Temperature sensitivity of cyclic-adenosine-3':5'-monophosphate-binding proteins, activity of protein kinases and the regulation of cell growth. A clone of neuroblastoma cells has been selected for its ability to survive and multiply at 40 degrees C. This temperature-resistant clone, like clones of neuroblastoma cells selected for resistance to dibutyryladenosine 3':5'-monophosphate (Bt2-Ado-3':5'-P) showed an increased tumorogenicity in animals and an increased saturation density at 37 degrees C. The Ado-3':5'-P-binding proteins and Ado-3':5'-P-dependent protein kinases from the temperature-resistant and non-resistant cells have been partially purified by chromatography on a DEAE-cellulose column. The Ado-3':5'-P-binding proteins from temperature-resistant cells were more sensitive to temperature than the binding proteins from non-resistant cells. After incubation of binding proteins from resistant cells at 37 degrees C, the specific activity of Ado-3':5'-P-binding to proteins was decreased about 50% and the apparent association constant (Ka) for Ado-3':5-p-binding was decreased from 7.4 X 10(7)M-1 to 4.4 x 10(7)M-1. There was no such decrease with binding proteins from non-resistant cells. A decrease in the activity of binding proteins from the temperature-resistant cells, but not of those from non-resistant cells, was also found when the proteins were stored at 2 degrees C. Treatment with 2-mercaptoethanol made binding proteins from the resistant cells less temperature-sensitive. In the absence of added Ado-3:5-P the protein kinase activity from the temperature-resistant cells was about 50% of the activity from non-resistant cells. Kinase activity was increased by addition of Ado-3:5-P and there was a greater increase with kinases from resistant cells. The maximum protein kinase activity was found in the presence of 10muM Ado-3':5'-P for the temperature-resistant cells and 0.1 muM Ado-3':5'-P for the non-resistant cells. The results indicate that the temperature sensitivity of Ado-3':5'-P-binding proteins, and the activity of protein kinase from cells selected for resistance to high temperature, are similar to those of cells selected for resistance to Bt2-Ado-3':5'-P. It is suggested that the temperature sensitivity of Ado-3':5'-P-binding proteins and the activity of Ado-3':5'-P-dependent protein kinases are involved in the regulation of malignancy and of cell growth at different temperatures.


The pyruvate-dehydrogenase complex from Azotobacter vinelandii. 3. Stoichiometry and function of the individual components. Labelling studies with N-ETHYLMALEIMIDE SHOW THAT EITHER IN THE PRESENCE OF Mg2+, thiamine pyrophosphate (TPP) and pyruvate or in the presence of NADH the overall activity of the pyruvate dehydrogenase complex from Azotobacter vinelandii is inhibited without much inhibition of the partial reactions. The complex undergoes a conformational change upon incubation with NADH. The inhibition by bromopyruvate is less specific. Specific incorporation of a fluorescent maleimide derivative was observed on the two transacetylase isoenzymes. Binding studies with a similar spin label analogue show that 3 molecules/FAD are incorporated by incubation of pyruvate, Mg2+ and TPP, whereas 2 molecules/FAD are incorporated via incubation with NADH. The spin label spectra support the idea that in the complex the active centres of the component enzymes are connected by rapid rotation of the lipoyl moiety. Three acetyl groups are incorporated in the complex by incubation with [2-14C]pyruvate. Time-dependent incorporation supports the view that the two transacetylase isoenzymes react in non-identical ways with the pyruvate dehydrogenase components of the complex. The results show that the complex contains 2 low-molecular-weight transacetylase molecules and 4 molecules of the high-molecular-weight isoenzyme. Mn2+-binding studies show that the complex binds 10 ions, with different affinities. 2 Mn2+ ions are bound with a 20-fold higher affinity than the remaining 8 Mn2+ ions. The latter 8 ions bind with equal affinities and are thought to reflect binding to the pyruvate dehydrogenase components of the complex. It is concluded that the complex contains 8 pyruvate dehydrogenase molecules, 4 high-molecular-weight transacetylase molecules, 2 low-molecular-weight transacetylase molecules and 1 dimeric (2-FAD-containing) symmetric molecule of lipoamide dehydrogenase. Evidence comes from pyruvate-dependent inactivation and labelling studies that the pyruvate dehydrogenase components contain either an - SH group or an S-S bridge which participates in the hydroxyethyl transfer to the transacetylase components.


The consequences of lipid peroxidation in isolated hepatocytes. Lipid peroxidation was initiated by the addition of either ADP-complexed Fe3+ or cumene hydroperoxide to isolated rat hepatocytes and the resultant biochemical and morphological alterations investigated. As previously observed with microsomes, malonaldehyde formation was associated with the inactivation of glucose-6-phosphatase. Inhibition of microsomal oxidative drug metabolism was correlated with the release and subsequent inactivation of NADPH-cytochrome c reductase, whereas cytochrome P-450 destruction occurred only in the presence of high concentrations of the organic hydroperoxide which were associated with extensive malonaldehyde formation. Under these conditions there were also marked ultrastructural alterations in the hepatocytes which were not apparent after incubation in the presence of iron (less than or equal to 187 muM Fe3+). The latter treatment was, however, associated with moderate biochemical effects such as glucose-6-phosphatase inactivation and increased membrane permeability. The cellular defence system against lipid peroxidation is discussed and it is concluded that the isolated liver cell system provides a valuable tool for the study of lipid peroxidation and its pathological implications.


Microbody of methanol-grown yeasts. Localization of catalase and flavin-dependent alcohol oxidase in the isolated microbody. Profuse appearance of microbodies was observed in the cells of methanol-utilizing yeasts in connection with the enhanced catalase activity. These microbodies were isolated successfully by means of sucrose gradient centrifugation from the methanol-grown cells of Kloeckera sp. no. 2201. Localization of a flavin-dependent alcohol oxidase as well as characteristic microbody enzymes (catalase and D-amino acid oxidase) were ascertained in the isolated microbodies, whereas formaldehyde and formate dehydrogenases were detected in the cytoplasmic region. Localization of catalase in the isolated microbody was also demonstrated by the cytochemical technique with 3,3'-diaminobenzidine.


Nuclear protein kinases from murine cells. Three protein kinase activities are found in nuclei from three different murine cells (Ehrlich ascites cells, mouse L cells and rat glioma cells). Two of these activities are soluble, one is bound to chromatin. The soluble enzymes are similar, if not identical, to the cytoplasmic protein kinases. The chromatin-bound, adenosine-cyclic-3':5'-monophosphate-independent enzyme is not found in the cytoplasm. This enzyme is composed of one subunit with a molecular weight of 80000-90000. Some biochemical properties of this enzyme are described. A brief description of a nuclear enzyme, which dephosphorylates phosphorylated histones, is also given.


ADP-ribosylation of DNA-dependent RNA polymerase of Escherichia coli by an NAD+: protein ADP-ribosyltransferase from bacteriophage T4. A protein from bacteriophage T4 responsible for the alteration of host DNA-dependent RNA polymerase and absent in T4 alt- phage was purified from T4 phage and enriched from T4-infected cells. It is injected during infection together with the known internal proteins. It has a molecular weight of about 70000 and catalyses the release of nicotinamide and the transfer of the ADP-ribosyl moiety from NAD+ to arginyl residues of various proteins including itself. RNA polymerase from Escherichia coli accepts ADP-ribosyl residues in all four subunits; the alpha subunit reacts with very high specificity. Only half of the alpha subunits are labelled, 45% with one, 5% with two residues. The main product shows the same electrophoretic mobility as alpha subunits altered or modified in vivo. The alpha subunit in modified RNA polymerase is no acceptor.


Procollagen biosynthesis by embryonic-chick-bone polysomes. Estimation of the relative numbers of active proalpha1 and proalpha2 messenger ribonucleic acids. Both total polysomes and polysomes of different size classes isolated from embryonic chick cranial bones were allowed to complete their nascent polypeptide chains in a cell-free system containing rabbit reticulocyte post-ribosomal supernatant fraction. In this system, no de novo initiation of polypeptide synthesis occurred. The product was analysed for relative content of proalpha1 and proalpha2, the precursors of the alpha chains of collagen, by dodecylsulphate-acrylamide gel electrophoresis as well as by paper electrophoresis following tryptic digestion. The results showed that the products of polysome protein synthesis contained proalpha1 and proalpha2 in the 2:1 ratio in which the corresponding alpha chains are present in native collagen, and that proalpha1 and proalpha2 synthesising polysomes are of the same size. These findings, in conjunction with results from a previous report (Vuust, J. and Piez, K.A. (1972) J. Biol. Chem. 247, 856-862) suggest that active messenger ribonucleic acids for the proalpha1 and proalpha2 chains, respectively, are present in the cells in a ratio of 2:1, and that the rates of initiation, elongation, termination and release from polysomes are all identical for the two chains.


Radiommunoassay for human and chick prolyl hydroxylases. A radioimmunoassay is reported for measuring prolyl hydroxylase. The assay is based on the displacement of radioactively-labelled prolyl hydroxylase from its antibody by the non-labelled enzyme, and on the subsequent precipitation of the enzyme-antibody complex by a cellulose-bound second antibody. Pure prolyl hydroxylase was isolated from foetal human or chick embryo tissues by an affinity column procedure usingpoly(L-proline). The enzyme was labelled with tritium using a technique of reductive alkylation with formaldehyde and sodium [3H]borohydride. No conversion of the enzyme tetramer to its monomers was found to take place during the tritiation reaction. Experiments on the dissociation of the non-labelled enzyme indicated that the degree of displacement of the labelled enzyme was similar regardless of whether the non-labelled enzyme was in the tetramer form or in that of the subunit monomers. The sensitivity of the radioimmunoassay is of the order of 5 -- 10 ng immunoreactive prolyl hydroxylase. The concentrations of the immunoreactive prolyl hydroxylase assayed with the present method in human serum and skin and in several chick embryo tissues are reported.


Kinetics and effect of salts and polyamines on T4 polynucleotide ligase. The kinetics of T4 polynucleotide ligase has been investigated at pH 8,20 degrees C and using the double-stranded DNA substrate (dA)n - [(dT)10]n/10. Double-reciprocal plots of initial rates vs substrate concentrations as well as product inhibition studies have indicated that the enzyme reacts according to a ping-pong mechanism. The overall mechanism was found to be non-processive. The true Km for the DNA substrate was 0.6 muM and that of ATP 100 muM. Several attempts were made to reverse the T4 polynucleotide ligase joining reaction using 32-p-labelled (dA)n - [(DT)40]n/40 as substrate. No breakdown of this DNA could be detected. The joining reaction was inhibited by high concentrations, i.e. above approximately 70mM, of salts such as KCl, NaCl, NH4Cl and CsCl. At a concentration of 200 mM almost 100% inhibition was observed. Polyamines also caused inhibition of the enzyme, the most efficient inhibitor being spermine followed by spermidine. At a concentration of 1 mM spermine, virtually no joining took place. Addition of salts or polyamines resulted in a large increase in the apparent Km for the DNA substrate whereas the apparent Km for ATP remained unchanged. It is suggested that the affinity of the enzyme for the DNA substrate is decreased in the presence of inhibiting agents.


