Role of coenzyme Q in the mitochondrial respiratory chain. Reconstitution of activity in coenzyme Q deficient mutants of yeast. The reduction of cytochrome c by the reduced form of the 6-decyl analogue of coenzyme Q follows first-order kinetics with respect to cytochrome c and increases in a linear manner with added mitochondrial protein. The activity is completely sensitive to antimycin A in whole cell extracts of yeast as well as in isolated mitochondria and fractionates with markers for the mitochondrial electron-transport chain. The presence of both cytochrome b and c1 in an approximately 2:1 ratio appears essential for enzymatic activity. Reduced coenzyme Q-cytochrome c reductase obeys Michaelis-Menten kinetics when assayed in mitochondria obtained from a yeast strain lacking coenzyme Q. Both reduced nitotinamide adenine dinucleotide and succinate:cytochrome c reductase activities were not detectable in six coenzyme Q deficient strains tested, but were restored after addition of the oxidized form of the coenzyme Q analogue. No marked difference in the concentration of the analogue required to restore the two activities was observed.


Pulse fluorimetry study of octopine dehydrogenase-reduced nicotinamide adenine dinucleotide complexes. We measured the transient fluorescence of NADH bound to octopine dehydrogenase in the binary octopine dehydrogenase-NADH complex and in the ternary complexes containing D-octopine, L-allooctopine, L-arginine, D-arginine, or 5-guanidinovaleric acid. The fluorescence decay in all these complexes is biexponential. This is explained by the presence of several conformations of the single NADH binding site. In addition, transietn anisotropy measurements show that the nicotinamide moiety is rigidly bound to the enzyme.


Effects of neuraminidase on lectin binding by wild-type and ricin-resistant strains of hamster fibroblasts. The nature of cell surface receptors for ricin on wild-type and ricin-resistant variants of baby hamster kidney fibroblasts has been studied. Neuraminidase stimulated ricin binding threefold by wild-type cells, and increased their susceptibility to ricin toxicity as measured by inhibition of [3H]leucine uptake (LD30 fell from 5.0 to 0.5 microgram/mL). Basal ricin binding by ricin-resistant variants (10-300% that of wild type) was also stimulated (2- to 17-fold) by neuraminidase in all seven clonal strains examined; susceptibility to ricin was greatly increased by neuraminidase in these variants. Neuraminidase did not affect the binding of concanavalin A by wild type or a ricin-resistant variant, but decreased the binding of wheat-germ agglutinin by 90% in both cell types. The trivial binding of peanut agglutinin by wild type and a ricin-resistant variant was markedly enhanced (14- to 22-fold) by neuraminidase. Neither collagenase (50 U/mL) nor Pronase (0.0001%) affected ricin binding by wild type or a ricin-resistant variant. These data suggest the existence of "exposed" and "cryptic" oligosaccharide receptors for ricin on the cell membrane glycoproteins of baby hamster kidney fibroblasts. The cryptic ricin receptors probably include at least the sequence D-galactosyl-beta-(1 replaced by 3)-N-acetylhexosamine substituted by sialic acid residues. Exposed and cryptic ricin receptors appear to be different and under separate genetic control.


Oxidative titrations of reduced cytochrome aa3: influence of cytochrome c and carbon monoxide on the midpoint potential values. Oxidative titrations were performed on the electrostatic complex formed between cytochrome c and cytochrome aa3 at low ionic strength. Midpoint potentials of the redox centers in the proteins in 1:1 and 2:1 complexes were compared with those in mixtures of the cytochromes at high ionic strength. Computer simulations of all titrations yielded midpoint potentials for the components of cytochrome aa3 which were consistent with literature values for isolated cytochrome aa3 or mixture of cytochromes c and aa3. However, the unequal heme extinction coefficients observed previously (Schroedl, N.A., and Hartzell, C.R. (1977), Biochemistry 16, 1327) during oxidative titrations of cytochrome aa3 became equal in magnitude under these experimental conditions. The binding of cytochrome c to cytochrome aa3 changed the midpoint potentials of cytochrome aa3 by 15-20 mV, while the midpoint potentials for cytochrome c were altered by 50-60 mV. Careful analysis of these titrations including computer simulation revealed that cytochrome c was able to bind to cytochrome aa3 only after cytochrome aL2+ had become oxidized. When bound to cytochrome aa3, the midpoint potential of cytochrome c was 210 7V. Titrations performed under a carbon monoxide atmosphere revealed cytochrome aa3 midpoint potentials unchanged from reported values. Cytochrome c again exhibited a midpoint potential of 210 mV after binding to cytochrome aa3.


Use of specific lysine modifications to locate the reaction site of cytochrome c with cytochrome oxidase. The reaction of cytochrome c with trifluoromethylphenyl isocyanate was carried out under conditions which led to the modification of a small number of the 19 lysines. Extensive ion-exchange chromatography was used to separate and purify six different derivatives, each modified at a single lysine residue, lysines 8, 13, 27, 72, 79, and 100, respectively. The only modifications which affected the activity of cytochrome c with cytochrome oxidase (EC 1.9.3.1) were those of lysines immediately surrounding the heme crevice, lysines 13, 27, 72, and 79, and also lysine 8 at the top of the heme crevice. In each case, the modified cytochrome c had the same maximum velocity as that of native cytochrome c, but an increased Michaelis constant for high affinity phase of the reaction. This supports the hypothesis that the cytochrome oxidase reaction site is located in the heme crevice region, and the highly conserved lysine residues surrounding the heme crevice are important in the binding.


3'-Phosphatase activity in T4 polynucleotide kinase. The purification of T4 polynucleotide kinase results in the copurification of an activity which will specifically remove the 3'-terminal phosphate from a variety of deoxyribonucleotides and ribonucleotides in the absence of ATP. This phosphatase activity requires magnesium, has a pH optiumum of 6.0, and is more active with deoxyribonucleotides than ribonucleotides. T4 polynucleotide kinase and the 3'-phosphatase activity copurify by gradient elution column chromatography on DEAE-cellulose, phosphocellulose, and hydroxylapatite. The two activities are included in and comigrate on Sephadex G-200. Polyacrylamide gel electrophoresis at PH 9.2 results in conigration of the two activities together with the major protein band. The two activities respond in parallel to heat inactivation at 35 degrees C and ATP, a substrate for the kinase only, protects both activities from heat inactivation. It is therefore suggested that the two activities are functions of the same protein molecule.


The anaerobic oxidation of dihydroorotate by Escherichia coli K-12. The oxidation of dihydroorotate under anaerobic conditions has been examined using various mutant strains of Escherichia coli K-12. This oxidation in cells grown anaerobically in a glucose minimal medium is linked via menaquinone to the fumarate reductase enzyme coded for by the frd gene and is independent of the cytochromes. The same dihydroorotate dehydrogenase protein functions in both the anaerobic and aerobic oxidation of dihydroorotate. Ferricyanide can act as an artificial electron acceptor for dihydroorotate dehydrogenase and the dihydroorotate-menaquinone-ferricyanide reductase activity can be solubilised by 2 M guanidine-HCl with little loss of activity.


The number of Fe atoms in the iron-sulphur centers of the respiratory chain. 1. From the 57Fe hyperfine interaction in EPR spectra of reduced submitochondrial particles from the yeast Candida utilis, grown with 57Fe, it is concluded that all Fe-S centers in these particles detectable in spectra at 35-80 K are [2Fe-2S]2-(2-; 3-) centers. These are the centers 1 of NADH and succinate dehydrogenase, the Rieske Fe-S center and possibly center 2 of succinate dehydrogenase. 2. The signals of the reduced particles detectable only at temperatures below 20 K are [4Fe-4S]2-(2-; 3-) clusters. These are the centers 2,3 and 4 of NADH dehydrogenase. 3. EPR spectra of the [2Fe-2S]3- centers of Complex I and II, but not that of Complex III, display a great inequality of the Fe nuclei in the effective hyperfine interaction in the x-y direction.


The structural transitions of erythrocyte membranes induced by cyclic AMP. The generalized structural transitions of erythrocyte membranes induced by cyclic AMP were registered by ESR, fluorescence, freeze-fracture and circular dichroism methods. Two transitions different in nature were revealed. One, which arises at 10-(11)--10-(10) M cyclic AMP, is cooperative and may be considered as a consequence of interaction of cyclic AMP with a receptor. It was calculated that a structural rearrangement in one erythrocyte ghost is induced by three cyclic AMP molecules. As a result of it the membranes are "loosened". The other transition arises at 10-(10)--10-(8) M cyclic AMP and depends on the activity of the protein kinase system. This transition was shown to be non-cooperative and due to phosphorylation of membranous proteins. During this rearrangement the membranes are "stiffened". Both transitions were demonstrated to relate to the membrane integrity.


Regulation of DNA chain elongation in SV3T3 cells in relation to growth rate. Regulation of DNA synthesis was investigated in SV40 transformed 3T3 cells exhibiting variable growth rates and residence times in S phase when cultured in the presence of different serum concentrations. Pulse-labeled DNA was chased into large molecular weight material in vivo much more slowly in slowly growing cells than in cells growing at the normal rate. Consistent with this, the joining of short (less than 10 S) chains to form long (greater than 10 S) chains by whole cell lysate system in vitro was greatly impaired in slowly growing cells compared to controls. Thus the lengthening of S phase in SV3T3 cells growing slowly in low serum is reflected in a reduced rate of DNA chain elongation. The presence of cycloheximide during chase in vivo reduced the rate of conversion of pulse-labeled molecules into large molecular weight DNA in both slowly growing and normally growing cells.


Effect of SV40 infection on [3H]uridine incorporation. More [3H]uridine was incorporated into RNA of SV40-infected than into uninfected cells 31 h after infection. When the specific activity of the uridine triphosphate pools in infected and uninfected cells was equated by the addition of appropriate amounts of exogenous unlabelled uridine, no difference in the rate of [3H]uridine incorporation into RNA was observed. Although no difference in [3H]uridine entry or phosphorylation was demonstrable, the apparently smaller pools of endogenous RNA precursors in infected cells resulted in less isotope dilution and thus to synthesis of uridine triphosphate and RNA of higher specific activity.


Synthesis and secretion of albumin by a synchronized rat hepatoma cell line. The rat hepatoma cell H4-12 which synthesizes and secretes albumin was synchronized by growth in isoleucine-deficient medium followed by a second block with excess thymidine. Albumin synthesis and secretion was measured in the synchronized cells at different time intervals representative of early S, late S, G2, mitosis, early G1 and late G1 phases of the cell cycle. Maximal albumin synthesis occurred during G1 although significant synthesis also occurred during the other cell cyle phases. Most (75--80%) of the radioactive albumin produced during a 15 min pulse incubation with L-[4,5-3H] leucine was found in the microsomal cell fraction and this nascent albumin was secreted into the incubation medium during a 160 min chase period. Fifty percent of the nascent albumin was secreted by 50--55 min and this pattern of secretion did not change during the cell cycle. These data indicate that albumin synthesis occurs throughout the cell cycle but that it is preferred during G1. The rate of intracellular transport and secretion of albumin does not vary during the different phase of the cell cycle.


