Regulation of aspartate aminotransferase activity associated with change of pyridoxal phosphate level.

The activities of aspartate aminotransferase (EC 2.6.1.1) in the cytosol fractions of the liver and kidney of rats fed pyridoxine-deficient or control diet for 3 weeks were determined. In the absence of pyridoxal phosphate, the activities in the liver and kidney preparations of deficient rats were both abnormally low. The activity in the kidney fraction of deficient rats was restored to almost the control level by addition of pyridoxal phosphate, whereas that of the liver was only partially restored. The antigen activity, however, measured using anti-aspartate aminotransferase, was similar in liver fractions from deficient and control rats. These findings suggest the existence of a form of transaminase with little or no activity in the liver of deficient rats. The properties of the crude enzymes from deficient and control rats were indistinguishable by immunodiffusion, and the enzymes had the same subunit size and heat stability under the conditions tested. However, purified enzyme from deficient rat liver had a different specific activity and absorption spectrum from purified enzyme from normal liver.     

Further studies of the selective inhibition by ouabain of antigen-induced proliferation of human lymphocytes

Cultures of human lymphocytes incubated for 48 hr in the presence of 2 × 10^?7M solutions of the cardiotonic steroid ouabain lose the proliferative response to antigens (SL-0, SK-SD) but can still proliferate when stimulated by nonspecific mitogens (PHA, Con A, pokeweed mitogen). The two-way mixed lymphocyte reaction was also irreversibly lost if cells of both donors were subjected to ouabain pretreatment. Neither cell counts nor cell viability (determined by dye exclusion) were significantly affected by the ouabain treatment. Pretreatment of a suspension of macrophages with the cardiac glycoside did not diminish their capacity to restore the proliferative response to antigen of macrophage-depleted lymphocyte suspensions; on the other hand, untreated macrophages could not restore the proliferative response of cultures of ouabain-pretreated lymphocytes. The ouabain treatment did not change the proportion of cells able to bind fluorescent anti-immunoglobulin nor did it modify the proportion of lymphocytes forming rosettes with either untreated, or antibody coated, red cells. Increased concentration of K⁺ in the medium, either during or after the ouabain treatment, did not reduce the ouabain effect. We conclude that the selective loss of certain lymphocyte functions caused by ouabain pretreatment was due to an effect on the lymphocyte and not on the macrophage; the effect was not due to the elimination of a relatively large fraction of the cells nor to a generalized disappearance of membrane antigens and receptors.     

Formation of a carboxyarabinitol bisphosphate complex with ribulose bisphosphate carboxylase/oxygenase and theoretical specific activity of the enzyme

¹⁴C-Labeled 2-carboxyarabinitol-1,5-bisphosphate was bound to both nonactivated and CO₂and Mg²⁺ activated forms of ribulose bisphosphate carboxylase/oxygenase. The complex could be precipitated with 20% polyethylene glycol and 20 mm MgCl₂ for quantitation of the moles of the affinity label bound per mole of enzyme. The [¹⁴C]carboxyarabinitol-P₂ bound to the nonactivated enzyme could be exchanged with a 100-fold excess of the unlabeled compound. With the activated enzyme the binding of [¹⁴C]carboxyarabinitol-P₂ was so tight that it did not exchange with the unlabeled compound and a binding stoichiometry of one molecule per active site was assumed. This tight binding was dependent upon pretreatment of the enzyme with both CO₂ and MgCl₂ in the same manner that enzyme activation depended on CO₂ and Mg²⁺ concentrations. Various enzyme preparations from spinach leaves tightly bound [¹⁴C]carboxyarabinitol-P₂ in proportion to their specific activities. By extrapolating to a maximum binding of 8 mol of [¹⁴C]carboxyarabinitol-P₂ per mole of this A₈B₈ enzyme a theoretical specific activity of 2.8 μmol · min^?1 · mg protein^?1 was indicated. Enzyme preparations purified from spinach leaves generally have a specific activity in the range of 1.0 to 2.3.     

The sequence of an atriopeptigen: A precursor of the bioactive atrial peptides

The high molecular weight fraction (atriopeptigen-APG) obtained by gel filtration chromatography of rat atrial extracts was fractionated by isoelectric focusing and reverse phase HPLC to obtain a pure APG. Purification of cyanogen bromide digests of the crude high molecular weight fraction resulted in the isolation of a single biologically active cyanogen bromide cleavage peptide. Sequence analyses of these peptides coupled with recent reports of sequence analyses of intermediate molecular weight atrial peptides (Thibault, et al. (1984) FEBS Letters 167, 352–356, and Kangwa, et al., Biochem. Biophys. Res. Commun 119, 933–940) provide the complete primary structure of an 111 residue APG.     