Nitrogenase from Azotobacter chroococcum. Purification and properties of the component proteins. 1. A large-scale purification of the nitrogenase components from Azotobacter chroococcum yielded two non-haem iron proteins, both of which were necessary for nitrogenase activity and each had a specific activity of approximately 2000 +/- 300 nmol of acetylene reduced/mg protein per min in the presence of sautrating amounts of the other. This procedure freed the Mo-Fe protein from a protein contaminant which had an electron paramagnetic resonance signal at g = 1.94. 2. Both proteins were purified to homogeneity as determined by disc gel electrophoresis and ultracentrifugal analysis. Both proteins were oxygen-sensitive but not cold-labile. Ultracentrifugal analysis indicated that both proteins dissociated to a slight degree at concentrations below 2 mg/ml. 3. The larger of the two proteins had a molecular weight of 227 000 and contained 1.9 +/- 0.3 atoms of Mo, 23 +/- 2 atoms of Fe, 20 +/- 2 acid-labile sulphide and 47 tryptophan residues/mol. The protein consists of 4 subunits of mol. wt 60 000 (approx.). The reduced protein showed electron paramagmetic resonance signals at g = 4.29, 3.65 and 2.013 but not in the area of g = 5 to 6. Upon oxidation abosrbance increased throughout the visible region of the ultraviolet visible spectrum, with a maximum difference between oxidised and reduced protein occurring at 430 nm. 4. The smaller protein had a molecular weight of 64 000 and contained 4 g-atoms of Fe and 4 acid-labile sulphide groups/mol but no tryptophan. It had two subunits of mol. wt 30 800. The reduced protein showed electron paramagnetic resonance signhe protein retained almost full activity after oxidation with phenazine methosulphate. The ultraviolet visible spectrum of oxidised protein was clearly different from that of the oxygen-inactivated protein: it had a sharp peak at 269 nm and a broad absorbance between 340 and 470 nm with a maximum difference between oxidised and reduced forms at 430 nm. Oxygen-inactivated protein showed a sharp peak at 277.5 nm and broad peaks from 305 to 360, 400 to 425 and 435 to 475 nm. 5. Amino acid analyses of both proteins showed that most common amino acids were present with a preponderance of acidic residues. Analyses of compositional relatedness showed that the nitrogenase proteins from A. chroococcum were most closely related to those from A. vinelandii and least so to those from Clostridium pasteurianum.


Independent regulation of the nuclear and mitochondrial DNA synthesis induced by either Simian virus 40 or serum in mouse fibroblast 3T3 cells. Resting cultures of 3T3 cells (an established line of mouse fibroblasts) were released from density inhibition by either infection with Simian virus 40 or addition of serum. The increased rate of thymidine incorporation into DNA, induced by these two agents, was measured in the presence and in the absence of three inhibitory conditions (cycloheximide or dibutyryladenosine 3':5'-monophosphate added to the medium, or lack of anchorage). The inhibition was found to be quite similar in cultures stimulated by virus or serum; under the same conditions, however, the incorporation into mitochondrial DNA was much less inhibited than that into nuclear DNA. The experiments also suggest that new protein synthesis may not be necessary, for either virus or serum, to start the inductive mechanism.


Characterization of calf-ovary adenosine;3':5'-monophosphate-dependent protein kinases and adenosine-3':5'-monophosphate-binding proteins. Protein phosphokinase activity from the calf ovary cytosol (105000 X g supernatant fraction) has been resolved by chromatography and polyacrylamide gel electrophoresis into two major protein kinases, PK-H1 and PK-H2, both dependent on adenosine 3':5'-monophosphate (cyclic AMP). The enzymes have similar molecular weights (230000) and substrate specificities but differ in their cyclic-AMP-dependency and stimulation by cyclic AMP. The differences have been explained by the presence in PK-H1 of a unique cyclic-AMP-binding protein which has little catalytic activity associated with it. The cyclic-AMP-binding protein has a high affinity for cyclic AMP and in addition is able to inhibit the activity of the isolated catalytic subunit. The ovarian cyclic-AMP-dependent protein kinases have properties similar to those found in other tissues. They can be dissociated into catalytic and regulatory subunits and are inhibited by a heat-stable protein inhibitor isolated from rabbit skeletal muscle. Preincubation of the cytosol with high levels of cyclic AMP resulted in additional cyclic-AMP-dependent protein kinases and cyclic-AMP-binding proteins which include protein kinases and binding proteins of greater than 400 000 molecular weight.


Cleavage map of the simian-virus-40 genome by the restriction endonuclease III of Haemopholus aegyptius. Enzymic digestion of Simian virus 40 (SV40) DNA with Haemophilus aegyptius restriction endonuclease Hae III results in 10 major and eight minor fragments. These were resolved by electrophoresis on graduated polyacrylamide slab gels. All fragments have been characterized with respect to the size relative to the Haemophilus influenzae Rd fragments (Hind). They were ordered on the SV40 DNA map by means of overlap analysis of the double cleavage products derived from sequential digestion of Hind fragments with Hae III endonuclease and Hae fragments with Hind II + III enzyme, as well as by other reciprocal cleavage experiments, including those involving Haemophilus para-influenzae fragments. In this way the 18 Hae III cleavage sites and the 13 Hind sites have been localized on the circular SV40 DNA map.


Specific cleavage and physical mapping of simian-virus-40 DNA by the restriction endonuclease of Arthrobacter luteus. Simian virus 40 (SV40) DNA (strain 776) is cleaved by the restriction endonuclease from Arthrobacter luteus into 32 specific fragments including 20 large pieces designated Alu-A through T as well as 12 minor products named Alu m1 through m8. These were mapped on the SV40 genome by double digestion experiments. Alu fragments were treated with Hind enzymes and vice versa. Similar reciprocal digestions were also carried out with Hae III enzyme. In this way a detailed cleavage map of the SV40 genome could be constructed.


Conformational changes in glycogen phosphorylase studied with a spin-label probe. Phosphorylase b and a were covalently modified on essentially one -- SH group per subunit by a spin label 4-(2-iodoacetamido)2,2,6,6-tetramethyl piperidinyloxyl. The labelled enzyme is fully active and exhibits all the characteristics of the native molecule. The electron spin resonance spectrum of the label depends on the nature of the ligand that is bound to the enzyme. This property of the spin label is used to study the interaction between the enzyme (both in the b and a forms) and activators (AMP, IMP, CMP), inhibitors (ADP, ATP, UDPG, glucose 6-phosphate), substrates (phosphate and glucose 1-phosphate) and other ligands (adenosine, beta-glycerol-2-phosphate). The interactions are analysed in terms of the apparent ligand dissociation constants and the multiplicity of conformations that this regulatory enzyme exhibits.


Heterotropic interactions of ligands with phosphorylase b. 1. The interaction of rabbit muscle glycogen phosphorylase b with pairs of ligands has been examined. 2. The electron spin resonance spectrum of a spin label, covalently attached to the protein, provided information about dissociation constants, formation of ternary complexes and both negative and positive interactions between different ligand pairs. 3. AMP competes with a series of nucleotides (ADP, ATP, CMP aand cytosine) but with adenosine a ternary enzyme - AMP - adenosine complex can be formed. 4. ADP binding is tight and ADP inhibits the AMP activation of phosphorylase b in a physiologically important concentration range. 5. The substrates glucose 1-phosphate and glycogen tighten AMP binding in the ternary complex as does the competitive inhibitor UDPG. Inorganic phosphate is different in this respect. Gluconolactone, a transition state analogue, competes with glucose 1-phosphate (but not with glycogen) but does not prevent completely the binding of the sugar phosphate. 6. The effect of glucose b-phosphate on phosphorylase is rather complex as it 'formally competes' with both AMP and UDPG probably mediated by a conformational changes and not by 'direct' interactions with these two ligands. Glycerol 2-phosphate, a commonly used buffer for phosphorylase, also shows complex interactions.


Localization in yeast mitochondrial DNA of mutations expressed in a deficiency of cytochrome oxidase and/or coenzyme QH2-cytochrome c reductase. 1. Three methods are described for the genetic analysis of yeast cytoplasmic mutants (mit- mutants) lacking cytochrome oxidase or coenzyme QH2-cytochrome c reductase. The procedures permit mutations in mitochondrial DNA to be mapped relative to each other and with respect to drug-resistant markers. The first method is based upon the finding that crosses of mit- mutants with some but not other isonuclear q- mutants lead to the restoration of respiratory functions. Thus a segment of mitochondrial DNA corresponding to a given mit- mutation or to a set of mutations can be delineated. The second method is based on the appearance of wild-type progeny in mit- X mit- crosses. The third one is based on the analysis of various recombinant classes issued from crosses between mit-, drug-sensitive and mit+, drug-resistant mutants. Representative genetic markers of the RIBI, OLII, OLI2 and PAR1 loci were used for this purpose. 2. The three methods when applied to the study of 48 mit- mutants gave coherent results. At least three distinct regions on mitochondrial DNA in which mutations cause loss of functional cytochrome oxidase have been established. A fourth region represented by closely clustered mutants lacking coenzyme QH2-cytochrome c reductase and spectrally detectable cytochrome b has also been studied. 3. The three genetic regions of cytochrome oxidase and the cytochrome b region were localized by the third method on the circular map, in spans of mitochondrial DNA defined by the drug-resistant markers. The results obtained by this method were confirmed by analysis of the crosses between selected mit- mutants and a large number of q- clones whose retained segments of mitochondrial DNA contained various combinations of drug-resistant markers. 4. All the genetic data indicate that the various regions studied are dispersed on the mitochondrial genome and in some instances regions or clusters of closely linked mutations involved in the same respiratory function (cytochrome oxidase) are separated by other regions which code for entirely different functions such as ribosomal RNA.