Different responses to some stimulators of prolyl hydroxylase activities in various rat organs. Prolyl hydroxylase (proline,2-oxoglutarate dioxygenase, EC 1.14.11.2) of soluble fraction (105 000 X g supernatant) of rat granulation tissues was markedly enhanced by addition of nucleoside triphosphates to the assay medium. But the stimulatory activities of nucleoside triphosphates were very different in fractions derived from tissues of rat. In skin, lung or whole fetal tissues other than granuloma, GTP enhanced the enzymatic activity by 3-4 fold. On the other hand, in kidney, liver and spleen tissues it brought about no enhancement. The same results were obtained even if ATP regenerating system was added in the assay medium. The stimulatory effect of nucleoside triphosphates was not seen with the soluble fraction of liver, but it appeared with the enzyme fraction purified by affinity column chromatography. The same phenomenon was observed by addition of bovine serum albumin instead of nucleoside triphosphates as stimulator. We discuss the possible reasons as to why the responses of the enzyme to stimulators were quite different among various tissues.


Cyclic 3':5'-nucleotide phosphodiesterase determined in various human tissues by DEAE-cellulose chromatography. Tissue extracts from human heart, lung, liver, kidney, skeletal muscle and cerebrum displayed at least 3 distinct cyclic 3':5'-nucleotide phosphodieterase (EC 3.1.4.17) activity peaks (FI, FII, FIII) on DEAE-cellulose chromatography and various properties of these forms were compared in each tissue. FI eluted at about 0.08 M sodium acetate, hydrolyzed cyclic GMP more rapidly than it did cyclic AMP, and cyclic GMP hydrolysis by FI in most tissues was enhanced by a protein activator in the presence of CaCl2. As only high concentrations of cyclic AMP inhibited cyclic GMP hydrolytic activity of FI, the enzyme probably has a low affinity for cyclic AMP. FII eluted at about 0.2 M sodium acetate, hydrolyzed both nucleotides at equal rates, and substrate affinities were relatively low. Cyclic GMP hydrolysis by FII was also stimulated by addition of a protein activator in the presence of CaCl2 and cyclic AMP hydrolysis in this fraction was accelerated by a micromolar fraction of cyclic GMP. FII eluted at about 0.35 M hydrolyzed cyclic AMP preferentially and was insensitive to protein activator. These two cyclic nucleotides act as mutual inhibitors of the hydrolysis in this fraction. Ratio of the cyclic GMP to cyclic AMP hydrolysis was in the order FI, FII, FIII. Four activity peaks were eluted from the cerebral extract and enzymes from this tissue exhibited much the same properties as observed in the other tissues examined herein.


Activation of low molecular weight acid phosphatase from bovine brain by purines and glycerol. Low molecular weight acid phosphatase (orthophosphoric monoester phosphophydrolase (acid optimum), EC 3.1.3.2) from bovine brain is activated up to 4-fold by guanosine, guanine, adenine, adenosine, and 6-ethylmercapto-purine. Several pyrimidines and other purines were tested and did not show any activation effect. The rate enhancement induced by purines is uncompetitive and not caused by transphosphorylation to the activator. Using transphosphorylation to glycerol as a probe, it is proposed that the activator binds to one of the phosphorylated intermediates in the reaction pathway. These findings are discussed in terms of the catalytic mechanism of low molecular weight acid phosphatase.


Evidence for mammalian collagenases as zinc ion metalloenzymes. Collagenases (EC 3.4.24.3) from human skin, rat skin and rat uterus were inhibited by the chelating agents EDTA, 1,10-phenanthroline and tetraethylene pentamine in the presence of excess Ca2+, suggesting that a second metal ion participates in the activity of the enzyme. Collagenase inhibition by 1,10-phenanthroline could be both prevented and reversed by a number of transition metal ions, specifically Zn2+, Co2+, Fe2+ and Cu2+. However, Zn2+ is effective in five-fold lower molar concentrations (1-10(-4) M) than the other ions. Furthermore, Zn2+ was the only ion tested able to prevent and reverse the inhibition of collagenase by EDTA in the presence of excess Ca2+. Atomic absorption analysis of purified collagenase for Zn2+ showed that Zn2+ was present in the enzyme preparations, and that the metal co-purifies with collagenase during column chromatography.


Protocatechuate 3,4-dioxygenase. Inhibitor studies and mechanistic implications. Protocatechuate 3,4-dioxygenase (EC 1.13.11.3) from Pseudomonas aeruginosa catalyzes the cleavage of 3,4-dihydroxybenzoate (protocatechuate) into beta-carboxy-cis,cis-muconate. The inhibition constants, Ki, of a series of substrate analogues were measured in order to assess the relative importance of the various functional groups on the substrate. Though important for binding, the carboxylate group is not essential for activity. Compounds with para hydroxy groups are better inhibitors than their meta isomers. Our studies of the enzyme-inhibitor complexes indicate that the 4-OH group of the substrate binds to the active-site iron. Taken together, Mössbauer, EPR, and kinetic data suggest a mechanism where substrate reaction with oxygen is preceded by metal activation of substrate.


Concentration of lipoproteins containing apolipoprotein B in human peripheral lymph. The concentration of apolipoprotein B (apoB) in human serum and peripheral lymph was measured by quantitative immunoelectrophoresis with anti-serum to human low-density lipoprotein. In four normal and six hyperlipidaemic subjects, total lymph apob/ml was 5-10% of total serum apoB/ml in the same subject. These ratios were equivalent to lymph apob concentrations of 60-120 microgram/ml. When the assays were carried out under conditions in which unmasking of immunoreactive sites on lymph and serum apoB was assumed to be maximal (delipidation with Nonidet P40), the lymph/serum apoB concentration ratios in three normal subjects were similar to those obtained with untreated lymph and serum.


Theta-toxin of Clostridium perfringens. I. Purification and some properties. Theta-Toxin, an oxygen-labile hemolysin produced by Clostridium perfringens, was purified 3300 fold from culture filtrate by successive chromatography on DEAE-Sephadex A-50 and Sephadex G-150. The purified toxin gave two distinct bands in disc electrophoresis, while the same material, after mild reduction with dithiothreitol, yielded a single band, indicating that the purified theta-toxin contained, as well as a reduced, active form, an oxidized, inactive form of toxin. These two forms of the toxin had a similar, if not identical molecular size. The purified preparation gave a single band in a sodium dodecyl sulfate polyacrylamide gel electrophoresis and formed a single precipitin line with National Standard gas gangrene (C. perfringens) antitoxin. By sodium dodecyl sulfate polyacrylamide gel electrophoresis, the molecular weight of theta-toxin was estimated to be 51 000, the value being in exact accordance with that obtained by amino acid analysis. The amino acid composition of theta-toxin was very close to that of cereolysin, an oxygen-labile hemolysin produced by Bacillus cereus. The amino-terminal residue of theta-toxin was lysine as determined by the Dansyl method.


Thermodynamic characterization of partially denatured states in the denaturation process of bovine alpha-lactalbumin by inorganic denaturants. In an attempt to understand the specific effect of inorganic protein denaturants, lithium cation and perchlorate anion, upon the molecular conformation of bovine alpha-lactalbumin and to characterize the denatured states of the protein and the denaturation processes, themodynamic studies on the reversible unfolding of the protein in the presence of lithium chloride, lithium perchlorate and sodium perchlorate were made by means of circular dichroism and ultraviolet absorption measurements. The denaturation reaction caused by lithium chloride was found to take place in a three-state type, while that caused by the two perchlorates in a two-state type. The latter produces the same denatured state as the acid does on the protein, the state where the helical structures remain unchanged. The former produces two kinds of the denatured state, one being a less unfolded state than the acid denatured one and the other a fully unfolded state which is identical with the finally denatured state induced by organic denaturants such as guanidinium chloride, guanidinium thiocyanate and urea.


Haemoglobin North Shore, beta134 Val replaced by Glu, a new unstable haemoglobin. A new mildly unstable haemoglobin, Hb North Shore (beta134 Val replaced by Glu) is described. It was found in a 4th generation Australian of Anglo-Celtic origin with a mild microcytic anaemia. The charge change involved is partially obscured as evidenced by the electrophoretic mobility of the native haemoglobin. The structural studies illustrate the usefulness of Staphylococcus aureus strain V8 protease as a specific cleavage enzyme at glutamyl residues.


Mössbauer studies of cytochrome c' from Rhodospirillum rubrum. Cytochrome c' from Rhodospirillum rubrum has been investigated in the ferric form with Mössbauer and EPR spectroscopy. In the pH range from 6 to 9.5, three species are observed which belong to two pH-dependent equilibria with pK values near 6 and 8.5. The pK = 6 transition is resolved only with high-field Mössbauer spectroscopy. For the three species we have determined the zero-field splitting parameters and the hyperfine coupling constants. The data were fitted to a spin Hamiltonian which takes into account a weak mixing of excited S = 3/2 states into the sextet ground manifold. The low temperature spectra clearly show that the quadruple coupling constant deltaEQ is positive for ferricytochrome c' and thus in accord with all other high-spin ferric heme proteins.


Phosphodiesterase from cultured tobacco cells. Physical and chemical properties. Phosphodiesterase isolated from suspension cultures of tobacco cells showed high affinity for concanavalin A-Sepharose and gave single superimposed bands of protein and carbohydrates on disc gel electrophoresis, suggesting that it is a glycoprotein. It contains 14% carbohydrate by weight, and has relatively high contents of basic and aromatic amino acids. Its isoelectric point is at pH 8.8, and the molecular weight of its subunits was estimated as 72 000 from a plot of the retardation coefficient on sodium dodecyl sulfate gel electrophoresis versus the molecular weight. The enzyme was catalytically active in an immobilized state on a concanavalin A-Sepharose column.


Effects of human chorionic gonadotropin and N6,O2'-dibutyryladenosine 3' ,5'-monophosphate on phosphofructokinase activity in isolated rat testis. Phosphofructokinase activity in rat testis is elevated by treatment in vitro with human chorionic godadotropin or N6,O2'-dibutyryladenosine 3' ,5'-monophosphate. Puromycin or actinomycin D suppresses the effect of the gonadotropin but does not affect the enzyme increase induced by the cyclic nucleotide. The possible cause for the divergent action of the two stimulatory agents are discussed.


[Formation of free radicals during photooxidation of bacterioviridin in its monomeric and aggregated state]. ESR study of photooxidation of bacterioviridin in polar and non-polar organic solvents containing monomeric and aggregated forms of the pigment was carried out. Light-indiced formation of free radicals was shown to proceed during photooxidation of bacterioviridin with oxygen. These free radicals were similar by the ESR-signal parameters (G-factor value and the line width) to the cation-radicals initiated during photooxidation of the pigment with p-benzoquinone and at the same time differing in kinetics and temperature relationship. A small yield of radical products in the presence of oxygen is observed in non-polar solvents where bacterioviridin forms the aggregated forms with the absorption maxima of 680 and 720 nm. The study of the kinetics of photoformation of radicals within the temperature range +20-- --110 degrees allowed to reveal different stages corresponding to the oxidation of the long wave and short wave forms of bacterioviridin, photooxidation of the long wave aggregated form being the first stage of photoprocess. It is concluded that aggregations contributs to the stabilization of the primary reversible products of bacterioviridin photooxidation having the nature of free radicals.


