of L-glutamate by neuroblastoma cells (clones M1 and M1NN) or of taurine by both glial or neuroblastoma cells were either smaller or absent. Some differences were found in kinetic parameters of uptake of L-glutamate and taurine by original and re-isolated cellls, respectively. It is possible that these differences resulted from co-cultivation of glial and neuronal cells. However, since some values reported in this paper differ to some extent from those published previously, some other factors, such as subcloning, may have been responsible.


Depolarization-induced decreases in fluroescence intensity of gastro-intestinal quinacrine-binding nerves. Quinacrine, a fluorescent antimalarial acridine derivative, selectively binds to a population of nerve cell bodies and nerve fibers in Auerbach's plexus and elsewhere in the gut. This quinacrine-binding, as measured by fluorescence intensity, is reduced if the nervous elements are depolarized by high K+ (80--150 MM) or veratridine (5 x 10(-5) M) during quinacrine incubation. A reduction of quinacrine-content in nerve terminals is also seen when depolarization (veratridine) takes place for 2 min after quinacrine-incubation, indicating a release of already bound quinacrine. If terminals are depolarized (high K+ or veratridine) before quinacrine incubation, a reduction of quinacrine content is also seen. The depolarization-induced reduction of quinacrine-binding is blocked by Ca2+-removal and, in the case of veratridine by tetrodotoxin. Our findings suggest that quinacrine binds to a compound that is released by nervous activity. Binding mechanism remains to be elucidated. The possibility that quinacrine visualized purine-rich structures is discussed.


Taurine: stimulation of pineal N-acetyltransferase activity and melatonin production via a beta-adrenergic mechanism. Pineal glands convert [3H]tryptophan to [3H]N-acetylserotonin and [3H]melatonin in organ culture. Taurine treatment increases the rate of production of these compounds 40- and 25-fold respectively by stimulating the activity of N-acetyltransferase. This stimulation is blocked stereospecifically by L-propranolol, indicating that taurine is probably acting via beta-adrenergic receptors. Taurine is active in stimulating N-acetyltransferase activity in denervated glands, suggesting that it might interact directly with the beta-adrenergic receptor, and not by causing the release of norepinephrine from nerve terminals.


Immunohistochemical mapping of enkephalin containing cell bodies, fibers and nerve terminals in the brain stem of the rat. Enkephalin immunoreactive perikarya, fibers and nerve terminals, visualized by the indirect immunohistofluorescent method in colchicine-pretreated animals, are localized in many discrete regions of the rat brain stem. These specific immunohistofluorescent patterns are similar after staining with selective primary antisera directed against either methionine-enkephalin or leucine-enkephalin. Cell bodies are found in the substantia gelatinosa and interpolaris zones of the trigeminal nuclear complex, the nucleus of the solitary tract, in the vicinity of the nucleus raphé magnus, in the dorsal cochlear, medial vestibular, and paraolivary nuclei and, dorsal to this last region, in the parabrachial nuclei and the dorsal tegmental nucleus of Gudden, in the periaqueductal gray matter and interpeduncular nucleus and along the borders of the lateral lemniscus and medial geniculate. In some areas, such as the parabrachial region, nucleus of the solitary tract and substantia gelatinosa of the trigeminal nucleus, these perikarya are associated with densities of fibers and terminals. Other regions, such as the dorsal cochlear nucleus and the vicinity of the nucleus raphé magnus, contain cell bodies associated with low densities of processes and terminals. In still other nuclei, such as the nucleus of the facial nerve and the locus coeruleus, fiber and terminal densities without associated cell bodies are evident. Many of these enkephalin localizations can be rationalized on the basis of known actions of opiate drugs and the brain stem distribution of opiate receptors.