Activity of the 30-S CsCl core in elongation-factor-dependent GTP hydrolysis. The activity of a 30-S CsCl core lacking proteins S1, S2, S3, S5, S9, S10, S14, S20 and S21 has been studied in the ribosome-dependent FTPase reactions in the presence of the 50-S subunit with and without methanol. Without methanol, the 30-S CsCl core was unable to sustain GTPase activity dependent on elongation factor G (EF-G), while it was only slightly active in the presence of elongation factor T (EF-T). With EF-T, addition of methanol induced in the presence of either 30-S subunits or 30-S CsCl cores an activity which was more than 10-fold higher than that observed with the 30-S subunit in the absence of methanol. Methanol lowered the Mg2+ optimum of the EF-T-dependent GTPase reaction from approximately 20 mM to approximately 10 mM. In the absence of methanol the EF-G-dependent (GTPase reaction at low concentration of monovalent cations depends on the 50-S subunit alone (30-S-uncoupled EF-G GTPase). Addition of the intact 30-S subunit but not of its CsCl core abolished inhibition of the 30-S-uncoupled EF-G-GTPase by NH4+. The 30-S CsCl core caused the same effect as the 30-S subunit when methanol was present. 30-S-uncoupled EF-G GTPase activity was lower than the GTPase activity dependent on 30-S plus 50-S subunits at [EF-G]/[50-S] below 5 but was considerably higher in the presence of a large excess of EF-G. In the presence of methanol the 30-S CsCl core behaved similarly to the 30-S subunit. Our results indicate that the action of the 30-S subunit in elongation-factor-dependent GTPases is supported by structural features that are preserved in the 30-S CsCl core. The 30-S split proteins are therefore not essential for EF-G and EF-T activities in the hydrolysis of GTP. With EF-T, in all conditions tested association of the ribosomal subunits appeared to accompany GTPase activity. Association seems also to be a prerequisite of the EF-G GTPase activity that depends on both ribosomal subunits.


The involvement of cytochrome P-450 in hepatic microsomal steroid hydroxylation reactions supported by sodium periodate, sodium chlorite, and organic hydroperoxides. The mechanism of steroid hydroxylation in rat liver microsomes has been investigated by employing NaIO4, NaClO2, and various organic hydroperoxides as hydroxylating agents and comparing the reaction rates and steroid products formed with those of the NADPH-dependent reaction. Androstenedione, testosterone, progesterone, and 17beta-estradiol were found to act as good substrates. NaIO4 was by far the most effective hydroxylating agent followed by cumene hydroperoxide, NADPH, NaClO2, pregnenolone 17alpha-hydroperoxide, tert-butyl hydroperoxide, and linoleic acid hydroperoxide. Androstenedione was chosen as the model substrate for inducer and inhibitor studies. The steroid was converted to its respective 6beta-, 7alpha, 15-, and 16alpha-hydroxy derivatives when incubated with microsomal fractions fortified with hydroxylating agent. Evidence for cytochrome P-450 involvement in androstenedione hydroxylation included a marked inhibition by substrates and modifiers of cytochrome P-450 and by reagents which convert cytochrome P-450 to cytochrome P-420. The ratios of the steroid products varied according to the type of hydroxylating agent used and were also modified by in vivo phenobarbital pretreatment. It was suggested that multiple forms of cytochrome P-450 exhibiting different affinities for hydroxylating agent are responsible for these different ratios. Horse-radish peroxidase, catalase, and metmyoglobin could not catalyze androstenedione hydroxylation. Addition of NaIO4, NaClO2, cumene hydroperoxide and other organic hydroperoxides to microsomal suspensions resulted in the appearance of a transient spectral change in the difference spectrum characterized by a peak at about 440 nm and a trough at 420 nm. The efficiency of these oxidizing agents in promoting steroid hydroxylation in microsomes appeared to be related to their effectiveness in eliciting the spectral complex. Electron donors, substrates, and modifiers of cytochrome P-450 greatly diminished the magnitude of the spectral change. It is proposed that NaIO4, NaClO2, and organic hydroperoxides promote steroid hydroxylation by forming a transient ferryl ion (compound I) of cytochrome P-450 which may be the common intermediate hydroxylating species involved in hydroxylations catalyzed by cytochrome P-450.


The mode of synaptic linkage in the cerebro-ponto-cerebellar pathway of the cat. I. Responses in the brachium pontis. Cerebrally-induced responses of pontine nuclei cells (PN cells) were studied in cats anesthetized with pentobarbitone sodium and with the midbrain transected bilaterally sparing only the cerebral penuncles. After stimulating the subcortical white matter, the internal capsule or the cerebral peduncle, mass potentials were recorded from the cut end of fibres in the brachium pontis (BP) and in the pyramid at the level of the trapezoid body. These potentials were regarded as indicating, respectively, the size of an output volley of PN cells and the size of its causative input volley through the pyramidal tract. BP responses consisted of short- and long-latency potentials which were caused by fast and slow conducting pyramidal tract volleys, respectively. The input-output relations for fast component responses took a characteristic S-shaped form resembling those obtained from the monosynaptic spinal reflex. The input-output relations for slow component responses were almost linear. Both fast and slow BP responses were remarkably potentiated after single or relatively brief repetitive peduncular stimulation, but were depressed after long-lasting high-frequency activation. During repetitive stimulation with varied frequencies, fast and slow BP responses showed different patterns of frequency-dependence of their amplitudes. These results suggest the existence of two separate transmission lines with different properties in cerebro-ponto-cerebellar pathways.


The mode of synaptic linkage in the cerebro-ponto-cerebellar pathway of the cat. II. Responses of single cells in the pontine nuclei. Extracellular and intracellular recordings were made from single cells in the pontine nuclei (PN) of the cat. PN cells were identified by antidromic invasion from the cerebellum by stimulating either the brachium pontis (BP) or the white matter near the cerebellar nuclei. The cerebrally-induced impulses excited PN cells postsynaptically with a monosynaptic latency. Both fast and slow conducting cortical fibres contributed to the corticopontine excitation, so that the latency varied over a wide range. Measurements of the latencies for antidromic and corticopontine excitation and of the distances between stimulated sites permitted the calcuation of conduction velocities of PN cell axons and of their cortical input fibres. PN cells with fast conducting axons received convergence from both fast and slow cortical fibres, whereas PN cells with slow axons were innervated only by slow cortical fibres. The majority of PN cells were also excited by stimulating the medullary pyramid through collaterals of the pyramidal tract. Evidence of abundant pyramidal collaterals was provided by the collision technique. The functional role of the PN is discussed in connection with the cerebro-cerebellar loop circuits.


The vestibulospinal tract: crossed and uncrossed effects on hindlimb motoneurones in the cat. Effects produced by stimulation of Deiters' nucleus on contralateral hindlimb motoneurones were investigated in the cat with intracellular and monosynaptic reflex recording. Polysynaptic PSPs were evoked in all species of motoneurone examined. The minimal synaptic linkage was found to be disynaptic in the excitatory path and prisynaptic in the inhibitory path. Experiments with various lesions to the spinal funiculus and those with mapping of the stimulated sites in the brain stem showed that the effects were evoked almost exclusively via the lateral vestibulospinal tract of the stimulated side. The crossed effects were excitatory in extensor motoneurones of all species studied except for hip extensors (ABSm) in which EPSPs and/or IPSPs were evoked. The effects on flexors, by contrast, were not simple and consisted of EPSPs, IPSPs or a mixture of both. The difference in effects depended, though not entirely, on the species of motor nucleus. Between the excitatory and inhibitory effects on knee flexor (PBSt) motoneurones the former predominated under chloralose and the latter under pentobarbital anaesthesia. By recording PSPs evoked from the two vestibulospinal tracts in the same motoneurone, the convergence pattern and the magnitude of effects from the two tracts were studied. The interneuronal organization of the crossed vestibulo-motoneuronel pathway and its functional significance are discussed.


Interaction between dehydroepiandrosterone, glucose-6-phosphate dehydrogenase, and cyclic adenosine-3',5'-monophosphate in neoplastic and normal human mammary tissue. When total DHEA, G-6-PDH activity, and c-AMP were determined in human neoplastic mammary tissue and corresponding normal tissue the G-6-PDH activity in the former tissue greatly exceeded that found in normal tissue. On the other hand, a remarkable decrease of total DHEA and c-AMP could be detected in cancer tissue, hinting at the participation of DHEA in the intracellular regulation of G-6-PDH and c-AMP levels.


Changes in the apoprotein composition of very low density lipoproteins in man following eating. The concentrations of certain of the very low density human serum lipoprotein apoproteins were found to increase following a meal, while the concentrations of the majority of the apoproteins were found to be unaffected.


Polycationic modified polypeptides enhancing poly I:C induced viral resistance. Polycationic modified derivatives of polyglutamic acid are at least as good enhancers of poly I:C induced viral resistance in various cell cultures as are DEAE-dextran or poly-L-lysine.


Depression of humoral and cell-mediated immune responses by coxsackieviruses in mice. Adult mice infected with coxsackieviruses A-15, B-1, B-2, B-4 and B-6 showed depressed antibody responses to unrelated antigens; mice infected with coxsackievirus B-3 developed reduced humoral and cell-mediated immune responses. These findings might have clinical and epidemiological implications.


Distribution of glycerophosphorylcholine diesterase in rat brain. The distribution of glycerylphosphorylcholine diesterase in the rat brain has been examined. The enzyme was evenly distributed throughout the brain but was localized in the synaptosome (nerve ending) fraction which was prepared by ultracentrifugation.


Creatinine metabolism by Clostridium welchii isolated from human faeces. A Clostridium welchii has been isolated from human faeces which can deaminate creatinine to N-methyl hydantoin. Evidence suggests the reaction is inducible since the rate of conversion is increased by growth of the organism in creatinine-rich media.


Effect of vitamin A on tumor development in burned, unburned, and glucocorticoid-treated mice inoculated with an oncogenic virus. High doses of vitamin A decreased the severity of tumor development in mice inoculated with a murine sarcoma virus; the same doses of vitamin A had no effect on the increased tumorigenesis seen in animals severely stressed with thermal injury or the increased tumorigenesis induced by exogenous glucocorticoid administration.


Variations of cyclic nucleotide monophosphate levels during spontaneous uterine contractions. Oscillations on the concentrations of cyclic nucliotides occurred during spontaneous rhythmic contractions in rat uterus. The levels of cyclic GMP and cyclic AMP were highest at the beginning of the contraction respectively the relaxation.


Respiratory inhibition and reversible fusion of frog blastomeres. Rotenone and high doses of chloramphenicol, both of which specifically inhibit electron transport between NADH and flavoprotein in the respiratory chain, caused fully separated Rana pipiens blastomeres to refuse, as shown by syncytium counts on embryos reconstructed from serial sections. With chloramphenicol, the effect was completely reversible: re-cleavage and normal development followed drug removal. The blastomere fusion effect was not produced by the succinic dehydrogenase-specific respiratory inhibitor, thenoyltrifluoroacetone, nor by a non-mitochondrial protein synthesis inhibitor, cycloheximide, both of which instead produced simple arrest of cleavage.


A technical improvement in the radioimmunoassay of cyclic-AMP. An improvement in the technique for the radioimmunoassay of cyclic-AMP, wherein ammonium sulfate precipitation is replaced with zirconyl phosphate gel, is presented. This substitution produces a more stable pellet than that obtained with ammonium sulfate. This greatly reduces a potential source of error due to pellet instability.