[Solubility of adenosine in concentrated salt solutions]. The solubility of adenosine has been measured in concentrated neutral salt solutions of NaClO4 (salt-destabilizer) and Na2SO4 (salt-stabilizer) as a function of salt concentration and temperature. The thermodynamic functions of adenosine transfer from water to salt solutions have been estimated. The values of delta Ftr, delta Htr, delta Str adenosine transfer in NaClO4 solutions are negative, in Na2SO4--positive. The results are considered in connection with the mechanism of high concentrations of salt action on DNA in the water solutions.


[Study of electron transport in the photosynthetic systems of higher plants by the EPR method. V. Interaction of paramagnetic prob I(12,3) with the membranes of bean chloroplasts]. Lipid-soluble paramagnetic proble I(12,3) was subjected to partially reversible photoinduced redox transformations while interacting with the electron-transport chains of chloroplasts. Incorporation of I(12,3) into the chloroplast membrane does not affect the kinetic characteristics of the EPR I signal (from P700+). The parameters of the EPR spectrum from I(12,3) are not changed under illumination, however, they are sensitive to temperature and different additions (Mg2+, ATP, atebrin).


[Different cooperativity of some oxidoreductases to specialized and nonspecialized regulators]. The activation of 2 different mouse liver enzymes: cytozolic disulfide reductase (DSR) and mitochondrial NAD-isocitrate dehydrogenase (ICDH), by catecholamines and especially by 3',5'-AMP is characterized by negative cooperativity; substrate (both enzymes), protamine and EDTA (DSR) produce the positive cooperativity type of activation; DSR activation by isopropyl noradrenaline and serotonine is characterized by hyperbolic kinetics. Consequently, one and the same enzyme can combine positive cooperativity to non-specialized regulators (substrate, protamine, EDTA) with negative cooperativity to specialized regulators (3',5'-AMP, catecholamines). The systems, switching on by catecholamines and 3',5'-AMP, are oligomeric, and the degree and even the type of cooperativity can modify depending on the kind of catecholamine. The negative cooperativity is revealed in literature for many effects of catecholamines and 3',5'-AMP. Probably, it guarantees the broad range of regulations. Dose effect curves for 3',5'-AMP, catecholamines and other hormones should be analyzed on the basis of allosteric protein kinetics. A simple nomogram is given to estimate nH less than 1.


[Preparative isolation and some properties of SV40 T-antigen]. SV40 T-antigen was isolated from hamster tumors and purified about 1600-fold by the procedure including successive ammonium sulfate precipitation, chromatography on DEAE-cellulose, preparative polyacrylamide gel electrophoresis and elimination of the bulk of contaminating cell proteins by the interaction with antibodies to the tissues of normal hamsters. The resulting preparation was not quite homogenous being contaminated with some of cell proteins left. T-antigen in the tumor extract was revealed at least in three distinct forms with molecular weight of 100 000, 200 000, and 400 000. It is proposed that these forms correspond to mono-, di-, and tetramers of the basal protein of T-antigen, although the alternative explanation, the existence of complex of T-antigen with cell proteins, cannot be ruled out.


[Role of superoxide anion-radicals and superoxide dismutase in the lipid photoperoxidation reactions of isolated chloroplasts]. Exogenous superoxide dismutase (SOD, erythrocuprein) inhibits the chlorophyll bleaching and photoperoxidation of polyunsaturated lipids in pea isolated chloroplasts. As SOD did not produce this effect in tris-HCl buffer, it is suggested that peroxidation is mediated by the OH radicals which may appear in illuminated chloroplasts with the participation of superoxide radicals and hydrogen peroxide. The physiological role of SOD is discussed in relation to the stability of cells photosynthesizing organisms towards light and oxygen effect.


[Effect of NAD recirculation on the mechanism of ATP stabilization in cytoplasm. Mathematical models]. A mathematical model of the glycolytic system with the cytoplasmic coenzymes NAD+ and NADH as essential variables is proposed. It has been shown that any increase in the steady-state concentration of NADH will reduce the range of activity of the "generalized" ATPase, wherein the level of ATP is stabilized. Such a reduction in the range of ATP stabilization may be caused by an increasing rate of the pyruvate loss into non-glycolytic pathways, in particular, into mitochondria. This effect may be compensated by increasing oxidation of NADH by the dehydrogenases of H+-transferring cytosol-mitochondrial shuttles (malate-aspartate or alpha-glycerophosphate). The properties of the complete model were compared with those of its simplified version, which takes account only of the phosphotransferase reactions of glycolysis. The effects of various factors, which do not alter the level of NADH in the system, may be studied within the scope of the simplified model.


[Factors influencing formation of dinitrosyl complexes of non-heme iron in the organs of animals in vivo]. The formation of paramagnetic non-haem iron dinitrozyl complexes (2,03 complexes) in mouse liver has been detected, when these animals were exposed to a diet with high content of nitrite or nitrite and iron salt. These complexes appeared after 3-5 days and their content was kept at the same level during a month. The 2,03 complexes content in mouse liver was 3 times higher for mice which were exposed to the diet with nitrite and iron salt than for those on the diet only with nitrite. While the liver preparations of experimental mice were treated with NO in vitro the 2,03 complexes content increased in the liver and reached the same level for all experimental mice. It has been supposed that the 2.03 complexes formation was limited in mouse liver in vivo, both by NO production in the liver and by the content of the more labile form of non-haem iron, which was primarily incorporated into the complexes.


[Polyphosphate kinase activity in yeast vacuoles]. The enzyme polyphosphate kinase (ATP: Polyphosphate phosphotransferase EC 2.7.4.1) relating to the class of transferases was detected in the vacuoles of Saccharomyces carlsbergensis yeast. The direct ATP: Polyphosphate phosphotransferase reaction resulting in the synthesis of polyphosphates from ATP was shown to occur mainly in vacuoles. The localization of the reverse polyphosphate: ADP phosphatransferase reaction was not established in any of the subcellular yeast fractions studied. The activity of the direct reaction in the yeast protoplasts makes up about 1% of the reverse one, but in vacuoles it is significantly higher and makes up to 19%. Under activation of biochemical processes involved in the production of cell wall components by protoplasts, vacuolar polyphosphates work mainly in the direction of ATP synthesis at the expense of polyphosphates accumulated in vacuoles.


[Use of spin labels for studying the association of adenosine, adenosine-5'-mono-, di- and triphosphate in aqueous solutions]. Possibility of spin label studies as a method of investigation of different molecular associations is considered with adenosine, adenosine-5'-mono, di- and triphosphate as examples. The proposed method is based on dependence of the parameters of spin-labelled derivatives ESR-spectra on concentration of corresponding unlabelled compounds. Being highly sensitive the method takes the advantage of obtaining not only thermodynamic characteristics of association but dynamic parameters of molecular mobility of the associates as well. It is shown by this method that base-phosphate interaction participates in nucleotide association, together with the stacking interaction.


[Activities of some yeast flavogenic enzymes in situ]. Effects of digitonin, dimethylsulfoxide and protamine sulfate on yeast Pichia guilliermondii were studied in order to produce cells with increased permeability and possessing the GTP-cyclohydrolase, riboflavinsynthetase and riboflavinkinase activities. The digitonin-treated cells exhibited a higher cyclohydrolase activity than the cell-free extracts; the activities of riboflavinsynthetase and riboflavinkinase in the cells and cell-free extracts were found to be similar. Treatment of cells with dimethylsulfoxide proved to be most effective to determine the activity of GTP-cyclohydrolase and also helpful to determine that of riboflavinsynthetase. Protamine sulfate had no effect on the cells of P. guilliermondii. The methods developed were used to determine the activities of GTP-cyclohydrolase, riboflavinsynthetase and riboflavinkinase in the cells of flavinogenic (P. guiller-mondii, Torulopsis candida) and non-flavinogenic (Candida utilis, Candida pulcherrima) yeasts grown in iron-rich and iron-deficient media. Derepression of riboflavinsynthetase and GTP-cyclohydrolase syntheses under conditions of Fe deficiency in the flavinogenic yeast cells confirmed previously made assumptions.


[Effect of aminazin on changes in the electrical activity of the cerebral cortex under the influence of the toxin C1. perfringens type A]. Experiments on cats revealed that injection of chlorpromazine (3 mg/kg of body weight, intramuscular) one hour prior to the Cl. perfingens toxin type A injection prevented the appearance of desynchronization of the electrical activity of the cerebral cortex usually observed during the first phase of intoxication. Chlorpromazine injection postpones the appearance of the electrical activity depression phase during the second phase and prolonged the animals' life span from 1.5 to 2 times. The effect of chlorpromazine was apparently connected with the block of the adrenergic structures of the brain stem reticular formation.


[Effect of epidermal chalones on the growth of transplantable tumors]. The ethanolic extract of rat skin contains epidermal G1- and G2-chalones which having been added to cell suspension of transplantable squamous-cell cornified carcinoma of mice cervix, inhibits its growth in recipients by 72,6 per cent. The same extract failed to inhibit the growth of transplantable mouse tumours of other histogenesis (hepatoma 22a, leukemia L-1210 and sarcoma 180). When added to cell suspension of transplantable carcinoma of mouse skin which had been anaplized during a long period of transplantation (over 10 years) this extract inhibits its growth by 39,2 per cent only.


[Concentration and metabolism of cyclic AMP during the early stages of hepatoma 22a growth]. Intracellular levels of cyclic AMP (cAMP), adenylate cyclase, and cAMP-phosphodiesterase activities at lag-period, exponential and stationary growth phases of hepatoma 22a were determined. It was shown that the transition of tumour cells from the lag-period to the exponential phase of growth was accompanied by the two-fold decrease of intracellular cAMP level on account of drastic activation of cAMP phosphodiesterase. Subsequently the cAMP level lowered more slowly until the cells entered the stationary phase of growth. In view of the fact that the adenylate cyclase activity failed to change at different growth phases of hepatoma 22a, it seems very proballe that the rise of cAMP phosphodiesterase activity could be a signal for the exit of tumour cells from the lag-period and their entrance into the mitotic cycle.


[Mechanism of disorders in energy formation in acute hepatic ischemia]. Respiration of hepatic mitochondria of rats after addition of ischemic toxin to the incubation medium was compared with respiration of mitochondria isolated from an ischemic rat liver. Its change could be prevented in both cases by the preliminary administration of dithiotreitol and could be decreased by the subsequent addition of cytochrome C or dithiotreitol into the incubation medium. Similarity of the mechanisms of disturbed energy formation in vivo and in vitro is supposed.