Regulation of plasma membrane Ca2+ ATPases by lipids of the phosphatidylinositol cycle

When the erythrocyte plasma membrane Ca2+ pump is reconstituted into phosphatidylcholine liposomes, the inclusion of small amounts of phosphatidic acid or phosphatidylinositol 4,5-bisphosphate stimulates the enzyme's activity. Other lipids of the phosphatidylinositol cycle (diacylglycerol, phosphatidylinositol) have little effect. The stimulatory effect of phosphatidylinositol 4,5-bisphosphate is greater than that of calmodulin; this lipid also stimulates the plasma membrane Ca2+ ATPase from rat brain.     

DNA sequence alterations in Hr-t deletion mutants of polyoma virus.

We have investigated the DNA sequence alterations in several hr-t mutants of polyoma virus. These mutants are defective in one of the two known viral functions essential for transformation and are altered with respect to several minor T antigen species. The lesions in some of these mutants have been mapped previously by marker rescue experiments to Hpa II fragment 4 (Hpa II-4, 78.4--91.7 map units) in the proximal part of the early region of the viral DNA. Thirteen of sixteen hr-t mutants examined carry deletions 2 to 5 map units (100--250 bp) long in Hpa 11-4. Three mutants carry either point mutations or very small deletions/insertions. Eight of the deletion mutants were mapped closely with restriction enzymes. Seven of them have deletions located entirely within the Hae III subfragment A of Hpa II-4 (the Hae A subfragment, 78.4--85.2 map units), and one extends just beyond this subfragment, ending at 85.5 map units. The complete sequence of the wild-type Hae A subfragment was determined and compared with those of four deletion mutants, NG-18, A-8, 6B5 and B-2. The deletion in each of these mutants is out-of-phase: NG-18, 187 bp; A-8, 127 bp; 6B-5, 179 bp; B-2, 241 bp. All are expected to remove protein sequences in the C terminal part of the small t antigen.     

Mapping unintegrated avian sarcoma virus DNA: termini of linear DNA bear 300 nucleotides present once or twice in two species of circular DNA.

Three major species of viral DNA have been observed in cells infected by retroviruses: a linear, double-stranded copy of a subunit of viral RNA; closed circular DNA; and proviral DNA inserted covalently into the genome of the host cell. We have studied the structures of the unintegrated forms of avian sarcoma virus (ASA) DNA using agarose gel electrophoresis in conjunction with restriction endonucleases and molecular hybridization techniques. The linear duplex DNA is approximately the same length as a subunit of viral RNA (approximately 10 kb) and it bears natural repeats of approximately 300 nucleotides at its termini. The repeats are composed of sequences derived from both the 3' and 5' termini of viral RNA in a manner suggesting that the viral DNA polymerase is transferred twice between templates. Thus the first end begins with a sequence from the 5' terminus of viral RNA and is permuted by about 100 nucleotides with respect to the 3' terminus of viral RNA; the linear DNA terminates with a sequence of about 200 nucleotides derived from the 3' end of viral RNA. We represent this structure, synthesized from right to left, as 3'5'-----3'5'. Two closed circular species of approximately monomeric size have been identified. The less abundant species contain all the sequences identified in linear DNA, including two copies in tandem of the 300 nucleotide 3'5' repeat. The major species lacks about 300 base pairs (bp) mapped to the region of the repeated sequence; thus it presumably contains only a single copy of that sequence. The strategies used to determine these structures involved the assignment of over 20 cleavage sites for restriction endonucleases on the physical maps of ASV DNA. Several strains of ASV were compared with respect to these sites, and the sites have been located in relation to deletions frequently observed in the env and src genes of ASV.     