Aspects of the biochemical toxicology of cadmium. Cadmium, in addition to producing a variety of toxic manifestations, is known to accumulate in certain "target" organs which include liver and kidney where histological and functional damage becomes apparent. The daily intraperitoneal injection of cadmium chloride for 21 or 45 days stimulated the activities of hepatic pyruvate carboxylase, phosphoenolpyruvate carboxykinase, fructose-1, 6-diphosphatase and glucose-6-phosphatase elevated blood glucose and urea, and lowered hepatic glycogen in rats. Whereas chronic Cd treatment failed to alter adenosine-3', 5'-monophosphate phosphodiesterase (PDE) activity, cyclic AMP (cAMY and the activity of basal and fluoride-stimulated forms of hepatic adenylate cyclase (AC) were markedly increased. However, the cAMP binding to hepatic protein kinase was decreased as was the kinase activity ration. An acute dose of Cd decreased hepatic glycogen content and increased blood glucose, serum urea, and hepatic cAMP. Chronic exposure to Cd induced adrenal hypertrophy and augmented adrenal norepinephrine and epinephrine as well as the activity of adrenal tyrosine hydroxylase. This treatment decreased prostatic and testicular weights of mature rats. Although cAMP as well as AC activity of the prostate gland were reduced, cAMP binding to the prostatic protein kinase was increased as was the activity of the cAMP-dependent form of the enzyme. Testicular AC and PDE activities, however, were stimulated, although cAMP remained unaffected. Whereas the activities of the cAMP-dependent and the independent forms of testicular protein kinase were significantly depressed, the binding of cAMP to protein kinase from testes of Cd-treated rats was not affected. In most cases, the observed metabolic alterations persisted up to 28 days on cessation of Cd administration. Subacute Cd treatment suppressed pancreatic function as evidenced by lowered serum immunoreactive insulin (IRI) in presence of hyperglycemia, as well as by partial inhibition of phentolamine-stimulated increases in serum IRI. Although chronic Cd treatment failed to alter the concentration of brain stem norepinephrine and cerebrocortical acetylcholine esterase activity, serotonin levels of brain stem were depressed and the concentration of striatal dopamine and cerebrocortical acetylcholine were significantly elevated when compared with the values seen in control nonexposed animals.


A case of xanthomatosis and hyperlipoproteinemia type V propably induced by overdosage of insulin. A woman, aged 46 years, with a hyperlipoproteinemia phenotype V, an unusual type of xanthomatosis, as well as lipemia retinalis, diabetes mellitus, and nephropathia, is discussed. The withdrawal of 64 IU insulin and the institution of a hypocaloric diabetes diet resulted in the disappearance of the skin lesions and a regression of the eye lesion. During this treatment the total lipid-level became almost normal (6,965 mg% before treatment and 947 mg% after 10 weeks). The chylomicrons disappeared but the VLDL content remained relatively and absolutely elevated, as in type IV hyperlipoproteinemia.


Differentiation between steroid hormone receptors CBG and SHBG in human target organ extracts by a single-step assay. The steroid complexes of (plasma) corticosteroid-binding globulin can be distinguished from intracellular steroid-receptor complexes by agar electrophoresis at low temperature in neuraminidase-treated tissue extracts. With this method, the presence of progesterone receptor has been demonstrated in heavily plasma-protein-contaminated human uterus "cytosol", but not in human mammary carcinoma extracts. SHBG and "basic" receptors for estradiol and dihydrotestosterone in human uterus cytosol could also be assayed simultaneously.


Testicular inhibition of fetal Mullerian ducts in vitro: effect of dibutyryl cyclic AMP. The effect of dibutyryl cyclic AMP (dibutyryl cAMP) on Mullerian ducts of 141/2-day-old rat fetuses in vitro was investigated. Dibutyryl cAMP was unable to produce the typical regression of Müllerian ducts which is observed in vivo in male fetuses and in vitro with Müllerian ducts in the presence of testes. Effects of dibutyryl cAMP and theophylline on regeneration of Müllerian ducts induced by fetal testes were also investigated, and it was found that 10(-3) dibutyryl cAMP added to the culture medium prevented the normal regression. Lower concentrations and theophyline (10(-3)M) were ineffective. An effect of lower doses of dibutyryl cAMP was obtained only when theophylline was also present. Because pretreatment of testes with dibutyryl cAMP did not prevent the inhibiting action of testes on Müllerian ducts, it is concluded that an interaction between dibutyryl cAMP and the inhibiting factor at the level of the target organs must be considered.


Oestrogen receptor in mammary gland cytosol of virgin, pregnant and lactating mice. A macromolecular component binding 3H-labelled 17 beta-oestradiol in a specific manner and sedimenting in the 8-10-S region on sucrose gradient has been detected in the mammary gland cytosol of ovariectomized adult virgin mice. The dissociation constant of the macromolecule-oestradiol complex is 4.2 times 10(-10)M at 4 degrees C. The binding sites for 17beta-oestradiol of cytosol are 3.7 times 10(-14) mole/mg of protein. Incubation of cytosol with different enzymes suggests that the oestrogen-binding cytosol component is proteinaceous. The binding activity is destroyed by incubation at high temperatures and by some but not all SH-reagents tested. Competition studies show a specificity for oestrogens relative to other steroid hormones. The conclusion is that mammary gland cytosol of virgin mice contains oestradiol receptor. The receptor content does not increase in a specific manner during pregnancy and lactation but rather proportionally to total mammary gland protein.


Infectivity studies of hepatitis A and B in non-human primates. We have induced hepatitis A in marmosets of the subspecies Saguinus mystax following primary inoculation with human serum containing the MS-1 strain of hepatitis A virus (HAV) and in 3 further marmoset subpassages using infective marmoset serum from each preceding passage. In each passage acquisition of serum antibody against 17 nm virus-like particles recovered from acute illness stools of human volunteers who developed hepatitis following inoculation with the MS-1 strain of HAV, as well as from acute illness stools of hepatitis A cases from a common source epidemic of heaptitis A in Arizona could be demonstrated by immune electron microscopy (IEM). Particle-containing stool filtrates from the latter epidemic also induced hepatitis in chimpanzees after intravenous inoculation. Inoculation of partially purified particles from a single banding in CsCl2 has further resulted in the induction of hepatitis in S. mystax marmosets. We have successfully induced hepatitis in a series of chimpanzees inoculated with sera containing hepatitis B surface antigen (HBsAg) of varying subspecificities. Susceptibility appears universal in animals who are initially lacking in serum antibody (anti-HBs) and antibody induced following experimental infection is homologous in subspecificity to the subspecificities of the antigenic coat components of the HBsAg in the inoculum. Results of cross challenge experiments indicate that animals developing hepatitis following inoculation of HBsAg of one subspecificity set in the d/y-w/r system do not again develop hepatitis following inoculation with HBsAg of the alternate subspecificity set.


Immune adherence and complement-fixation tests for human hepatitis A. Diagnostic and epidemiologic investigations. The reliable propagation of CR326 strain of human hepatitis A virus in Saguinus mystax marmosets has permitted the development of specific serum neutralization, complement-fixation (CF), and immune adherence (IA) assays for hepatitis A antigen and antibody. The CF and IA assays were made possible by the use of livers of CR326-infected marmosets as a source of hepatitis A antigen. All assays were shown to be specific for hepatitis A. Cases of hepatitis B did not show development of hepatitis A antibody. Hepatitis A antibody appeared following onset of illness, and, in the longest time period studied, has persisted for 7 years. Epidemiologic studies have been performed on several Costa Rican families with outbreaks of hepatitis, using the IA and CF assays. Also, several populations in the U.S.A. were studied. These indicated a high incidence of hepatitis A at an early age in Costa Rica and a relatively low incidence of hepatitis A antibody among adults in the U.S.A. It was shown that human immune globulin can be standardized for hepatitis A antibody content by the IA assay. Finally, the IA assay indicated probable hepatitis A antibody in uninoculated chimpanzees, grivet monkeys, and rhesus monkeys.


The mythology of various hepatitis A virus isolates. Several types of viral hepatitis may exist. Hepatitis A (MS-1 type) can be transmitted to marmosets and chimpanzees. Virus-like particles, which may be parvo- or enteroviruses and which have been demonstrated in feces of this type of hepatitis, do not share cross-reacting antigens with hepatitis B but do cross-react with fecal hepatitis A antigen. Hepatitis A (GB type), which also does not cross-react with hepatitis B, is not antigenically identical with MS-1; it can be transmitted to marmosets and it may be similar to non-type A/non-type B post-transfusion hepatitis. Hepatitits B does not cross-react either with HA particles, the faecal hepatitis type A antigen or with the MS-1 or GB strains; it can be transmitted to chimpanzees and rhesus monkeys but not to marmosets.


Virus-like particles in hepatitis A.

Virus-like particles in hepatitis A. Distinctive 27 nm virus-like particles have been found by immune electron microscopy in faecal extracts obtained during the late incubation of naturally occurring hepatitis A infection. A variety of particles measuring 22 nm and 30 nm have also been observed. However, the predominance of the 27 nm particles suggests that these small cubic virus-like structures are closely associated with hepatitis A.


Characterization of CR326 human hepatitis A virus, a probable enterovirus. CR326 human hepatitis A virus purified by isopycnic banding from infected marmoset sera was shown to consist of 27 mmu spherical particles on electron microscopic examination. The particles were identified as hepatitis A virus by tests for infectivity and by specific neutralization of infectivity with convalescent human hepatitis A serum. Also, identical 27 mmu viruses in liver extracts gave specific reactions with hepatitis A antisera when tested by immune electron microscopy. The buoyant density of the virus in CsCl was 1.34 and it was heat (60 degrees C), ether and acid stable but was destroyed by heat (100 degrees C), formalin (1:4000) and ultraviolet irradiation. Electron microscopic studies of sections of infected marmoset liver showed intracytoplasmic virus particles, usually in vesicles. Presumptive findings for RNA, together with the intracytoplasmic location of the virus, indicated the virus to be of RNA-type. The attributes of the virus indicate it is closely related to the enterovirus family and not to hepatitis B virus.


Attempts to differential hepatitis B from hepatitis A infection by newly developed serological tests. An immune Indian ink micro-agglutination method has been evolved for the detection of an antigen present in the blood associated with infectious hepatitis (called IHxAg). In previous studies 86% of serum samples taken from children with hepatitis A proved to be positive by this technique. Present studies were related to 239 adult in-patients with a clinical diagnosis of hepatitis A (123 cases) or hepatitis B (116 cases). Blood samples taken serially during the illness were tested for IHxAg, HBsAg and anti-HBsAg. The results were in accordance with the clinical diagnosis in 60% in contradiction in 30%, whilst all tests brought negative results in 10%. The clinical laboratory findings (SGPT, thymol turbidity) were more in harmony with our laboratory results than with the clinical diagnoses. Rheumatoid factor did not disturb the immune Indian ink reaction, labile serum proteins caused, however, non-specific reaction in 30% of serum samples. When durocytes were used instead of Indian ink the rate of false positive results dropped to 10%. Sera taken in convalescent phase from patients with IHx antigenemia in the acute phase of illness contained an antibody against IHxAg. A crude gammaglobulin preparation from a pool of convalescent sera gave a precipitation line in agarose gel with an antigen present in the fecal extract of children with hepatitis A. This precipitation line proved to contain virus-like particles with an approximate diameter of 25 nm when tested by electronmicroscopy. No precipitation could be seen when sera of the same patients were tested against the same gammaglobulin preparation.