[Effect of benz(a)pyrene on a monolayer culture of normal and malignant mouse liver cells]. Both the cells of monolayer culture of mouse embryonic liver and that of highly malignant hepatoma 22A transplanted for 20 years actively metabolized the carcinogenic hydrocarbon benz(a)pyrene and were highly sensitive to iits toxic action. Since hepatic tissue was resistant in vivo to carcinogenic carbohydrates it is suggested that the resistance depended on factors acting at the organ or the organism but not as the cellular level. The mechanism of retention of hepatoma 22A sensitivity to the toxic action of benz(a)pyrene is also discussed.


[Pathologic anatomy of experimental diseases of small laboratory animals caused by encephalomyocarditis virus (strain EMK-70)]. The results of pathomorphological investigation of the disease in small laboratory animals experimentally induced by the EMC-70 strain of encephalomyocarditis virus isolated from monkeys are presented. Irrespective of the mode of virus injection, the newborn and juvenile mice developed some lesions in the brown fat, transverse-striated muscles, as well as in the brain and heart. In guinea pigs the changes were characterized by the development of severe myocarditis and encephalitis accompanied by viral antigen accumulation. The disease induced by the EMC-70 strain could not be differentiated from the Coxsackie infection by the pathomorphological data. This fact should be taken into consideration in solving some problems pertinent to pathoanatomical diagnosis of viral diseases.


Effect of atropine on vascular adrenergic neuroeffector transmission. Atropine, homatropine, scopolamine, procaine, lidocaine and phentolamine inhibited the contractile response of rabbit isolated pulmonary artery elicited by electrical-field stimulation. Methylatropine had no effect. The inhibition induced by atropine (2 x 10(-6)-2 x 10(-4) M) had a rapid onset of action and then remained almost constant. The inhibition was slowly reversible. The potency of atropine as an inhibitor of responses to field stimulation was very much less than the potency of phentolamine. The inhibition was not antagonized by cocaine or (+)-amphetamine. Atropine (3 x 10(-5) and 3 x 10(-4) M) enhanced the electrical-field-stimulation-induced outflow of tritium from the pulmonary artery preloaded with 3H-(-)-noradrenaline. In contrast, atropine in a concentration-dependent manner either had no effect or slightly decreased the tyramine-induced outflow of tritium. Atropine reduced the contractile response of the pulmonary artery evoked by tyramine. Atropine (10(-4) and 3 x 10(-4) M) and phentolamine inhibited the arterial contractions elicited by exogenous (-)-noradrenaline in an apparently competitive manner. The contractions of rabbit isolated aorta elicited by (-)-noradrenaline, serotonin and histamine were inhibited by atropine (10(-5) and 10(-4) M). Atropine was very much less potent in antagonizing noradrenaline, histamine and serotonin than in antagonizing acetylcholine. tthe inhibotory potency of atropine, procaine and lidocaine on the accumulation of 3H-(-)-noradrenaline by rabbit aorta in vitro was much less than that of cocaine. The relationship between the aortic concentration of 3H-atropine and in vitro accumulation was almost linear. The accumulation was slightly higher at 37 degrees C than at 1 degree C. The results suggest that atropine blocks alpha-adrenoceptors, both presynaptically at the adrenergic neurone terminals and postsynaptically at the smooth muscle. In addition, atropine may possibly act in a nonspecific manner at postsynaptic sites.


Pharmacological and certain chemical properties of AH 10407, an unusually short-acting, competitive neuromuscular blocking drug, and some related compounds. 1,1-Azobis[3-methyl-2-phenylbenzimidazolinium]dimethanesulphonate (AH 10407) has an ultrashort, competitive neuromuscular blocking action in the mouse, cat, dog, Cynamolgus monkey and cotton-eared marmoset. 2 AH 10407 is chemically unstable in bicarbonate-containing solutions and is degraded to inactive products. The half-life of AH 10407 in vitro in dog and human whole blood and in Krebs physiological solution is about 1.0 minute. In distilled water and in HCO-3-deficient Krebs solution AH 10407 is much more stable. Base catalyzed degradation is shown to be the prime determinant of the duration of action of the drug. 3 Some pharmacological properties of AH 11244 and AH 11056, close analogues of AH 10407, are briefly described and the duration of their neuromuscular blocking actions rationalized by reference to their chemical stabilities.


Suppression of transmission of nociceptive impulses by morphine: selective effects of morphine administered in the region of the substantia gelatinosa. 1 In spinal cats anaesthetized with alpha-chloralose, a study was made of the effects of morphine and naloxone, administered electrophoretically from micropipettes, on the responses of dorsal horn neurones to noxious (raising of skin temperature above 45 degrees C) and innocuous (deflection of hairs) peripheral stimuli.2 Administered near cell bodies, morphine reduced the nociceptive responses of only 2 of 37 cells. Excitation occurred more commonly than depression and abnormalities in action potentials were commonly observed following ejection of morphine. None of these effects of morphine was antagonized by electrophoretically applied naloxone.3 Administered in the substantia gelatinosa from one micropipette while recording responses of deeper neurones with a second micropipette, morphine reduced the nociceptive responses of 15 of 19 neurones. Firing in response to deflection of hairs was not reduced by morphine. Depression of nociceptive responses by morphine was long lasting (>20 minutes). Naloxone ejected into the substantia gelatinosa or given intravenously in doses as low as 0.1 mg/kg antagonized the effects of morphine. The effectiveness of this dose of intravenous naloxone suggests that the concentrations of morphine in the substantia gelatinosa which reduced nociceptive responses were not unlike those present after analgesic doses of systemic morphine. Naloxone alone, and excitant and depressant amino acids ejected into the substantia gelatinosa had little effect on cell firing.4 Both the selective action of morphine on nociceptive responses and the reversal of this action by intravenous naloxone suggest that the opiate receptor present in the substantia gelatinosa is relevant to analgesia produced by opiates given systemically.


Studies of hydrolytic enzymes and isoenzymes of normal and neoplastic childhood renal tissues and their tissue cultured cells. The hydrolases of acid and alkaline phosphatase and non-specific esterases were investigated by histochemical and electrophoretic techniques in normal and neoplastic renal tissues and their cultured cells. A distinct tumour specific pattern of non-specific esterases was demonstrated in Wilms' tumour. The reasons for the quantitative differences found in the distribution of alkaline phosphatase in Wilms' tumour and their cultured cells are discussed.


Glomerulosclerosis following irradiation and cytotoxic therapy for nephroblastoma. A 6-year-old child had a nephrectomy for nephroblastoma followed by radiotherapy and chemotherapy with actinomycin D and vincristine. He died of metastatic disease 13 months after the operation. Autopsy showed a diffuse glomerulosclerosis in the remaining kidney which is attributed to the postoperative therapy.


Axonal regeneration in the rat sciatic nerve: effect of a conditioning lesion and of dbcAMP. After the sciatic nerve had been crushed at the level of the mid-thigh, the rate of outgrowth of the regenerating axons was measured by using the pinch test to locate the leading sensory axons. This standard crush lesion ("testing" lesion) elicited axonal outgrowth at a rate of 4.3 +/- 0.1 mm/day, with an initial delay (before the axons entered the degenerating distal stump) of 1.6 days. A "conditioning" lesion (transection of the tibial nerve at the ankle), made two weeks before the testing lesion, caused an increase of 23% in the outgrowth rat (P less than 0.02), with no appreciable change in the initial delay. Dibutyryl cyclic AMP (dbcAMP) was found to have no effect on the rate of axonal outgrowth measured by the pinch test. Histological examination of the pinch-tested nerves showed that the drug also had no effect on the numbers of regenerating silver-stained axons or fluorescent noradrenergic axons seen at various levels distal to the testing lesion.


The mechanism of actions of collagenase on the inhibition of release of acetylcholine from synaptosomal preparations. The release of acetylcholine from synaptosomal preparations from bovine superior cervical ganglia and rat cortex was inhibited when the preparations were pretreated with collagenase. The inhibition of release could be overcome with the calcium ionophore A23187. Collagenase treatment was shown to inhibit the uptake of calcium into the preparations. In addition, gel electrophoresis of synaptosomal membranes revealed two missing high molecular weight proteins when either synaptosomes or synaptosomal membranes were incubated with collagenase.


Paradoxical sleep and brain catecholamines in the rat after single and repeated administration of alpha-methyl-paratyrosine. This study is concerned with the role of catecholamines (CA) in the regulation of paradoxical sleep (PS) in the rat. Alpha-methyl-paratyrosine (alphaMPT), an inhibitor of tyrosine hydroxylase, was used in a multiple administration schedule in order to avoid toxic effects. From 2 to 10 doses of 75 mg/kg of alphaMPT, a progressive reduction of green fluorescence occurred in CA cell bodies as well as in terminals. The green fluorescence reduction was faster in dopaminergic than in noradrenergic neurons, and faster in cell bodies than in terminals. Sleep recordings showed a slight increase of PS after one or two doses of 75 mg/kg of alphaMPT, and a dose-related decrease after 3-7 injections. After two doses of 75 mg/kg, the number of PS phases was significantly increased. This can be due to the release of a non-CA PS primer mechanism. These experiments support the view that an intact synaptic transmission in CA neurons is necessary for the realization of PS.


Post-synaptic potentials recorded from afferent nerve fibres of the posterior semicircular canal in the frog. Glass microelectrode recordings were made from single fibres of the posterior ampullary nerve in the isolated labyrinth of the frog (Rana esculenta). Potentials were recorded both at rest and during rotatory stimulation of the canal. At rest, the tracings revealed an intense background of small, largely summated potentials (0.5-10 mV amplitude, 3-6 msec duration), which underlay the discharge of spikes in all the impaled units. The frequency of the subthreshold events was related to the frequency of the propagated spikes, the latter ranging from 0 to 40/sec. Stimulation modulated the frequency of both spikes and subthreshold potentials, whose summation during excitation led to a positive shift of the fibre membrane potential. The small potentials proved to be dependent on Ca2+ and Mg2+ levels in the bath. Antidromic-stimulation of the posterior ampullary nerve indicated that the observed events do not represent an artifact due to extracellular field interference related to spike activity in the neighbouring fibres. Tetrodotoxin (10(-7)-10(-6) g/ml) applied externally to the preparation or previously perfused through the frog vessels abolishes the propagated spikes but left unaffected the small potentials which, even under drug treatment, were normally modulated by the stimulus. The subthreshold potentials thus appear to be EPSPs generated at the cyto-neural junction between the hair cells and the endings of the ampullary nerve fibres.


Responses of the immature fowl to a single injection of adrenocorticotrophic hormone. Chicks, aged 1 or 21 d, were injected with long-acting ACTH (6, 12, 30 or 60 IU/kg) and changes in plasma glucose and cholesterol and in adrenal weight and cholesterol measured. 2. No consistent response was observed in the 1-d-old chicks. 3. Hyperglycaemia and adrenal cholesterol depletion could be demonstrated in the 3-week-old bird at all doses. 4. The duration of the hyperglycaemic response was dose-dependent but there was only limited evidence that adrenal cholesterol depletion was similarly dependent. 5. Starving the 3-week-old bird overnight led to a significant hypercholesteraemia which was reduced after ACTH. In contrast no change in plasma cholesterol concentration was noted in fully fed birds.