The postulated role of feeder swine in the perpetuation of the transmissible gastroenteritis virus. Clinical, immunofluorescence and histopathological observations were found to be an efficient approach for the confirmation of the diagnosis of transmissible gastroenteritis in feeder swine. Two cases are reported to exemplify how feeder swine exposed to points of concentration such as holding areas, sales barns and auctions can play an important role in the epizootiology of transmissible gastroenteritis. A third field case is reported as an example of an outbreak of transmissible gastroenteritis beginning in feeder swine and then spreading to baby pigs on the farm. All baby pigs died that were born during the acute phase of the outbreak in the feeder swine. Baby pigs born shortly after the clinical signs had abated in the herd, and from sows that had been exposed orally to virulent transmissible gastroenteritis virus and vaccinated with a commercial transmissible gastroenteritis vaccine ten days before farrowing, survived. This was explained by a combination of a decrease in the amount of virus shed in the environment and the immunity induced in the sows. These observations of field outbreaks of transmissible gastroenteritis combined with recently reported experimental studies lend strong support to the hypothesis of a reservoir for transmissible gastroenteritis virus in feeder pigs. This reservoir would be based principally on the transmission of the virus on a continuous basis from the feces of recently infected pigs to susceptible pigs. Clinical signs of transmissible gastroenteritis in such pigs are difficult to recognize or absent and this contributes to the importance of the reservoir in the field.


The use of immunofluorescence techniques for the laboratory diagnosis of transmissible gastroenteritis of swine. Over a four year period, 74 of 250 field outbreaks of enteric disease (30%) and 110 of 440 swine (25%) were positive for transmissible gastroenteritis by immunofluorescence procedures. Of 141 swine from herds positive for transmissible gastroenteritis 110 (78%) were positive by fluorescent antibody techniques. The fastest, easiest to perform and most effective procedure was the examination of frozen sections of the jejunum from acutely ill animals by the fluorescent antibody tissue section technique. Only two herds were found to be positive by the fluorescent antibody tissue culture technique which were negative by fluorescent antibody tissue section technique. A considerable number of outbreaks, 21 of 74 (28%), of transmissible gastroenteritis were detected by immunofluorescence in swine over two weeks of age. The majority of outbreaks of transmissible gastroenteritis, 50 of 74 (68%), occurred in Missouri during the months of January through April and 63 of 74 (85%) during the months of December through May. The recurrence of the disease in a number of counties over a four-year period suggest the possibility of endemic foci.


Mechanisms of recovery from Herpesvirus infections -a review. A variety of specific immunological mechanisms have been shown to be effective at neutralizing herpesviruses or destroying herpesvirus infected cells. These include both humoral and cell mediated immune responses or combinations thereof. Thus, it is genarlly accepted that humoral immunity is probably responsible for preventing reinfection whereas cellular immunity, mediated by T lymphocytes or by the interaction of antibody and Fc receptor bearing cells, is more important in recovery from infections. In addition to these specific responses to herpesvirus infection, a number of nonspecific cellular and humoral components have been shown to inhibit the progression of virus replication and therefore, have been implicated in assisting the host in the recovery process. The various interactions and counteractions between the various nonspecific and specific components of the immune response are discussed with respect to their role in recovery from both primary and recurrent disease as well as how they may eventually be manipulated so as to control herpesvirus recrudescent disease.


A simple, rapid syncytial-inhibition test for antibodies to bovine leukemia virus. Cocultivation of equal numbers of cells from a fetal lamb kidney line infected with bovine leukemia virus and African green monkey (Vero) cells results in the rapid production of syncytia. The effect was blocked or inhibited by serum containing antibodies to bovine leukemia virus. A serological test based on syncytial inhibition was compared to the agar gel immunodiffusion test and the modified direct complement fixation test for the detection of bovine leukemia virus antibodies in sera from leukosis-free cattle, cases of adult enzootic bovine lymphosarcoma and cattle from herds in contact with enzootic lymphosarcoma. The results showed the syncytial inhibition test to react positively with sera from all cases of adult enzootic lymphosarcoma, but to be much less sensitive than the other tests in detecting bovine leukemia virus antibodies in sera of exposed animals.