Incorporation of 75Se-selenomethionine into human apoproteins. I. Characterization of specificity in very-low-density and low-density lipoproteins. Characterization of specificity of incorporation of 75Se-selenomethionine into human apoproteins following in-vivo injection of the radioactive amino acid precursor is reported. Apoprotein fractionation was attained by serial ultracentrifugation, anhydrous partial delipidization, decyl sulfate solubilization with Sephadex G-150 gel filtration into polypeptide fractions, Sf-I, and Sf-II, for VLDL, and LDL subfractions. Similarity of the Sf-I fractions isolated from VLDL, LDL1, and LDL2 was defined by gel exclusion volume, disk-gel electrophoresis, immunodiffusion, and amino acid composition. Enrichment of the Sf-I fractions with 75Se-SM in VLDL and LDL subfractions was observed, suggesting a possible use for this isotopic method for the investigation of apoprotein metabolism.


Incorporation of 75Se-selenomethionine into human apoproteins. II. Characterization of metabolism of very-low-density and low-density lipoproteins in vivo and in vitro. This investigation is designed to explore the potential role of apo VLDL as a precursor of a polypeptide component of human LDL. Attention was directed to the chromatography-defined Sf-I polypeptide fraction of apo VLDL, which has been previously shown to be immunologically and chemically indistinguishable from the major component of apoLDL.1-3 In VLDL isolated from bloow drawn within two hours following 75Se-SM injection, the Sf-I polypeptide fraction of apo VLDL was highly enriched with isotope, providing an appropriate preparation for in-vitro tracer studies. Conversion of 75Se-VLDL to 75Se-LDL occurred in vitro in the presence of normal plasma at 37 degrees C., and this conversion was augmented by post-heparin plasma. No conversion to HDL lipoproteins could be detected. Injection of heparin in vivo resulted in acute reciprocal changes in the radioactivity contained within serum apo VLDL and apoLDL. These findings suggest that a component of the Sf-I polypeptide fraction of apo VLDL can be metabolized into the apoprotein of LDL in man. Thus, the biochemical and immunologic similarities between the Sf-I fractions of apoVLDL and apoLDL may result from a physiologic "precursor-product" relationship between the apoprotein moieties of these two lipoprotein species. A method for further investigation of the metabolism of human apoprotein is suggested.


The control of insulin receptors in the New Zealand obese mouse. Insulin receptor concentrations in liver plasma membranes of New Zealand Obese (NZO) mice have been studied. When NZO mice were implanted with normal islets of Langerhans their blood glucose and plasma insulin declined. When the implanted islets were removed these changes were reversed and the mice reverted to their insulin resistant state. Changes were observed in the binding of 125I-insulin to liver plasma membranes of implanted NZO mice. Binding increased when the plasma insulin  was decreased and conversely insulin binding decreased when the plasma insulin levels became elevated. The increased insulin binding was not accompanied by any changes in the affinity of NZO liver receptors for insulin.


Effect of methylxanthines on alloxan inhibition of insulin release. Isloated rat islets were maintained in vitro in a perifusion system, exposed to alloxan (20 mg/100 ml) for 5 minutes in the presence of agents which affect cAMP metabolism and subsequently stimulated with glucose. The rate of insulin secretion was monitored throughout the period of perifusion. Exposure to alloxan alone produces complete inhibition of glucose-induced insulin release [18] whereas concomitant exposure to carreine and theophylline for this brief interval provided almost complete protection of the islets from the inhibitory action of alloxan. Glucagon, cAMP and CBcAMP did not protect the islets form alloxan. Pre-treatment of the islets with either theophylline or glucagon and DBcAMP did not provide protection. These findings indicate that the protective action of theophylline and carreine against alloxan is unrelated to the effect of these agents on cAMP metabolism in the beta cell.


Increased sensitivity of diabetic rat adipose tissue towards the lipolytic action of epinephrine. Adipose tissue from streptozotocin-diabetic rats exhibits half-maximal lipolytic responses (FFA, glycerol release, increase in tissue FFA) to epinephrine at hormone concentrations 5-10 times lowere than those required for half-maximal stimulation of lipolysis in adipose tissue from normal rats. The lipolytic response to epinephrine also occurs more promptly and the antilipolytic effect of insulin in the presence of submaximal epinephrine conceptrations is much less pronounced than in normal tissue. In contrast, diabetic adipose tissue is less responsive to ACTH and glucagon than normal tissue. Half-maximal lipolytic responses are elicited by similar dibutyryl cyclic AMP concentrations in both tissues. Insulin treatment of diabetic rats during 24 hrs restores the lipolytic response of their adipose tissue to epinephrine to nearly normal. Our findings point to an abnormality of diabetic adipose tissue possibly related to the hypersensitivity of catecholamines encountered in denervated organs which are adrenergically innvervated. They are consistent with present concept of different hormone discriminators on the fat cell membrane and offer a further explanation for increased FFA mobilization in the diabetic state.


Human islet cell adenoma: metabolic analysis of the patient and of tumor cells in monolayer culture. Cell cultures were established from a benign pancreatic islet adenoma. Over 200 muU/culture/day immunoreactive insulin were found in culture media. Cultures with medium 199 released insulin for about 2 months; those with medium F12K were maintained for over 7 months, and have been successfully subcultured. Increasing culture medium glucose to 326 mg per 100 ml, alone or with leucine (10 mM) or theophylline (2 mM), failed to increase insulin release above baseline. Studies in the patient prior to surgery using oral glucose, leucine, beef meal, intravenous tolbutamide, and glucagon failed to increase plasma insulin and thus were consistent with cell culture responses. Extracts of tumor tissue contained 23% proinsulin-like material; high insulin containing samples of culture medium had 5% proinsulin and less than 40 pg glucagon/ml. Aldehyde fuchsin positive granulation was sparse in both cultured cells and the original tumor. These studies demonstrate long term viability, in monolayer culture, of cells derived from this islet cell adenoma, with retention of secretory characteristics consistent with data obtained prior to removal of the adenoma from the patient.


Function of microdissected pancreatic islets cultured in a chemically defined medium. I. Insulin content and release. Microdissected pancreatic islets from non-inbred ob/ob-mice were cultured for 6 or 7 days in serum-free tissue culture medium 199. The insulin content of the islets decreased 60% during culture in 17 mM or 28 mM glucose and about 70% in the presence of 3.3 mM or 5.6 mM glucose. At the end of a culture period in high glucose, the sum of the insulin in the islet plus that in the culture medium was almost twice as high as the insulin content of fresh islets, indicating an active insulin biosynthesis. The maximal insulin response to glucose after culture in 17 mM or 28 mM glucose was about 40% of that in fresh islets; after culture in 3.3 mM glucose it was 10%. Half-maximal stimulation was observed at a glucose concentration of 5 mM for islets cultured with high glucose as compared to 9 mM for fresh islets. Like glucose, glibenclamide was a more effective insulin stimulator after culture with a high glucose concentration than with a low one. However, leucine-induced insulin release was not affected by the glucose concentration in the preceding culture medium. Whereas potentiation of glucose-stimulated release by arginine or dibutyryl-cAMP was independent of glucose concentration during the culture, theophylline released three times more insulin when the islets had been cultured with high glucose.


Cyclic AMP in pancreatic acinar cells: effects of gastrointestinal hormones. In the presence of 5 mM theophylline, secretin and vasoactive intestinal peptide (VIP) each increased cyclic adenosine 3':5'-monophosphate (cyclic AMP) in acinar cells isolated from guinea pig pancreas. Without theophylline, neither peptide altered cellular cyclic AMP. Glucagon, which is similar to secretin and VIP both in chemical structure and spectrum of biologic activities, neither stimulated cellular cyclic AMP nor inhibited the stimulation produced by secretin or by VIP. Other agents which were tested and found not to increase cellular cyclic AMP were cholecystokinin, carboxyl-terminal octapeptide of cholecystokinin, gastrin I, gastrin II, bovine pancreatic polypeptide, carbamylcholine, and prostaglandin E1. Neither carboxyl-terminal octapeptide nor gastrin I altered the stimulation of cellular cyclic AMP produced by secretin or VIP. With natural secretin a significant increase in cellular cyclic AMP could be detected at concentrations as low as 3 x 10(-10) M and maximal stimulation occurred at 10(-8) M. VIP was approximately 1% as potent as natural secretin and maximal concentrations of secretin plus VIP increased cellular cyclic AMP to the same value as was obtained with a maximal concentration of secretin alone.


Colonic blood flow in cat and man as analyzed by an inert gas washout technique. An inert gas elimination technique for studying blood flow and flow distribution in the colon was developed on the cat and applied to patients during abdominal surgery. The method involves recording of the elimination of intra-arterially injected 85Kr from a colonic segment whereby the gamma- and beta-radiation of the tracer is registered simultaneously by a scintillation detector and Geiger-Muller tube, respectively. Total blood flow was determined from the recording of gamma-radioactivity using a modification of Zierler's formula, and muscularis blood flow as calculated according to Kety from the mono-exponential elimination recorded by the Geiger-Muller tube. The relative weights of the muscularis and mucosa-submucosa were determined from dissections or histological sections. With these weights and total and muscularis blood flows, flow in the mucosa-submucosa was calculated. Total colonic blood flow was 18 +/- 2 ml per min and 100 g of colonic tissue (mean +/- SE; n = 21). In the muscularis layer blood flow amounted to 11 +/- 1 ml per min and 100 g of muscularis tissue (n = 12), and flow in the mucosa-submucosa was calculated to be 28 +/- 5 ml per min and 100 g of mucosal-submucosal tissue. A major fraction (66 +/- 6%) of total blood flow was distributed to the mucosa-submucosa. As total colonic blood flow was enhanced the increase in flow was diverted only to the mucosa-submucosa. The feline and human large bowel exhibited great qualitative and quantitative circulatory similarities.


Blood flow in the small intestine of cat and man as analyzed by an inert gas washout technique. Using a recently developed 85Kr-elimination technique, blood flow and flow distribution of the human small intestine have been investigated in patients during abdominal surgery. Total intestinal blood flow was estimated to be 38 +/- 4 ml per min and 100 g of intestinal tissue (mean +/- SE: n = 19), jejunal blood flow being slightly higher than ileal. In 14 of these patients muscularis blood flow was determined to be 21 +/- 2 ml per min and 100 g of muscularis tissue and mucosal-submucosal blood flow was calculated to be 51 +/- 6 ml per min and 100 g of mucosal-submucosal tissue. Seventy-five +/- 3% of total blood flow was distributed to the mucosa-submucosa and the remaining 25 +/- 3% to the muscularis. It was demonstrated that an increasing fraction of flow was diverted to the mucosa-submucosa with enhanced total intestinal blood flow. The human gut exhibited great qualitative and quantitative circulatory similarities with the feline small intestine.