[Effect of cyclic AMP on retrogression of the Mullerian ducts in chick embryos]. Theophyllin and puromycine, inhibitors of the enzyme phosphodiesterase and AMPc are all able to inhibit the retrogression of mullerian ducts in the female chick embryo, grafted with an embryonic testis. We can think that these results are explained by an inhibitory action of AMPc on the mechanisms responsible for the mullerian retrogression. So the chick embryo reacts similarly as do the mammalian embryo.


Regional distribution of beta-lipotropin converting enzymes in rat pituitary and brain. Among the brain areas studied only pars distalis and pars intermedia are found to contain beta-lipotropin activating enzyme indicating the these may be the exclusive organs for a physiologically significant conversion of beta-lipotropin into beta-endorphin. beta-Endorphin inactivating enzyme is found to be rather uniformly distributed in all the pituitary and brain regions. alpha-and gamma-endorphins are presumably formed by the action of the enzyme on beta-endorphin.


Virions associated with acute gastroenteritis in Vancouver, 1976. Virions were identified by electron microscopic observation of unconcentrated extracts of feces obtained from 55 (33%) of 167 children aged 3 weeks to 5 years who were hospitalized with acute gastroenteritis at Children's Hospital, Vancouver between January and December 1976. Morphologic types included 30 rotavirus, 15 adenovirus, 5 astrovirus and 5 picornavirus-like particles.


Primary mucinous carcinoma of skin: histochemistry and electron microscopy. Two primary mucinous carcinomas of skin were studied by histochemistry and by light and electron microscopy. Enzyme histochemistry showed a pattern of reactivity similar to that found in eccrine secretory epithelium. Mucin histochemistry substantiated previous reports of probable sialomucin formation. Electron microscopy revealed a highly differentiated neoplasm with a mode of mucin secretion similar to that observed in the dark (mucinous) cell of the eccrine coil. The natural history of mucinous carcinoma of skin indicates that although local growth is the rule, lymph node metastasis may occur.


Erythrophagocytosis by epithelial cells of a breast carcinoma. Erytrophagocytosis by epithelial tumor cells has been observed in metastases of ductal carcinoma of the female breast. Some malignant cells of this tumor seem to be capable of phagocytizing and digesting extravasated red blood cells with for formation of residual hemosiderin probably from their hemoglobin content. Erythrophagocytosis has been observed only in hemorrhagic areas of the tumor. Erythrophagocytosis has been observed only in hemorrhagic areas of the tumor. Although the nature of this phenomenon is unknown, it is postulated that acquired hematological disturbancess during the natural course of the malignant disease affect the surface of the red blood cells making them vulnerable to phagocytosis by the malignant cells. This case seems to represent the second time such a phenomenon has been reported in an epithelial neoplasm in man. However, it has been more frequently observed in reticulo-endothelial malignancies. The possible occurence of this phenomenon should alert pathologists to search for it in primary and metastatic epithelial tumors and in living patients to correlate pertinent hematological studies in an attempt to elucidate its possible significance.


Observation of nervous tissue in a Wilms' tumor: its histogenetic significance. A case of Wilms' tumor occurring in a horseshoe kidney is reported in which bona fide nervous tissue containing ganglion cells was observed. This finding cannot be reconciled with the currently held "metanephrogenic blastema" theory of the histogenesis of this tumor. Masson's concept of "neuroepithelial origin" of the Wilms' tumor should be re-evaluated.


The histogenesis and development of pulmonary tumorlets. A lung, which was surgically removed from a patient who had oat cell carcinoma, contained multiple tumorlets and showed extensive Kultschitsky-type cell proliferation of bronchial and bronchiolar mucosa. On the basis of light and electron microscopic observations, it is shown that pulmonary tumorlets arise from focal areas of bronchial and bronchiolar Kultschitsky cell proliferation which may advance to luminal obliteration. Involvement of the pulmonary parenchyma takes place by extension of these newly proliferated cells along the terminal branches of the bronchiolar tree or by penetration of the bronchial or bronchiolar wall; the latter process evokes a striking proliferation of connective tissue which forms the matrix in which the cells of some fully developed tumorlets are embedded. Because of striking morphologic similarities between tumorlets and spindle cell carcinoid tumors, and the proven origin of tumorlets from pulmonary Kultschitsky-type cells, it is suggested that the more complete and histogenetically acceptable term "carcinoid tumor-let" be used to distinguish this lesion from other forms of epithelial proliferations in the lung.


Tendosynovial sarcoma: a clinicopathological study of 136 cases. A series of 136 cases of tendosynovial sarcomas were studied from the histologic point of view. All of the primary, recurrent and metastatic tumors were reclassified according to histologic type, anatomic site and size; age and sex of patients and modality of surgical treatment. The overall 5-year survival rate was 40%. Among the factors which most favorably influenced the prognosis were: small size (74%, 5-year survival), "exposed" primary site (53%, 5-year survival), and histologic type (biphasic form: 55%, 5-year survival and epithelioid sarcoma: 58%, 5-year survival). Children and elderly patients had also better than the average prognosis. Sixty percent of the sarcomas recurred after wide local excision. Twelve percent of the tumors metastasized to regional lymph nodes and 10% to bones. Almost all of the 24 patients who were autopsied died because of cardio-pulmonary insufficiency due to massive pleuropulmonary metastasis. It is hoped that better understanding of the natural history of these tumors will lead to optimal local and systemic therapy and better survival.


Perianal mucinous adenocarcinoma.

Perianal mucinous adenocarcinoma. Perianal mucinous adenocarcinoma, also known as anal duct or anal gland carcinoma, is a rare tumor usually associated with chronic fistulae-in-ano. Whether the tumor occurs primarily and the fistula is a secondary manifestation, or whether this tumor arises in a chronic fistulous tract is debatable. Diagnosis can be made only by open deep biopsy of the buttock mass, and, due to the low grade histologic appearance of the malignancy, an incorrect diagnosis is frequently made. Treatment is abdominoperineal resection with wide removal of the buttock mass.


A minute hepatocellular carcinoma found in a liver with clonorchis sinensis infection: report of two cases. Two cases of minute hepatocellular carcinoma (HCC) found in a liver infested with Clonorchis sinensis are described. One had mild infestation with hepatic changes suggestive of posthepatitic cirrhosis, and the other heavy infestation exhibiting secondary biliary cirrhosis with dilated intrahepatic bile ducts and periductal fibrosis. None had evidence of hepatitis B infection. The tumor nodule was solitary, measuring 5 X 7 mm and 10 X 11 mm, respectively, and the cells were differentiated to be classified as Grade I of Edmondson-Steiner's scale of anaplasia. It is not clear whether or not clonorchiasis was etiologically related to HCC, but it was of interest that in both cases the tumor nodule was very small representing the primary lesion without metastasis.


Metastatic infantile Wilms' tumor and hydrocephalus; a case report with review of the literature. A case of infantile metastasizing Wilms' tumor is described clinically and pathologically. The presenting feature was congenital hydrocephalus due to cerebral metastases. The literature is reviewed and four similar examples are found. The unusual pattern of clinical presentation and metastases is emphasized as it may be characteristic for this rare entity.


Cystic nephroma.

Cystic nephroma. The clinical and pathologic findings of four cases of cystic nephroma (so-called "renal multilocular cyst") in four infants nephrectomized with the diagnosis of Wilms' tumor are presented. Each lesion consisted of an encapsulated mass composed of cysts separated by thin septa. Histologically the cysts were lined by epithelium, and the septa contained foci of immature metanephric blastema (abundant in one case and sparse in another). rhabdomyocytes and mature tubules. Neither embryonic ducts nor cartilage were seen. One cystic lesion was adjacent to a nephroblastoma which formed a separate nodule. No recurrences of metastases developed, and the only death was due to postoperative sepsis. These lesions are considered by some authors to be dysplasia, while others interpret them as benign neoplasms. Our findings and a review of the literature probably represent the differentiated counterpart of nephroblastoma.


Intracerebral delayed hypersensitivity reactions in glioblastoma multiforme patients. Patients with malignant gliomas who had undergone BCG inoculation were injected intratumorally with PPD to induce an intratumoral delayed hypersensitivity reaction. Histopathological examination of the tumor before and after PPD injections revealed that chronic inflammatory responses were increased after injection in four of the five patients. In no case, however, was the response more than moderate, and in no case did the inflammatory response encompass the tumor at its peripheral margins. Intracerebral delayed hypersensitivity reactions were evoked in humans with acceptable morbidity. The mild reactions evoked did not cause detectable tumor regression.


A possible involvement of cya gene in the synthesis of cyclic guanosine 3':5'-monophosphate in E. coli. Based on the following genetical experiments, the cya gene in E. coli was shown to be involved in the synthesis of both cyclic AMP and cyclic GMP. First, all five independent cya-deficient mutants accumulated exceedingly low amounts of cyclic GMP. Second, the ability to form both cyclic AMP and cyclic GMP was simultaneously restored by transduction of an intact cya locus to one of the above cya-deficient mutants. Third, a spontaneous revertant from one of the above mutants regained the synthetic activity for cyclic GMP as well as for cyclic AMP. Fourth, the characteristic of a strain overproducing cyclic GMP was co-transduced with the cya locus. These results suggest that the synthesis of both cyclic GMP and cyclic AMP is mediated by the same enzyme, adenylate cyclase, Interestingly, a reciprocal effect of glucose starvation was observed on the accumulation of both cyclic nucleotides. The formation of cyclic AMP was greatly enhanced on glucose starvation, whereas that of cyclic GMP proceeded at a slower rate than in the presence of glucose. This effect was observed only in cells carrying normal cya and crp genes, but not in a cya-altered or a crp-deficient strain.


Overlapping of the VP2-VP3 gene and the VP1 gene in the SV40 genome. The nucleotide sequence of the SV40 Hind E fragment has been determined mainly by the partial chemical degradation procedure of Maxam and Gilbert (1977). The sequence of the strand with the same polarity as the late messenger RNA shows only one open reading frame for translation. Considering that VP3 corresponds to the carbosyl terminal part of VP2, and considering various evidence which indicates that the SV40 Hind E segment is part of the amino acid sequence of VP2-VP3. It continues clockwise in Hind K, where it terminates with a UAA signal. The latter is located 110 nucleotides beyond the initiation signal for the major structural protein VP1 (Fiers et al., 1975; Van de Voorde et al., 1976). Hence this small overlapping region of the genome codes for the synthesis of three different proteins in two different reading frames. The deduced amino acid sequence covers a major part of the vp3 poly peptide, and the amino acid composition is in good agreement with published values (Greenaway and Levine, 1973).