A bridged Mo(VI) complex containing six and five coordinate Mo: Synthesis, 95Mo N.m.r. spectroscopy and X-ray crystal structure of Mo2O5(C17H17N2O)2

The kinetics of the reaction between Carcinus maenas hemocyanin and cyanide has been studied at various KCN concentrations and a different temperatures (21° and 4°C) by following the decrease of the copper-peroxide absorption band, centered at 337 nm, of the copper still bound to the protein and the intrinsic fluorescence changes as functions of time. In all conditions used, the absorption band completely disappears and KCN concentration affects only the rate of the process. The reaction is kinetically homogeneous indicating no site-site interaction. The apparent rate constant increases with the square of cyanide concentration and the inverse of O₂ concentration. The copper still bound decreases at a rate slower than the 337 nm absorption and the process is not kinetically homogeneous. The fluorescence of the protein increases after an induction period showing an inflection point at about 50% of the total effect. A kinetic model has been proposed on the assumption that the two metal ions are removed sequentially from the active site. The experimental data are in agreement with the theoretical equations derived from the model. The equilibrium constants for the formation of the complex between the first and the second copper ion with cyanide and the rate constants of their decomposition have been calculated. The rate-limiting process for the removal of the second copper ion is the formation of the complex with cyanide.     

Inhibition of the vaccinia virus associated ribonuclease by HeLa S3 cytosol

A ribonuclease associated with purified vaccinia virus is able to degrade into 3S fragments the viral 8–12S mRNAs synthesized $\text{in}\text{vitro}$. This RNase not detected on purified viral cores was shown to be located on the outer side of the viral envelope. When added to reaction mixture, a partially purified HeLa S₃ cytosol fully inhibits the vaccinia virus associated RNase. Other cytosols from very common mammalian cell lines also contain RNase inhibitor and are of interest in order to prepare large amounts of undegraded vaccinia virus mRNAs.     

On the nature of nuclear envelope-associated RNA

Nuclear envelopes were isolated from rat-liver nuclei. Nuclear envelope-associated RNA was isolated and hybridized to filter-bound DNA in the presence of competing RNA populations. Cytoplasmic RNA did not effectively compete for DNA binding sites, while nuclear RNA did. The results indicate a high degree of complexity for nuclear envelope-associated RNA, and are compatible with the idea that hnRNA may be processed after attachment to the nuclear envelope (or nuclear matrix).     

Requirement of human chromosomes 19, 6 and possibly 3 for infection of hamster x human hybrid cells with baboon M7 type C virus.

The replication pattern of the endogenous baboon type C virus M7 was studied in 29 primary Chinese hamster × human hybrid clones generated with leukemic cells from two different patients with acute lymphoblastic or myeloblastic leukemia. There was no evidence of viral particulate RDDP or M7 antigen before viral infection. M7 virus replicated in human and some hybrid cells but not in Chinese hamster cells, indicating that M7 requires dominantly expressed human gene(s) for replication. Enzyme and cytogenetic analyses show that a gene(s) coded for by human chromosome 19 is necessary for M7 infection of these hybrids. Detailed cytogenetic correlations revealed, however, that the chromosome 19+/M7 + hybrid clones with intact chromosomes also had copies of chromosomes 3 and 6. Previously, Bevi, the putative integration site for M7 virus, has been assigned to human chromosome 6. Many clones with various combinations of chromosomes 3 and 6 lacked chromosome 19, however, and failed to replicate exogenously applied M7 virus, while tests of 27 secondary clones showed that M7 markers co-segregated with chromosome 19 markers. These findings all confirm the need for a chromosome 19-coded function in Chinese hamster × human hybrids. In addition, the yield of viral particulate RDDP produced into the culture fluid was 50–100 fold less per viral antigen-positive cell in the hybrids compared with human cells. Thus some form of regulation of viral components exists in the hybrid cells. When the virus replicating in hybrid cells was transferred back to human cells, this regulation was relaxed and the yield of RDDP per FA(+) cell greatly increased. We conclude that human chromosomes 6 and 19 code for functions involved in M7 virus metabolism, and we cannot exclude a function coded for by chromosome 3.     

Terminal deoxynucleotidyl transferase activity in a cell line (molt-4) derived from the peripheral blood of a patient with acute lymphoblastic leukemia

A terminal deoxynucleotidyl transferase having a sedimentation coefficient of 3–4S has been found associated with the chromatin from a cell line (Molt-4) derived from the peripheral blood of a patient with acute lymphoblastic leukemia.     

Temperature-sensitive mutants of Balb/3T3 cells: description of a mutant affected in cellular and polyoma virus DNA synthesis.

A temperature-sensitive Dna- mutant (ts-2) of the mouse cell Balb/3T3 is characterized. Studies with synchronized cells indicate that the defect is in DNA synthesis itself, rather than in progress toward its initiation. ts-2 supports polyoma DNA synthesis after infection at 33degreesC but not at 38degreesC. Viral DNA synthesis begun at 33degreesC is inhibited upon shift to 38degreesC. A procedure is proposed by which viral DNA synthesis can be used to distinguish different classes of cell Dna- mutants.     