[Differential ultrastructural elements in human cytomegalovirus and Herpes simplex virus replication in cultures of human cells]. Differential ultrastructural features in the replicative cycle between a strain of human cytomegalovirus (CMV) and two strains (types 1 and 2) of herpes simplex virus (HSV) have been studied by electron microscopy. Either on the same or different host cell system four differential aspects have been constantly observed: a) presence in CMV infected cells of a large nuclear inclusion, lacking in HSV infected cells; b) envelopment of CMV capsids by microvescicular systems near the nuclear membrane, whereas HSV (types 1 and 2) takes envelope from the inner lamina of the nuclear membrane; c) presence in the cytoplasm of CMV infected cells of homogeneous dense material and dense microbodies, lacking in HSV infected cells; d) in extracellular space homogenous dense microbodies are present only in CMV infected cell cultures. Diagnostic usefulness of the above reported ultrastructural differences and their meaning on the basis of most recent data of the literature are discussed, with special concern to envelopment of two viruses and to the nature of homogeneous dense material observed in CMV infected cells.


A deletion map of cyc1 mutants and its correspondence to mutationally altered iso-1-cytochromes c of yeast. Mutants arising spontaneously from sporulated cultures of certain strains of yeast, Saccharomyces cerevisiae, contained deletions of the CYC1 gene which controls the primary structure of iso-1-cytochrome c. At least 60 different kinds of deletions were uncovered among the 104 deletions examined and these ranged in length from those encompassing only two adjacent point mutants to those encompassing at least the entire CYC1 gene. X-ray-induced recombination rates of crosses involving these deletions and cyc1 point mutants resulted in the assignment of 211 point mutants to 47 mutational sites and made it possible to unambiguously order 40 of these 47 sites. Except for one mutant, cyc1-15, there was a strict colinear relationship between the deletion map and the positions of 13 sites that were previously determined by amino acid alterations in iso-1-cytochromes c from intragenic revertants.


Lipid deficiencies, leukocytosis, brittle skin--a lethal syndrome caused by a recessive mutation, edematous (oed), in the mouse. A new neonatal lethal mutation in the mouse with pleiotropic effects, edematous (oed), arose spontaneoutosomal recessive mode of inheritance. The external phenotypic characteristics include a generally bloated appearance, shiny celophane-like skin, and distal hematomata of the extremities. Internally, no gross or histological abnormalities could be identified, with the exception of a striking leukocytosis. Biochemical analysis revealed a severe disturbance of lipid metabolism. Deficiencies in the VLDL, LDL, and the HDL lipoprotein fractions have been found in the mutant plasma. In addition, all serum lipids are markedly decreased. However, in the mutant liver, only triglycerides are significantly decreased; total liver cholesterol and phospholipid values are within normal limits. The primary biochemical defect as well as the causal relationship between the striking abnormalities of lipid metabolism and those of skin and blood are unknown at this time.


Interrelationships of dental maturity, skeletal maturity, height and weight from age 4 to 14 years. The correlations of dental mineralization stages, skeletal mineralization, body-height and weight were determined for each year between age 4 and 14 for 121 boys and 111 girls of the serial experimental group of the Burlington Growth Centre. Dental development related more strongly to morphological development than to skeletal development in both sexes, and skeletal age was more strongly related to morphological age than to dental stage. The relationship of individual teeth to skeletal age, height and weight were different but consistent in a sex-specific pattern. In males, both skeletal mineralization and dental mineralization, particularly of the first molars, were more closely related to height than to weight. In the females, skeletal and dental mineralization, especially of the second molars, were more closely related to weight from age 7 than to height. These relationships were significant in both pre-adolescent and adolescent years. In both these periods, skeletal age and body size related most strongly to late stages of tooth formation. Hence, from an early age, the stages of dental, skeletal and morphological maturation were significantly interrelated in a pattern that was specific for individual teeth and for sex.


Stimulation of water and sodium secretion and inhibition of glucose absorption from the rat jejunum during intraarterial infusions of prostglandins. The effect of prostaglandins, isoprenaline and dibutyryl cyclic adenosine monophosphate (dibut cAMP) on the net transfer of water and Na+ and glucose absorption have been studied in the anaesthetized rat. The lumen of the jejunum was recirculated with a solution of normal saline containing D (+) glucose and phenosulphonphthalein. The superior mesenteric artery was perfused extracorporeally and drugs were infused into the arterial blood. Prostaglandin E1 (PGE1) at infusion rates of 2-5 X 10(-9) and 10(-8) mol/min induced a net secretion of water and Na+ which was significantly different from the infused control mean. Both water and Na+ secretion were dose-related. PGE1 induced slight but significant inhibition of glucose absorption at 10(-8) mol/min. This could either be a direct effect or secondary to solvent drag. PGE2 (2-5 X 10(-9) mol/min) and PGF2alpha (8 X 10(-9) mol/min) induced water and Na+ secretion, and inhibited glucose absorption but not significantly. Isoprenaline (10(-9) mol/min) and dibut cAMP (8 X 10(-9) mol/min) did not alter water, Na+ or glucose absorption.


Oxidative enzymes in the development of Fasciola hepatica L. IV. The activity of oxidases and dehydrogenases in redia. The object of the study was to investigate the occurrence and localization of oxidative enzymes in the redia -- the third larval stage of Fasciola hepatica L. The author detected cytochrome oxidase, peroxidase, NADH and NADPH tetrazolium reductases (diaphorases), as well as succinate, isocitrate, malate, lactate, alpha-glycerophosphate, glyceraldehyde phosphate, glucose-6-phosphate, 6-phosphogluconate, beta-hydroxybutyrate, L-glutamate, and alcohol dehydrogenases. The presence and localization of the enzymes in various periods of development of the redia were detected with histochemical methods. Out of the studied oxidases and dehydrogenases only cytochrome oxidase was found to be absent from the stages of young rediae. It was ascertained that the redia uses all three paths of release of energy i.e. the glycolytic, Krebs, and pentose cycles, glycolysis being presumably the principal mode of energy production.


Oxidative enzymes in the development of Fasciola hepatica L. V. Activity of oxidases and dehydrogenases in the Cercaria and Metacercaria. The object of the study was the investigation of the occurrence and localization of oxidative enzymes in the 4th and 5th larval stages of the liver fluke, i. e. in the cercaria and metacercaria. The following enzymes were detected: cytochrome oxidase, peroxidase, NADH and NADPH tetrazolium reductases/diaphorases) as well as succinate, isocitrate, malate, lactate, alpha-glycerophosphate, glyceraldehyde phosphate, glucose-6-phosphate, 6-phosphogluconate, beta-hydroxybutyrate, L-glutamate and alcohol dehydrogenases. The occurrence and localization of the enzymes were investigated histochemically in the cercaria still unreleased from the snail tissues, in the free natatorial cercaria, and in the encysted specimen, i.e. metacercaria. Among the enzymes studied only peroxidase was found to be absent from the cercaria and metacercaria, the latter larva being deprived of alcohol and L-glutamate dehydrogenases as well. The aerobic path, i.e. the Krebs cycle, was ascertained as the principal mode of obtaining energy in the free natatorial cercaria and metacercaria, and glycolysis as the main energy path for the undetached larva. The analysis of the above-mentioned oxidative enzymes was the basis for the description, within the available range, of the function and metabolism of individual organs of cercaria in different periods of its development. In the recapitulation the author discusses the effect of the parasitic way of life of the larval forms of Fasciola hepatica on their energy metabolism.


Specific and non-specific phosphatases in the miracidium of Fasciola hepatica L. Localization and activities of alkaline phosphatase, ATPase, 5-nucleotidase, glucose-6-phosphatase, thiamine pyrophosphatase and nucleoside diphosphatase were studied in the miracidium of Fasciola hepatica L. Except for nucleoside diphosphatase whose activity in the miracidium was not observed, all the enzymes were most active in the archenteron, protonephridia and nerve ganglion. This localization of the reaction intensity allows the inference that the three organs mentioned are sites of both intense carbohydrate metabolism and lively active transport. The role of phosphatases in carbohydrate metabolism is discussed.


[The angiographic appearances of liver haemangiomas (author's transl)]. Thirteen cases of cavernous haemangiomas of the liver are described, and the clinical features, diagnosis and treatment are discussed. The purpose of this paper is to show the value of selective coeliac angiography in the differential diagnosis. The most important angiographic findings in cavernous haemangiomas of the liver are: supplying arteries of normal width, prolonged stasis of the contrast medium producing the appearance of spotty contrast depots, frequently, sharp delineation of the tumours; no A-V fistulae and no tumour vessels. Other hypervascular liver tumours must be distinguished from these, particularly hepatocellular carcinomas and sarcomas, as well as the rare haemangioendotheliomas.


[Angiographic findings in juvenile angiofibroma of the nasopharynx (author's transl)]. Juvenile angiofibroma of the nasopharynx has a specific tendency to grow through anatomical fissures and foramina with invasion of the surroundings. Angiography by selective techniques, detail angiography and the use of subtraction permits accurate delineation of the tumour and of its vascular supply. The angiographic appearances are highly characteristic and equal in value to a histological diagnosis. Consequently a possibly difficult biopsy can be avoided and radical surgery carried out. As a result, the recurrence rate can be reduced. Post-operative control and the demonstration of recurrences is also possible with angiography.


Epithelial basement membrane of bovine renal tubuli. Isolation and analysis of the carbohydrate chains. The carbohydrate chains present in the tubular basement membrane of bovine kidney were studied. Digestion with collagenase followed with pronase resulted in a complete solubilization of the basement membrane. The different glycopeptides were purified by gel filtration and ion-exchange chromatography. Two kinds of carbohydrate chains could be characterized: oligosaccharides composed of glucosamine, mannose, galactose, fucose and sialic acid, and glucosylgalactose disaccharides. A very small portion of the oligosaccharide chains (ca. 4%) appeared to be free of sialic acid. The bulk of these chains contained sialic acid and fucose, although in small amounts. Only traces of galactosamine were found.