Further characterization of Sendai virus DI-RNAs: a model for their generation. Sendai virus DI-RNAs which contain complementary ends have been characterized as follows. First, the complementary ends of three DI-RNAs, although somewhat different in size (110-150 base pairs), contain sequences that are both identical to each other and to the 5' end of the nondefective (ND) genome. Second, almost all the sequences contained sequences that are both identical to each other and to the 5' end of the nondefective (ND) genome. Second, almost all the sequences contained in the DI-RNAs derive from sequences that are contiguous to the 5' end of the ND genome. The ND genome, on the other hand, does not contain any sequences that are complementary to its 5' end. A genetic map and a model for the generation of the Sendai DI-RNAs are presented.


Adenylyl cyclase and the control of cell differentiation in Dictyostelium dicoideum. Adenylyl cyclase is part of a biochemical network that controls cell differentiation in Dictyostelium discoideum. At a certain stage of development the enzyme is rhythmically activated, with periods of about 8 min. These oscillations are superimposed upon an increase of the basal activity extending over a period of hours. The basal activity remains low in a mutant blocked at an early stage of development. In strain Ax-2 two periods of strongly increasing basal activity have been found: the first from 2 to 4 h after the end of the growth phase, the other beginning at about 8 h. Based on the periodic regulation of adenylyl cyclase, cyclic AMP is released into the extracellular space in the form of pulses. Application of cyclic-AMP pulses, but not its continuous influx, stimulates the increase of basal adenylyl cyclase activity. Two other constituents of the cyclic-AMP signal system cyclic-AMP receptors and cell-surface phosphodiesterase, are similarly controlled. The principal importance of positive feedback loops in a network controlling cell differentiation is discussed.


[Cellular contaminants and structural proteins of Rous sarcoma virus (RSV), studied by polyacrylamide gel electrophoresis]. The number of polypeptides in highly purified preparations of RSV, of two different subgroups, produced in culture, has been compared to the polypeptides present in the supernatant of uninfected cultures and processed in identical manner. The analysis of PAGE-SDS shows that from 13 to 18 polypeptides present in viral preparations may be cellular contaminants. Fewer contaminating polypeptides are found in the myeloblastosis virus purified from plasma of Chicken.


[Measurement of cyclic AMP in the islets of Langerhans of newborn rats. Effect of amino acids]. Radioimmunoassay of cyclic AMP (cAMP) in the islets of Langerhans from 48-64 h old Rats was performed after succinylation of the samples. cAMP was detected at 0.03 nM. The cAMP content of islets increases when L-arginine, L-lysine and L alanine are added together in the incubation medium at a concentration of 5-10 mM each. When phosphodiesterase is inhibited by theophylline the three amino acids considerably increase the cAMP content of islets. Thus an increase in cAMP content of the islets was observed with a concentration of amino acids which is efficient in stimulating the insulin and glucagon secretion.


[Variation in the toxicity of various silicas in relation to the duration of presence in the body]. The toxic effect of two forms of sand on the lung was investigated by tracheal injection in Rats. After a latent period with low toxicity, more and more extensive pulmonary lesions, comparable to typical silicotic lesions, appear progressively. On the basis of some observations, the hypothesis is put forward that the impurities present in the dust have a retardative effect.


[Receptors for cytomegaloviruses on the surface of human lymphocytes]. The relationships between cytomegalovirus (CMV) and lymphocytes have already been noted because of: (a) the immunological abnormalities induced by this virus, and (b) activation of latent CMV in the course of lymphocyte reactions associated with anti-histocompatibility antigen immune response. The present work shows that the lymphocyte surface may have specific receptors for CMV. Cultured fibroblasts infected with DMV were incubated with lymphocytes isolated from the blood of human immune subjects. Rosettes defined by the adherence of three or more lymphocytes around a fibroblast were formed in infected preparation while no rosettes were seen with normal control fibroblasts. Approximately 1.2 per cent of lymphocytes were involved in the formation of these rosettes. Rosette formation is inhibited when infected fibroblasts have been incubated with anti-CMV antibodies prior to the addition of lymphocytes.


[15 new serotypes of Salmonella of subgenus I isolated in Africa]. Filteen new serotypes of Salmonella belonging to the sub-genus I have been isolated in Africa. Isolated in Ivory Coast: S. dabou (8,20:Z4,Z23,l,W), S. elokate (9,12:c:1,7), S. assinie (3,10:1,w:Z5), S. anna (13,23:Z35:e,n,Z16), S. mpouto (16:m,t:-), S. banco (28:r:1,7), S. abidjan (38:b:l,w); in Senegal: S. bignona (17:b:e,n,Z15), S. lode (17:r:1,2), S. derkle (52,e,h:1,7); in Tchad: S. moussoro (1,6,14,25:i:e,n,Z15), S. drogana (1,4,12,27:r,i:e,n,Z15); in Algeria: S. hydra (21:c:1,6); in Haute-Volta S. farakan (28:Z10:1,5); in Republique Centrafricaine: S. babili (28:Z35:1,7).


[Role of acoustic stress on the development of polyoma tumors in immunized golden hamsters]. An acoustic stress increases the metastatic spread of polyoma virus-induced tumors in syrian Hamsters. To try to elucidate the mechanism of this action, we studied the effect of stress on the protection afforded by the virus against tumour challenge. Exposure to ultrasonics during the immunization period markedly decreased the protective effect of the virus.


[Ultrastructural aspects of the cell fusion induced by Visna virus on sheep choroid plexus cell in culture]. Sheep choroid plexus cells infected with low multiplicities of infection of Visna Virus were stellate and had long and thin processes containing filaments and forming cytoplasmic bridges between adjacent cells. Enlargement of the bridges resulted in the formation of multinucleated cells. Some glycoproteins were clustered on filaments outside the cell. The cytoplasmic changes showed : an intensive protein synthesis; numerous mitochondria closely associated with filaments and some lysosomes and numerous vesicules near the plasma membrane.


[Oxidation of malate, NADH and glycine in C3 and C4 plant mitochondria]. Spinach leaf mitochondria (C3 plant) were capable of oxidizing Glycine. This oxidation was linked to the mitochondrial electron transport chain, was coupled to three phosphorylation sites and was sensitive to electron transport inhibitors. In marked contrast however, neither mitochondria from dark grown plants nor mitochondria from C4 leaf plants were capable of oxidizing this amino acid.


[Early effect of poliomyelitis virus on 5'-nucleotidase activity in HeLa cells]. One of the earlier effects of poliovirus was the maintaining of a high level of Hela cells 5'-nucleotidase activity (maximal efficiency for a virus/cell ratio = 500 ID 50/cell) (ID50:50% Infections Dose). The activity decreased when cells were suspended in the presence of EDTA(EDTA:Ethylene Diamine Tetracetic Acid) and was partially restored by adding 10 mM Mg++ interactions are discussed.


[Effect of the inhibition of "NADH-diaphorase" activity on the ultrastructural localization of 3 beta-hydroxysteroid dehydrogenase]. The NADH-ferricyanide oxidoreductase ("NADH-diaphorase") activity can be inhibited selectively with 0.5 mM p-chloromercuribenzoate, and the reduction of the potassium ferricyanide can be restored with an exogenous electron carrier (PMS). These operations do not affect the localization of the final precipitate of copper ferrocyanide which results from 3beta-hydroxysteroid dehydrogenase activity.


[Tissue antagonist of interferon : murine substance similar to plant lectins]. A tissue antagonist of interferon (TAI) extracted from mouse costal cartilage contains a substance which has many properties characteristic of plant lectins. After binding to the cell membrane receptors, it agglutines normal and transformed murine cells. In interferon treated cells, it restores virus sensitivity probably through a modification in the distribution of membrane bound cellular antigens.


New micromethod for measuring cholesterol in plasma lipoprotein fractions. A method is described for the reliable, fast, and relatively inexpensive fractionation of plasma lipoproteins and quantitation of their cholesterol content. This procedure requires 350 microliter of plasma and can be completed within 3 h. Plasma lipoproteins (175 microliter of plasma) were prestained with Fat Red 7B and centrifuged (Beckman Airfuge) at plasma density (d = 1.006 kg/liter) and at a solvent density of 1.060 kg/liter, adjusted by adding solid KBr. Prestained centrifuged samples demonstrated the characteristic elevation of chylomicrons in phenotypes I and V, low-density lipoproteins of phenotype II, very-low-density lipoproteins in phenotype IV and V, and continuum of pink color throughout the centrifuge tube, diagnostic of the floating beta lipoprotein of type III. Centrifuged samples were separated into top and bottom fractions by aspiration. Cholesterol was quantitated with an enzymic oxygen-electrode analyzer (Beckman Cholesterol Analyzer). Correlation coefficients between cholesterol values for plasma from normal hyperlipidemic individuals obtained with the Beckman Analyzer vs. the Technicon AutoAnalyzer II and SMAC systems were 0.977 and 0.973, respectively.


The effect of temperature on the kinetic constants of human lactate dehydrogenase 1 and 5. The kinetic constants of human lactate dehydrogenase 1 and 5 (L-lactate: NAD+ oxidoreductase, EC 1.1.1.27), assayed lactate-to-pyruvate increase with temperature. The reaction mechanism is ordered sequential as has been found with lactate dehydrogenase from other sources. The KM values for each substrate are larger for isoenzyme 5 than for 1. For lactate dehydrogenase 1 the KM(lactate) increases from 1.07 mM at 25 degrees C to 3.95 mM at 37 degrees C and for lactate dehydrogenase 5 it increases from 5.37 mM at 25 degrees C to 6.88 mM at 37 degrees C. The KM(NAD+) for lactate dehydrogenase 5 is 0.14 mM at 25 degrees C and 0.29 mM at 37 degrees C. The increase in the KM for each substrate with increasing temperature confirms that additional substrate is required for optimal reaction conditions at higher temperatures.


Urinary pregnanetriol excretion in hirsutism. Eighteen consecutive hirsute women attending an endocrine clinic have been studied by measurement of the urinary pregnanetriol excretion before and following the concurrent administration of corticotrophin and metyrapone. An abnormal increment in pregnanetriol excretion was observed in 11 of these 18 patients. It is suggested that this is evidence that an adrenal abnormality, probably operative at the 21-hydroxylase level, might be a factor responsible for the hirsutism in these 11 patients. Five adrenalectomized women who were also studied showed no significant increase in urinary pregnanetriol excretion in response to concurrent corticotrophin and metyrapone administration.


How to collect blood for the measurement of HGH, LH, FSH, cyclic AMP, folic acid, cortisol and testosterone. In order to estimate peptide hormones, steroid hormones, folic acid and cyclic AMP in the same blood sample we investigated the influences of different anticoagulants on the measurement. It could be demonstrated that blood collected in EDTA-coated tubes can be for the measurement of each of the mentioned constituents.