Stress-induced inhibition of sexual behavior: Corticosterone inhibits courtship behaviors of a male amphibian (Taricha granulosa)

When male rough-skinned newts (Taricha granulosa) are exposed to presumptive stressors, the incidence of courtship decreases and plasma corticosterone concentration increases. When sexually active males are injected intraperitoneally with corticosterone (1, 5, 10, 15, 20, or 25 μg), the incidence of courtship decreases rapidly and in proportion to the dose of corticosterone. Intracerebroventricular infusion of synthetic corticotropin-releasing factor (CRF) elevates plasma corticosterone levels and suppresses courtship. When male newts receive an injection of metyrapone, a drug that interferes with corticosterone synthesis, the inhibitory effects of stress or CRF infusion on courtship are reduced. These results support the hypothesis that, in this amphibian, elevated levels of corticosterone associated with exposure to stressful stimuli inhibit sexual behaviors.     

A comparison of alternative measures of mutagenic potency in the Salmonella (Ames) test

Both the spontaneous and the induced mutation rates in Salmonella tester strains vary among different laboratories, and also within the same laboratory over time. If there is an association between spontaneous and induced mutagenesis, a measure of mutagenic potency that incorporates the background may be more consistent than the simple measure of the induced slope. We have used the statistical procedures recently described by Bernstein et al. (1982), and a large data-base of Salmonella test results to examine the association between spontaneous and induced mutation and to compare several alternative measures of mutagenic potency. A correlation analysis indicated an association between spontaneous and induced mutation for TA98, TA1537 and TA1535; TA1538 was close to being significant. This was observed over a wide range of chemicals. In addition, for TA98, for which we observed the strongest association, we obtained a rough estimate of the relationship between slope and intercept by using least squares to fit K and p in the power curve beta = k alpha p. We then chose 3 simple potency measures: the slope, the ratio of slope to spontaneous background, and the ratio of slope to the square-root of spontaneous background. These corresponded to the range of p's estimated from the least-squares fit procedure. The reproducibility of these measures was compared and no significant differences were found. Though there were some differences in the relative potency ranking of chemicals using the different measures, they were highly correlated.     

Dry matter,protein, energy and fibre intake by dairy heifers grazing a Rhodes grass (Chloris gayana) pasture

Dry matter (DM), protein, energy and fibre (ADF) intakes of Friesian and Ayrshire dairy heifers grazing a Rhodes grass pasture in Central Kenya were studied by indicator techniques for ten 22-day periods, each comprising 10 preliminary days and 12 collection days.Digestibility coefficients of DM, CP, GE and fibre were higher (P < 0.05) in the wetter periods and generally declined during the drier periods, as pasture matured. Intakes of DM, DCP and DE decreased (P < 0.05) as herbage matured. Average daily intake per kgW^0.75 was 73.9-g DM, 4.1-g DCP, 31.8-g ADF and 167-kcal DE. Average daily gain (ADG) also varied (P < 0.05) with period and was positively related to intake of digestible dry matter, protein and energy. When conditions were wet, ADG was satisfactory at 480 g. In dry conditions, the heifers ingested 26.1-g DDM, 1.6-g DCP and 110-Kcal DE per kgW^0.75 daily and gained at 200 g/day, and supplementation was deemed necessary to sustain an optimum ADG of 550 g. The factors which may have limited pasture intake by the grazing heifers are discussed. In general, a Chloris gayana pasture, grazed at an appropriate stocking-rate, will meet the requirements of dairy heifers for maintenance and, to a variable degree, those for growth in areas of Kenya with an annual rainfall of 1400 mm and with a high potential for increased cattle production.     

A simple, automated apparatus for the rapid multiple flushing of reaction (assay) vessels with gases

Folin and Ciocalteu's phenol reagent may be reduced by a variety of compounds, which therefore interfere with the Lowry method of protein determination (1,2). Peters and Fouts (3) reported that two of the zwitterionic biological buffers described by Good et al. (4) reacted strongly with the Folin reagent and thus seriously interfered with protein determinations. The zwitterionic buffers have many properties which lead to their use in biological work, where protein estimations will be required. Since the publication of Good et al. (4), the range of zwitterionic buffers has been increased. The possibility that these other buffers may also produce artefacts has therefore been investigated.