[Studies on the metabolism of oestrone and oestradiol-17 beta in the liver of minipigs of different ages and sexes (author's transl)]. After incubation of [4-14C]oestrone (E1) with liver slices from minipigs, the ether-soluble fraction contained [4-14C]oestradiol-17 beta (E2). In the protein-bound fractions, only polar metabolites were found, whereas in the water-soluble fraction the 3-monoglucuronide of oestriol (E1) was the preferred conjugate. When E2 was used as a substrate, E1 was present as main metabolite in the ether-soluble fraction. The radioactive metabolites in the protein-bound and water-soluble fractions were similar to those in the experiments with E1. The metabolism of E1 and E2 was dependent on age. Thus, the rate of conversion of oestrogens was greater in liver tissue of infertile male animals than in fertile males. In contrast, the two steroids were metabolised more rapidly in liver of fertile female minipigs than in infertile female animals. In fertile animals, the metabolic pattern of oestrogens in the ether-soluble, the protein-bound and the water-soluble fractions showed sex dependence: In females, E1 and E2 were metabolised to a greater extent by liver slices than in males. On the other hand, in experiments with male minipigs, E3-3-monoglucuronide was the only metabolite in the water-soluble fraction, whereas liver slices of female animals not only form E3-3-monoglucuronide, but also the 3-glucuronides of E1 and E2. The results described here show that, in liver tissue of minipigs, the oxidoreduction of E2 and E1 is the predominant reaction; in contrast to human liver, hydroxylation reactions play only a minor role. It may be concluded that there are differences in the metabolism of steroid hormones in man and minipig.


[Studies on the metabolism of oestrone and oestradiol-17 beta in the kidneys of minipigs of different ages and sexes (author's transl)]. After incubation of [4-14C]oestrone (E1) with kidney cortex slices of minipigs, [4-14C]oestradiol-17 beta (E2) and small amounts of a polar metabolite were detected in the ether-soluble fraction. E1, E2 and polar metabolites were found in the protein-bound fraction. The water-soluble fraction contained E1-3-glucuronide (80% of total glucuronides), E2-3-glucuronide and trace amounts of the 3-monoglucuronide of oestriol (E3). When E2 was used as substrate, the main product formed was E1; it was detected in both the ether-soluble and protein-bound fractions. E1-3-Glucuronide was the main metabolite in the water-soluble fraction, which also contained some E2-3-Glucuronide. In male minipigs, the mast, the rate of conversion of E1 and E2 as well as the formation of glucuronides were significantly greater in fertile females than in infertile females. Whereas no sex difference was observed in the metabolism of oestrogens in kidneys of infertile minipigs, the rate of oxidoreduction and glucuronidation was more pronounced in fertile female animals than in the corresponding males. The present results suggest that, in the kidneys of minipigs, the ratio of E1 to E2 is shifted towards the former; furthermore, by a comparatively rapid metabolism of the oestrogenic hormone, the renal tissue contributes to the maintenance of hormonal equilibrium.


Psychiatric liaison with private facilities caring for discharged patients. Motivated by concern about the fate of psychiatric hospital patients after discharge to the community, a community mental health center decided to send a psychiatrist into the community to establish liaison with private-care facilities and to determine the needs of such discharged patients. As a result of the psychiatrist's efforts, owners of the area's private-care facilities acquired a better understanding of the needs of their residents and learned where they could turn for help in caring for discharge psychiatric patients. A study of one board-and-care facility revealed a marked decrease in the use of the center's emergency service and in the number of hospital admissions after one year of this extended service.


[Quantitative and virus specific serum IgM detection in newborns (author's transl)]. Serum IgM concentration was determined in umbilical cord blood samples from 1000 newborns. 34 cases presenting elevated IgM values (greater than or equal to 30 mg%) were tested for virus specific IgM antibodies. We could demonstrate such virus specific IgM antibodies against Cytomegalovirus (4), against Rubella, Influenza A and Influenza B (2 each) and against Coxsackie virus (1). The quantitation of IgM in blood samples of child bearing women showed no elevated values compared to those of non-pregnant women of similar age groups.


Passive immunization in experimental Herpesvirus hominis infection of newborn mice. Infection of newborn mice with Herpesvirus hominis type 2(HVH-2) was used as an experimental model of disseminated HVH infection in newborn humans. Mice were challenged with 103 plaque-forming units of HVH-2 intranasally and were given 0.2 ml of rabbit serum intraperitoneally. Passive immunizations with rabbit anti-HVH-2 serum resulted in a significant decrease in mortality and prolongation of survival time. This effect correlated with the neutralizing antibody titer of the serum against HVH-2 and was more pronounced when immune serum was administered 1 h after infection as compared with 24 h. These results suggest that administration of high-titer anti-HVH-2 immunoglobulins shortly after delivery could afford significant protection to the newborn of a mother with genital HVH-2 infection.


Effect of anti-herpesvirus drugs on human and bovine lymphoid function in vitro. Lymphocyte proliferative responses to phytohemagglutinin, pokeweed mitogen, and herpesvirus (herpes simplex virus and infectious bovine rhinotracheitis virus) antigens were evaluated in the presence of various concentrations of the anti-herpesvirus drugs, methylmethoxydeoxyuridine (OCH3CH2UdR), cytosine arabinoside was inhibitory at 25 mug/ml and cytosine arabinoside was inhibitory OCH3CH2UdR per ml, lymphocyte proliferative responses were unaffected and were not abolished even by concentrations of 2,500 mug/ml. In contrast, adenine arabinocide was inhibitory at 25 mug/ml and cytosine was inhibitory at 5 mug/ml. Both direct and antibody-dependent lymphocyte cytotoxicity of herpesvirus-infected target cells were highly resistant to OCH3CH2UdR; concentrations of 1,000 mug/ml were only marginally inhibitory. The anti-herpesvirus activity of all three drugs is similar (0.5 to 8 mug/ml) (L.A. Babiuk, B. Meldrum, V.S. Gupta, and B. T. Rouse, submitted for publication). The drug OCH3CH2UdR should be given a careful, well-controlled evaluation of its effectiveness in the treatment of herpes simplex infections in animals, perhaps including humans, since our results using in vitro models show that cellular responses important for recovery from herpesvirus infections are unaffected by doses far in excess of those found be be antiviral.


Increased susceptibility to bacterial infection as a sequela of exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin. The effects of subclinical levels of 2,3,7,8-tetrachloridibenzo-p-dioxin (TCDD) on the response of mice to infection with either Salmonella bern or Herpesvirus suis, also known as pseudorabies virus, are reported. TCDD is a contaminant of certain commercially useful chemicals, such as chlorinated phenols or herbicides. It has been shown to cause thymic atrophy and to suppress cell-mediated immunity in laboratory animals. Sublethal levels of TCDD were used: 0.5, 1,5, 10, or 20 mug/kg, given through a gastric tube once weekly for 4 weeks. A significant decrease in weight gain compared with control mice occurred at the 20-mug dosage. Dose schedules of 1 mug or more, followed by salmonella infection, resulted in significant increases in mortality and decreases in the time from infection to dealth. However, TCDD had no significant effect on mortality in the pseudorabies-infected mice. The most important finding in this study is that extremely low levels of TCDD, which do not produce clinical or pathological change, still have the capacity to affect host defense.


Influence of fatty acids on pressor responses to catecholamines. Lauric, Myristic and Palmitic acids had no appreciable effect whereas Stearic, Oleic and Linoleic acids caused some reduction in dog blood pressure. Pressor responses to epinephrine and nor-epinephrine were potentiated whereas the depressor response to isoproterenol was reduced during the infusion of fatty acids in dogs. ACTH alone, which causes mobilization of free fatty acids had no appreciable effect on blood pressure responses to catecholamines, however, its administration followed by salicylate produced marked potentiation of the pressor responses to epinephrine and nor-epinephrine; the depressor response to isoproterenol was reduced.


Determination of subgroup-specific feline oncornavirus-neutralizing antibody. A microneutralization assay was developed for antibody-to-subgroup-specific feline oncornaviruses. This study combines the economic advantage of a microtiter system and the quantitative focus reduction method which permits contruction of multiplicity curves for determination of virus-neutralizing titers. A twofold increase in Synder-Theilen feline sarcoma virus (ST-FeSV) on feline embryo cells decreased by approximately twofold the titer of reference goat serum prepared against Kawakami-Theilen feline leukemia virus. Similar dose effects with FeLV serotype virus preparations were not observed. An assay system utilizing FeLV serotypes on sarcoma-positive leukemia-negative cells demonstrated slightly greater sensitivity than one employing ST-FeSV on FE cells. Differential antibody responses to the three subgroup-specific feline oncornaviruses (A, B ,and C) were observed in reference goat sera. This test demonstrated good reproducibility as well as sensitivity and constitutes a significant improvement over end point dilution assay systems.


Induction of herpes simplex virus immunity in newborn mice. It was demonstrated that newborn mice, surviving an infection with attenuated herpes simplex virus, become resistant to challenge with a pathogenic strain of herpes simplex virus. This resistance did not seem to be mediated by antibody.


An improved specific IgM antibody assay for Epstein-Barr virus. A new method for detecting specific IgM antibodies to the Epstein-Barr virus using indirect fluorescence is presented. The procedure is simple and reproducible, and of great value for the diagnosis of an acute infection. The number of positive cells is higher than in the Epstein-Barr IgG test. It would seem that it is possible to detect a positive IgM-specific antigen with this new test.


Radical formation in salts of pyrimidines. II. Cytosine. HCl crystals. Radicals produced by X-irradiation at 77 K and at 300 K of cytosine. HCl crystals have been analysed by electron spin resonance spectroscopy. Four radicals have been identified: the anion radical of the cytosine molecule, the radical resulting from H-addition at position C6, the radical resulting from H-addition at position O2, and finally a radical resulting from addition of a Cl- to nitrogen N3. Hückel molecular orbital calculations are presented, which support the hypothesis according to which in unsaturated pyrimidines the site of hydrogenation or protonation depends on the state of the molecule.


Reassociation kinetics of DNA from x-irradiated ascites hepatoma cells of the rat. The kinetics of the reassociation fo DNA from ascites hepatoma cells has been studied. The curve exhibited three zones corresponding to 'fast', 'intermediate' and 'slow' speeds of DNA reassociation. The difference was observed in the DNA reassociation curves of the control and irradiated (1500 rad) cells which was particularly expressed in the 'slow' zone (10(2) less than C0t less than 10(4). The same dose, however, does not qualitatively effect the secondary DNA structure, which was estimated by the method of thermal elution from the hydroxyapatite column.


E.s.r. studies of halogenated pyrimidines in gamma-irradiated alkaline glasses. The reactions of mobile electrons (em-) and oxygen radical anions (O--) with halogenated bases and nucleosides have been studies in gamma-irradiated alkaline glasses by e.s.r. and specific halogen-ion electrode techniques. It is shown that electrons react with halogenated uracil bases (XUr where X = Cl, Br. I but not F) by dissociative electron attachment to form uracil-5-yl radicals (U-) and halogen anions. The relative rates of reaction of em- with XUr decrease in the sequence BrUr greater than ClUr greater than FUr greater than IUr. Thermal annealing studies carried out on U- in H2O and D2O matrices support the hypothesis that U- in H2O hydrates across the 5-6 double bond in the temperature region 135 degrees-155 degrees K, and deuterates to a much smaller extent in D2O at temperatures above 155 degrees K. Studies on bromouridine and bromodeoxyurinde suggest that em- reacts with the base moieties to form U- type radicals which abstract H- from the sugar moieties of adjacent nucleosides.