Changes in serum high density lipoproteins in women on oral contraceptive drugs. Serum lipids, lipoproteins, and lipoprotein subfractions were measured in a group of 18 women aged 20 through 39 who were users of oral contraceptive drugs, and in 19 age-matched controls. Concentrations of the major lipid and lipoprotein classes were higher in the users, but the elevation was statistically significant only in the case of the high density lipoproteins. This increase was shown to be due to a highly significant increase (275 +/- 9 vs. 223 +/- 9 mg/100 ml, (p less than 0.005) in the denser high density lipoprotein subfraction (HDL3). Levels of the other subfraction (HDL2) were similar in users and controls. Thus, anovulatory steroids have selective effects on individual types of high density lipoproteins. Studies of such specific effects may help to further define the functional properties of the high density lipoproteins such as their apparent protective role in atherosclerosis.


Isolation and characterization of lipoprotein-X from the pig. In vitro incubation of pig bile with pig whole serum resulted in the formation of "LP-X like" material migrating towards the cathode on agar-gel electrophoresis. As in all other species studied so far, LP-X occurred also in the serum of bile duct ligated pigs, however, relatively late (48-72h) after the operation. In parallel, alterations of some serum parameters and of the protein-lipid composition of the different lipoporotein fractions were observed. While the electrophoretic behaviour, the protein-lipid and apoprotein composition of pig LP-X were comparable to those found in man, dog and rat, it sedimated in most instances at the density 1.063 g/ml.


Genetic study of hyperlipoproteinaemia types IV and V. In a family, of which 32 members belonging to three generations could be examined, 21 subjects were normal, while 11 were affected by hyperlipoproteinaemia types IV and V or related biological disorders. Neither an autosomal dominant inheritance nor an autosomal recessive inheritance nor a multiple-type hyperlipoproteinaemia can unreservedly be accepted.


A family with syndactyly type II (synpolydactyly). Syndactyly Type II is reported in eight members of a family in four generations. Affected individuals show two distinctive patterns of variation in the expression of the gene. Distortion of dermatoglyphic patterns is associated with the severe but not the mild manifestation of the malformation. The diagnostic significance of minimal features of the condition is discussed. Linkage data suggest that loci for Syndactyly II and for blood-group antigens ABO, MNSs, P, Rh and Kell are not closely linked.


Plasma concentration of pyridostigmine and effects in myastenia gravis. The relation between the plasma concentration of pyridostigmine and its effects was studied in 5 patients with myasthenia gravis. In 4 patients with typical electromyographic decrement in the adductor pollicis, there was a positive correlation between the concentration  of pyridostigmine in plasma and the effect on neuromuscular transmission. The plasma concentration of pyridostigmine required to restore transmission to normal (as calculated from the regression line relating plasma concentration to neuromuscular function) varied over a 5-fold range, reflecting the variable severity of the disease. In another myasthenic patient with purely ocular symptoms, there was a significant correlation between the plasma concentration of the drug and the diameter of the palpebral fissure. It is suggested that the routine measurement of the plasma concentration of pyridostigmine may be of value in the management of myasthenia gravis. A method to calculate the optimal daily dose of pyridostigmine in individual myasthenic patients is described.


The passage of apoproteins from plasma lipoproteins into the lipoproteins of peripheral lymph in man. 1. The transport of apoprotein B from the lipoprotein of plasma into the lipoproteins of lymph draining the foot has been studied in four men with type III hyperlipoproteinaemia. 2. Three subjects were given autologous 125I-labelled very-low-density lipoprotein (VLDL) and 131I-labelled low-density lipoprotein (LDL) by intravenous injection; the fourth was given autologous 125I-labelled VLDL and 131I-labelled intermediate-density lipoprotein (IDL) plus LDL. 3. The 125I/131I ratios in serum and lymph apoprotein B, and the 125I and 131I specific radioactivities of apoprotein B in VLDL, IDL and LDL from serum and lymph, indicate that apoprotein B in the circulating VLDL can reach peripherallymph without the intermediacy of circulating LDL.


[Studies on the development and therapy of scoliosis]. Inadequately treated cases of scoliosis with deterioration were studied. Biomechanics and pathology are discussed. It has been observed that permanent lateral deviation of the vertebral column continues irreversibly if not treated in time. A study of the evolution of scoliosis in relation to vertebral growth is reported. It is followed by a description of therapeutic management based on fundamental factors: --aetiology, the severity of the deformity, bone age and potential deterioration. A series of cases treated conservatively and by surgical fixation is illustrated.


Pathological and therapeutic consequences of sleep loss: a review. Until recently sleep deprivation has been studied from the point of view of determining whether or not it produces deleterious effects. Evidence, however, has begun to accumulate indicating that both REM deprivation and single night sleep deprivation may have antidepressant effects. Although beneficial effects are found primarily in endogenously depressed patients, variable results have been obtained in both "endogenous" and "reactive" depressions, suggesting that these diagnostic categories include biologically heterogeneous populations.


Modification of glycoprotein residues as Ca2+ receptor sites after chronic ethanol exposure. Previous studies have demonstrated that chronic low-dose administration of ethanol in the drinking fluid of rats produces an increase in content of calcium on synaptic membranes. In the present study, ethanol was studied in vitro and in vivo for its effects on Ca2+ binding to a high affinity receptor site on synaptic membranes. Ethanol (1-50 micrometer) significantly inhibits the binding of Ca2+ in non-competitive fashion, together with inhibiton of a cooperative mechanism for Ca2+ binding. Ruthenium red and neuraminidase were used to probe surface glycoprotein sites as receptors for Ca2+. Use of these probes together with ethanol in vitro or in vivo suggests that increases in Ca2+ receptor sites after chronic ethanol exposure are related to an increase in sialic acid exposure, possibly as a result of conformational changes in membrane protein. These findings are consistent with the hypothesis that chronic low-dose exposure to ethanol causes changes in proteins of synaptic membranes, which may in turn regulate the cellular adaptation to ethanol.


[Primary carcinoma of the liver and long-term administration of oral contraceptives followed by pregnancy (author's transl)]. A 32-year-old woman who had taken oral contraceptives for seven years, became pregnant after omitting the drug and had a normal child. Soon after a malignant hepatic tumour became apparent of which she died after three months. The tumour was a hepatocellular carcinoma, of low differentiation under the electron microscope. The tumour cells contained fibrils similar to alcoholic hyaline. A review of published reports suggests the possibility that the oral contraceptives and the hormonal influence of the pregnancy together played an important role in the development of the malignant tumour.


[Familial hypobetalipoproteinaemia and diabetes mellitus (author's transl)]. In a 60-year-old patient with manifest diabetes mellitus and in his 63-year-old brother with latent diabetes mellitus hypobeta-lipoproteinaemia was diagnosed. Cholesterol values were around 1,8 mmol/1 in whole serum samples. The LDL-cholesterol fraction was 1,04 mmol/1. The beta-lipoprotein band in the lipoprotein electrophoresis was markedly reduced. Apolipoprotein B measured by radial immuno-diffusion was about 30% of the normal for age. The components of LDL were normal. Values of hepatic triglyceride lipase and lipoprotein lipase in heparinised plasma were within the normal range. The simultaneous occurrence of hypobetalipoproteinaemia and diabetes mellitus is described here for the first time.


Relationship between levels of cyclic nucleotides and gamma-aminobutyric acid during ethanol withdrawal in rats. Cyclic nucleotide and gamma-aminobutyric acid (GABA) levels were measured in four brain areas of control rats and rats undergoing ethanol withdrawal. Cyclic adenosine 3',5' monophosphate (cyclic AMP) was increased in the cerebral cortex and pons-medulla oblongata; cyclic guanosine 3',5'-monophosphate (cyclic GMP) was increased in the cerebellum and cerebral cortex; and GABA was decreased in the cerebellum, subcortex and pons-medulla oblongata. Individual values in withdrawn animals were correlated and subjected to a factor analysis. One of the common factors identified by this analysis was important for all but one of the levels which changed during ethanol withdrawal. Significant correlations of levels correlated with this factor included a negative correlation between cyclic GMP and GABA levels in the cerebellum, a positive correlation between cyclic GMP levels in the cerebellum and cyclic AMP levels in the pons-medulla oblongata and a positive correlation between cyclic AMP levels in the pons-medulla oblongata and cerebral cortex. These results indicate that multiple brain areas and neurotransmitters are involved in ethanol withdrawal.


[Demonstration of the agent of bovine viral diarrhea-mucosal disease (BVD/MD) in comparison between organ fluoresence freezing method and the direct cultivation in tissue culture]. Organs of 100 calves whose clinical symptoms indicated a BVD/MD viral infection have been examined in the laboratory by different methods on BVD/MD virus in order to compare the effectivity of the single test-systems. By inoculation of organ material into tissue culture from foetal calf kidneys and additional staining with swine-fever conjugate in the CCSC-system 59 calves were detected as BVD/MD virus carriers. Taking this result for comparative purposes equal to 100% there could be stated an effectivity of 73% by inoculation of tissue cultures and judging cpe instead of staining with fluorescent antibody as above. Only 34% reactions could be demonstrated by means of heterotypic immunofluorescence in organ tissues. For diagnostic purposes a combination of the easy and quick method of heterotypic immunofluorescence in organ tissues and a cultural virus isolation from organ material with additional immunofluorescence is recommended.


Cyclic adenosine 3',5'-monophosphate, adenylate cyclase and physical dependence on ethanol: studies with tranylcypromine. Tranylcypromine, a monoamine oxidase inhititor, was administered to rats during chronic ethanol treatment. The severity of the hyperactive withdrawal behavior observed upon removal of ethanol was, during the first 60 hours of treatment, similar in both ethanol and ethanol + tranylcypromine treated animals. However, after 84 hours of ethanol treatment, tranylcypromine slightly depressed the withdrawal severity. Rises in cerebral cortical cyclic adenosine 3',5'-monophosphate (cAMP) levels and adenylate cyclase activity associated with withdrawal behavior in ethanol-treated rats, though, were not observed. (Adenylate cyclase activity used throughout this paper refers to % conversion of 3H-adenine into 3H-cAMP). Based on this and previous data, a model invoking two neurochemical adaptations--one in adenylate cyclase and one, as yet, unknown-is proposed for the mechanism of physical dependence.


[3 gastrointestinal neoplasms in the dog]. In the last five years, 3 carcinomas of the intestine were found in a total of 1961 necropsies of dogs. One proved to be a primary gastric carcinoma situated in the area of the Curvatura minor, another a primary carcinoma of the ileocaecal valve and the third one a signet-cell carcinoma in the end of the jejunum. The morphological characteristics of the carcinomas are discussed.