Thoraco-abdominal approach in tumour nephrectomy. In order to prevent intraoperative metastasis, venous invasion and infilitration of the regional lymph nodes by renal carcinoma, the thoraco-abdominal surgical approach is recommended. The vascular pedicle can in this way be explored and ligated before manipulation of the tumour. After total nephrectomy en bloc with the fatty capsule and adrenal gland, paracaval, paraaortal and intravasal lymphadenectomy is performed. Tumorous protrusions into the vena cava can mostly be removed at the same time. Inoperability rate could be reduced to 2.1 per cent as opposed to an earlier 11.3 per cent with the lumbar approach. TNM classification followed the recommendations of the UICC and was supplemented with a four-stage system. No improvement over the lumbar approach in respect of survival has been achieved in the first three postoperative years. However, local recurrence is expected to diminish as a result of improved radicality.


Experimental studies with Staphylococcus aureus in M-K media. This study reviewed the potential for survival of a pathogenic bacteria when inoculated into McCarey-Kaufman modified tissue culture media 199. A clinically isolated specimen of Staphylococcus aureus was selected and rabbit eyes inoculated with 0.1 ml. of the organism in a suspension of 1 to 3 x 10(-5). Upon enucleation 12 hours later no clinical signs of infection were noted. Limbal-conjunctival cultures were obtained on all eyes before and after application of the antibiotic. Corneas were stored in M-K media with standard penicillin-streptomycin added and cultured up to 48 hours. Our studies showed some recovery of the infecting organism from the media with topical antibiotic application but none following complete immersion of eyes in antibiotic. Direct cultures from corneal buttons taken 48 to 72 hours after antibiotic application by either method showed 1 to 3 colonies per plate in 25 per cent of eyes. In this study improperly applied antibiotic allowed some survival of S. aureus in the media.


Cytopathic effects in primary epithelial cultures derived from the human prostate. Normal, benign, and malignant human prostatic tissue was cultivated in vitro. Cytopathic effects in derived epithelial cells were examined. Light microscopy revealed polykaryocyte formation, vacuolation, cytoplasmic bridges and processes, nuclear inclusions, increased acid phosphatase activity at the cell membrane of polykaryocytes as compared to mononuclear cells, and cell rounding and clumping. Electron microscopy of the polykaryocytes showed nuclear membrane proliferation and protrusion, scarlike nuclear inclusions containing microfibrils, and virus-like particles similiar to viral nucleoids and nucleocapsids. The latter were also observed in the cytoplasm. The above alterations are similar to those induced by known herpesviruses. The significance of these changes, the possibility of the presence of a latent herpesvirus in the prostate, and its role in neoplastic disease are postulated.


A human adrenocortical adenoma in tissue culture. Morphology and hormone secretion. A human adrenocortical adenoma was cultivated in vitro and the morphological characteristics, steroidogenic activity and response to ACTH of the cultured cells were studied over a period of about two months. At first, the morphology of the cultures was similar to that of the original tissue. This structure changed in the course of time and the cells underwent fibroblastoid transformation. Stimulation with ACTH evoked two types of morphological response: a) a short-term response, whereby an extensive but rapidly reversible vacuolization was followed by cell enlargement and granulation, and b) a long-term effect in which the differentiated cell morphology was maintained. The cultures showed a low steroidogenic activity throughout the experiment. ACTH stimulation increased the total 17-hydroxysteroid production up to 10-fold. After ACTH withdrawal the corticosteroid levels declined slowly and about 50% of the maximal activity persisted on the fifth day after ACTH withdrawal.


Sodium chloride restriction and extracellular fluid volume contraction in hyperphosphatiuric vitamin D resistant rickets in the Lowe syndrome. A patient with a Lowe syndrome was observed from birth. Progressive hyperchloraemic renal tubular acidosis, hypophosphataemia, hyperphosphaturia and generalized hyperaminoaciduria had developed in infancy. Supplementary vitamin D, alkali and a high intake of dietary phosphate were unsuccessful in controlling the severe phosphate diabetes and rickets. Contraction of the extracellular fluid volume by dietary sodium restriction resulted in correction of the acidosis, hypophosphataemia, hyperaminoaciduria, and hyperphosphaturia, and healing of the rickets.


Genital herpes simplex virus type 1 infection--variability in modes of spread. Ever since the first report in 1967 of the association of herpes simplex virus type 2 (HSV-2) with urogenital infections, we have noted the occasional isolation of HSV-1 from such sites. Our most recent results of the typing of HSV isolates from 527 individuals with urogenital infections show that 10.9% of such infections in females and 3.4% in males are caused by HSV-1. We describe here the acquisition of a primary oral HSV-1 infection in a male after orogenital contact with his wife who had a primary genital HSV-1 infection, probably as a result of sexual contact with another male partner. The various modes of acquisition and spread of genital HSV-1, including nonvenereal routes, are reviewed. It is suggested that the influence of the microenvironment of the female genital tract on the selection of variants of microorganism--viruses, bacteria, etc--requires concerted study.


Effect of desdanine on nucleoside diphosphate kinase and pyruvate kinase of Escherichia coli. Kinetic analysis demonstrated that the irreversible inhibition of nucleoside diphosphate kinase of Escherichia coli by desdanine proceeds via a reversible Enzyme-Inhibitor complex. It is known that pyruvate kinase of E. coli becomes inactive upon prolonged dialysis in the absence of a reducing reagent, such as dithiothreitol and that the inactive enzyme is reactivated if dithiothretiol is added. Desdanine inhibits this reactivation process. The effect is discussed in relation to inhibition in relation to the inhibition of growth of E. coli by desdanine under anaerobic conditions. Pyruvate kinase of E. coli changes not only in intracellular quantity but in its kinetic characteristics depending on growth conditions, aerobic or anaerobic. The enzyme shows a broad specificity for nucleside diphosphates, especially in the presence of AMP, and thus resembles closely nucleoside diphosphate kinase. The in vivo role of pyruvate kinase in supplying nucleoside triphosphates under anaerobic conditions is discussed.


An application of neuroendocrinological studies in autistic children and Heller's syndrome. The response of plasma 11-hydroxycorticosteroids (11-OHCS) to intravenous pyrogen as well as the circadian rhythm of plasma 11-OHCS levels were investigated in seven autistic children and in two children with Heller's syndrome. In autistic children, the stress response, which is acquired in an earlier stage of development, was adequately sustained. However, the circadian rhythm, which seems to appear at a later stage with the maturity of the CNS, frequently revealed abnormal patterns. Similar findings were obtained in the Heller's syndrome cases, indicating organic changes in the brain. On the basis of these results, it is postulated that in early infantile autism there exist some functional changes in the CNS that show a close correlation to the regulatory mechanism of ACTH secretion.


Specific inhibition of phospholipid synthesis in plsA mutants of Escherichia coli. plsA mutants of Escherichia coli are temperature-sensitive strains which possess two enzymes of abnormal thermolability, sn-glycerol 3-phosphate acyltransferase and adenylate kinase. Phospholipid synthesis is inhibited after shift of plsA mutants to temperatures at the lower end of the nonpermissive temperature range. This inhibition is not due to inactivation of the adenylate kinase activity since nucleic acid (and hence adenosine 5'-triphosphate) synthesis is inhibited only slightly. These results show that in vivo inactivation of the sn-glycerol 3-phosphate acyltransferase can be observed under conditions which allow normal adenylate kinase function.


Spin-labeling studies on the lipids of psychrophilic, psychrotrophic, and mesophilic clostridia. Spin-labeling studies were conducted to elucidate the viscosity and phase transition temperatures of lipids isolated from psychrophilic, psychrotrophic, and mesophilic clostridia. Electron spin resonance spectroscopy indicated that the lipids, for all the growth temperatures tested, were in a fluid state and from 13 to 24 C higher than the corresponding lipid transition temperatures. When the organisms were grown at different temperatures, a psychrotropic and two mesophilic clostridia were shown to be able to adjust their lipid-phase transition temperature to the growth temperature. A psychrophilic Clostridium strain, when grown at different temperatures, synthesized lipids that had the same phase transition temperature. It is suggested that this lack of growth temperature-inducible regulation of lipid-phase transition temperature may be a molecular determinant for the psychrophily of this organism. It is proposed that the growth temperature range of an organism is dependent upon the ability of the organism to regulate its lipid fluidity within a specific range.


Ultrastructural study of polymyxin-resistant isolates of Pseudomonas aeruginosa. Upon exposure to 6,000 U of polymyxin B sulfate per ml, cells of the polymyxin-sensitive PAO 1 strain of Pseudomonas aeruginosa displayed in thin sections long projections arising from the outer membrane of the cell wall and extensive cytoplasmic degradation with accumulation of cytoplasmic membrane infoldings. Polymyxin-resistant isolates derived from the PAO 1 strain, however, grew well in the presence of 6,000 U of polymyxin per ml and exhibited none of these effects, having instead the appearance of a typically healthy cell. Freeze-etching of cells of the sensitive strain grown in basal medium without polymyxin revealed a concave cell wall layer studded with numerous particles. Freeze-etching of cells of the resistant isolates grown in basal medium containing 6,000 U of polymyxin per ml revealed a concave cell wall layer (i.e., the outer half of the outer membrane) in which most of these particles were absent. Thus, acquisition of resistance to polymyxin was correlated with an alteration in the architecture of the outer membrane. When the resistant isolates were grown in the basal medium lacking polymyxin and then freeze-etched, the particle distribution in the concave cell wall layer resembled that of the sensitive parent strain. The cells had regained sensitivity to polymyxin upon suspension in medium containing 6,000 U/ml as determined by their failure to grow and by internal damages seen in thin sections. These cells also had acquired increased sensitivity to ethylenediaminetetraacetate, whereas the polymyxin-resistant cells grown in the presence of polymyxin were resistant to lysis by ethylenediaminetetraacetate. The polymyxin-resistant isolates were not stable mutants but instead represented an adaptive response to the presence of polymyxin in the medium.


Mutants of Aspergillus nidulans lacking nicotinamide adenine dinucleotide-specific glutamate dehydrogenase. Ten mutants of Aspergillus nidulans lacking nicotinamide adenine dinucleotide-specific glutamate dehydrogenase (NAD-GDH) have been isolated, and their mutations (gdhB1 through gdhB10) have been shown to lie in the gdhB gene. In addition, a temperature-sensitive gdhB mutant (gdhB11) has been isolated. A revertant (designated R-5) of the mutant gdhB1 bears an additional lesion in the gdhB gene and has altered NAD-GDH activity with altered Km values for ammonia or ammonium ions and for alpha-ketoglutarate. These results suggest that gdhB specifies a structural component for NAD-GDH. The growth characteristics of gdhB mutants indicate the routes by which amino acids are utilized as nitrogen and carbon energy sources. The properties are described of the double mutants bearing the mutations gdhB1 and gdhA1 or tamA119, which have low NADP-GDH activity.