Characterization and follicle stimulating hormone activation of Sertoli cell cyclic AMP-dependent protein kinases. The Sertoli cell of the rat testis contains two cytoplasmic forms of adenosine 3',5' monophosphate (cyclic AMP)-dependent protein kinase, designated as Peak I and Peak II, which change in relative proportion during Sertoli cell maturation. Peak I and Peak II differ in their subunit interaction. Thus, while the substrates, ATP and histone, affect cyclic AMP binding to Peak I, neither of these compounds affect the binding of cyclic AMP to the Peak II enzyme. The effects of cyclic AMP binding to Peak I appear to be due to the fact that histone and high ionic strength cause dissociation of the Peak I holoenzyme, whereas ATP stabilizes the holoenzyme complex against dissociation. The Peak II holoenzyme is not affected by either salt of histone and is only dissociated by cyclic AMP. Once dissociated, the subunits of Peak II will rapidly reassociate under low salt conditions whereas the subunits of Peak I will not reassociate. By utilizing the distinct properties of Peak I and Peak II, it is possible to demonstrate the activation of both Peak I and Peak II Sertoli cell protein kinase in response to FSH.


Preparation and immunological characteristics of biologically active radioiodinated human calcitonin. Synthetic human calcitonin (hCT) was radioiodinated by electrolysis and chloramine-T oxidation. The electrolytic method was adapted for practicable quantities of hormone and isotope with no loss in efficiency of labeling. The immunological characterisitcs of the two hormone preparations were nearly identical using four calcitonin antisera with specificities for the regions 17-21, 17-29, 28-29, and 29-32 of hCT, respectively. However, only the electrolytically-labeled preparation retained full ability to produce hypocalcemia in vivo and stimulate cyclic AMP production in kidney homogenates of the rat. In addition, the electrolytically-iodinated hormone remained nearly homogeneous during 104 days of storage and the specific activity as measured by radioimmunoassay remained constant during one half-life of the isotope. These studies demonstrate that electrolytic iodination of hCT results in a tracer which is immunochemically stable and biologically active.


Studies on the mode of action of somatostatin on insulin secretion. The effect of somatostatin on insulin release by incubated slices of rat pancreas was studied. Somatostatin inhibited insulin release induced by arginine/glucose (A/G), glucagon, glibenclamide, pentoxifyllin, 3',5'-adenosine monophosphate (cAMP), phentolamine, and KCl. When A/G was used as a stimulus, the quantial inhibitory effect of somatostatin was not neutralized by progressively increasing glucose concentrations. The alpha adrenergic blocking agent phentolamine, the phosphodiesterase inhibitors theophylline (10 mM) or pentoxifyllin (10 mM), and KCl partially reversed the inhibitory effect of somatostatin on A/G stimulation. The maximal reversal of somatostatin inhibition was obtained when the slices of pancreas were stimulated with A/G in the presence of the calcium ioniphore A23187 plus ATP. These results suggest that the inhibitory effect of somatostatin on insulin secretion could result from calcium translocation in pancreatic beta cells.


Presence of corticotropin in human placenta: demonstration of in vitro synthesis. Immunoassayable (I) and bioassayable (B) ACTH activity is present in extracts of extensively washed human placental tissue and dispersed viable trophoblasts (Tr). Similar concentrations (I) (1.7-2.7 ng/g) are present in term placentas from elective cesarian section or vaginal delivery and from plancentas obtained by prostaglandin-induced abortion at 12, 15 and 18 weeks of gestation. Oral dexamethasone (8-12 mg) administered over an 8-48 h period prior to delivery does not significantly alter placental ACTH content. B-ACTH concentrations are 31-40% of I-ACTH. Sephadex G-50 filtration of term placental homogenates demonstrates two I-ACTH peaks, one eluting in the void volume and the other eluting in the region of synthetic hACTH1-39. When Tr are incubated in tissue culture medium, ACTH content of cells and of medium is significantly greater than pre-incubation levels.


Cyclic AMP production in rat calvaria in vitro: interaction prostaglandins with parathyroid hormone. We studied the cAMP response of cultured rat calvaria to parathyroid hormone (PTH) and prostaglandins (PG) in order to test the hypothesis that PTH action is mediated by PG. PTH and PGE1 each caused an 8-fold increase in tissue cAMP during 15 min incubation. There was an additive response to the combination of the two agonists at maximally effective individual concentrations. Similar results were obtained when adenylate cyclase activity in bone homogenates were measured. Calvaria incubated for 60 min with PGE1 were desensitized to further stimulation by PGE1, but responded normally to PTH. Indomethacin, aspirin and phenylbutazone, 10(-5)M, had no effect on the cAMP response to PTH. At millimolar concentration these agents did block the hormone response; however, the response to exogenous PGE1 and the fluoride-sensitive adenylate cyclase were also inhibited. Thus, it appears that the effects of high concentrations of the anti-inflammatory drugs on cAMP formation are non-specific. Our results suggest that receptors in bone for PTH and prostaglandins are separate and independent, and that the cAMP response to PTH is not mediated by prostaglandins.


Testicular binding of 125I-HCG in developing estrogenized and androgenized rats. The present work describes changes seen in the binding of 125I-HCG by testis homogenates of rats injected at the age of three days with 400 microgram estradiol-17beta-dipropionate, or 250 microgram testosterone propionate. Estrogenized and androgenized male rats showed a marked decrease of gonadotropin binding in testis at the 30th, 45th and 60th postnatal day. These results show that delayed sexual maturation of male rats treated with estrogen and androgen in the neonatal period is also related to pubertal decrease of testicular gonadotropin receptors.


Studies on protein-phosphorylation reactions in isolated chromatin. The endogenous protein-phosphorylating activity of isolated chromatin was tested. We have found that a group of high-molecular-weight proteins (Mr greater than 50 000) was preferentially phosphorylated when chromatin from mouse ascites cells or from bovine lymphocytes was incubated in the presence of ATP. After disintegration of chromatin by nuclease treatment or by high salt concentration, a larger spectrum of chromatin proteins becomes accessible for phosphorylation by the chromatin-bound protein kinase. Some observations described in this communication may help to partially explain this result. The protein kinase was not found in nucleosomal subunits, indicating a non-random distribution of the enzyme in chromatin. This suggests that enzyme and substrate have to be in close spatial contact for the phosphorylation reaction to occur. Furthermore, we have shown for one protein, histone H1, that phosphorylation sites for the endogenous protein kinase are available on the free but not on the DNA-bound protein, suggesting that phosphate-accepting sites in chromatin proteins may be blocked by protein-DNA or by protein-protein interactions. We also discuss the possibility that chromatin protein kinase occurs in stable complexes with its phosphate-accepting substrates, as has been suggested by the findings of other [Kish, V.M. & Kleinsmith, L.J. (1974) J. Biol. Chem. 249, 750-760].


The isolation of tubulin and actin from mouse 3T3 cells transformed by simian virus 40 (SV3T3 cells), an established cell line growing in culture. Tubulin can be purified from mouse SV3T3 cells (3T3 cells transformed by SV40 virus) by several cycles of temperature-dependent polymerization and depolymerization. Electron microscopical analysis of the final product reveals morphologically normal microtubules. Homogeneous actin can be isolated as a byproduct of the purification procedure. Mouse SV3T3 actin and skeletal muscle actin were compared by fingerprint analysis of the tryptic peptides obtained from performic-acid-oxidized protein. The two actins show a high degree of homology although apparently five of the twenty-five spots visualized by fluorescamine show a difference in chromatographic mobility. The purification procedure described allows the rapid isolation of both actin and tubulin from tissue culture in sufficient amounts for comparative biochemicals studies.


Purification and primary structure of cytochrome f from the cyanobacterium, Plectonema boryanum. The amino acid sequence of the soluble c-type cytochrome, cytochrome f, from the cyanobacterium Plectonema boryanum (also called Phormidium luridum or Schizothrix calcicola) has been determined. The proposed sequence consists of one polypeptide chain of 85 residues and has three Asn-Gly linkages. Partly due to the presence of these Asn-Gly bonds, which readily undergo rearrangement, proteolytic digestion on the small amount of protein available was unsatisfactory. The structure was determined partly by a combination of chemical cleavage and automatic sequencing techniques. A new technique for conserving material by cyanogen bromide cleavage of residual polypeptide after automatic degradation is described. The possible evolutionary significance of primary structure comparisons with other cytochromes f is discussed.


Induction of division synchrony in Tetrahymena pyriformis by a single hypoxic shock. Its use in elucidating control of the cell cycle by adenosine 3':5'-monophosphate. A single hypoxic shock was used to induce division synchrony in Tetrahymena pyriformis. Hypoxia results in accumulation of the cells in the G2 phase of the cell cycle. Cyclic AMP and adenyl cyclase activity were measured in this system. Cell-cycle blockade was associated with extraordinarily high levels of intracellular cyclic AMP. After release from hypoxia, the cells retain a characteristic pattern of modulation of cyclic AMP associated with division that is found in selection-synchronized cells. The results are discussed with reference to other methods of induction synchrony and related studies on the natural cell cycle in this organism.


Choline kinase and ethanolamine kinase are separate, soluble enzymes in rat liver. Choline kinase and ethanolamine kinase are located in the cytosol from rat liver and have been copurified more than 500-fold by affinity chromatography [P. J. Brophy and D. E. Vance (1976) FEBS Lett. 62, 123-125]. Kinetic properties of the two activities were determined. Choline kinase had a Km for choline of 0.033 mM and ethanolamine was a competitive inhibitor (Ki = 6.2 mM). Ethanolamine kinase had a Km for ethanolamine of 7.7 mM and choline was a 'mixed' type of inhibitor with a Ki of 0.037 mM. Both enzymes activities responded in a similar fashion to the adenylate energy charge. Betaine and choline phosphate partially inhibited both kinases with a 93% inhibition of the ethanolamine kinase by 5 mM choline phosphate. CTP and ethanolaminephosphate partially inhibited the ethanolamine kinase, but not the choline kinase. Other metabolites tested had negliglible effects on both kinases. The affinity-column-purified enzyme was analyzed by disc gel electrophoresis which resolved the two activities. Hence, although many of the properties of the two activities are similar, choline kinase and ethanolamine kinase must be separate enzymes. Analysis of rat liver cytosol by disc gel electrophoresis indicated four isoenzymes for choline kinase and ethanolamine kinase.


Inhibition of human erythrocyte lactate dehydrogenase by high concentrations of pyruvate. Evidence for the competitive substrate inhibition. The mechanism of the inhibitory effect of high concentrations of pyruvate on human erythrocyte lactate dehydrogenase has been studied by the use of a new parameter, delta, defined as the difference between the reciprocals of initial reaction rates obtained from experimental measurements and hypothetical linear Lineweaver-Burk plots. This parameter served as a method for differentiating between the competitive and umcompetitive substrate inhibition. Results of this study indicate that pyruvate is a competitive substrate inhibitor. It is suggested that the inhibitory effect of pyruvate is due to its competition with  NADH for binding to the free enzyme and formation of an inactive enzyme-pyruvate binary complex. The competitive nature of pyruvate inhibition is further supported by the results of a kinetic study with NADH as the variable substrate. The dissociation constnat of the inactive enzyme-pyruvate binary complex was determined to be 101 micrometer. The physiological significance of the inhibitory effect could be to preserve a level of NADH concentration necessary for other vital enzymic reactions of living cells despite the presence of a high concentration of pyruvate.