Inhibition of DNA Synthesis but not of Poly-dAT Synthesis by the Arabinose Analogue of Cytidine <Emphasis Type="Italic">in vitro</Emphasis>

Kimball and Wilson¹ reported that the arabinose analogue of cytidine (ara-C) inhibited DNA polymerase in a crude extract prepared from Ehrlich ascites cells. Furth and Cohen² observed cytosine arabinoside triphosphate (ara-CTP) inhibited DNA polymerase in extracts from either calf thymus or bovine lymphosarcoma tissue, although these investigators³ had already found no effect of ara-CTP on DNA polymerase from Escherichia coli. The inhibition in both of these cases could be substantially reversed by dCTP; but incorporation of the arabinose nucleotide (ara-CMP) into DNA could not be unequivocally demonstrated. Graham and Whitmore⁴ reported the incorporation of ara-C into DNA in vivo and the inhibition of a DNA polymerase from L cells by ara-CTP. They found that ara-CMP was initially incorporated into small DNA strands but subsequently appeared in long strands. Momparler⁵ has presented evidence that, in vitro, ara-C incorporation was limited to the 3′-hydroxyl end of DNA chains. Such incorporation might be expected to block further chain elongation but this expectation was not supported by the evidence presented by Graham and Whitmore.     

Endopeptidase Activity of Phage λ-Endolysin

JACOB and Fuerst^1,2 demonstrated the presence of a bacteriolytic enzyme (λ-endolysin) in the induced cultures of lysogenic Escherichia coli K12 (λ). The enzyme was later identified as the product of gene R; of phage λ³ which is involved in bacterial lysis at the end of a latent period. The enzyme is apt to form spheroplast-like structures in E. coli² and one would therefore expect its substrate to be murein.     

Reversed-phase boronate chromatography for the separation of O-methylribose nucleosides and aminoacyl-tRNAs

Boronate forms an anionic complex with the cis-2′,3′ hydroxyls of unsubstituted ribonucleosides and the 3′-terminal adenosine of unacylated tRNAs, but not with ribosesubstituted nucleosides such as 2′-O-methylnucleosides and aminoacyl-tRNAs. We have synthesized phenyl boronates with hydrophobic side chains of about 1-nm-long and coated inert 10-μm solid beads of polychlorotrifluoroethylene with this material. This matrix complexes easily with compounds containing free cis-hydroxyls, but not with their O-alkyl or O-acyl derivatives. This permits the separation of mammalian and bacterial amino-acyl-tRNAs from uncharged tRNAs and O-methyl nucleosides from ribose-unsubstituted nucleosides in one chromatographic step, as the substituted members of each group do not undergo boronate complex formation and are thus not as much retarded in passing through the column. Complex formation between ribofuranoses and the boronate matrix appears to be enhanced by the hydrophobic “tail” of the boronate compound, by the high ionic environment of the solvent, and by the hydrophobic nature of the inert support. This method of one-step purification of tRNAs on reversed-phase boronate columns has been tested for several tRNAs specific for amino acids of different hydrophobicity and ionic character. The results indicate that each tRNA tested can be purified with appreciable purity (70–95%) and high yield (80%). However, recovery of the queuine base containing aminoacyl-tRNAs is only about 6% of the applied material. Several other boronate matrices have also been synthesized using cellulose, agarose. Sepharose, or porous glass beads as the inert support with different lengths of the spacer arm. Cellulose with a 1-nm-long spacer arm is satisfactory not only for the separation of aminoacyl-tRNAs and O-methylribose nucleosides, but also for the separation as a group of tRNAs containing the base of Q, queuine. However, other inert supports are unsatisfactory because of a non-specific binding of the tRNAs.     

Protection against acute paraquat toxicity by ambroxol

An expression vector for G-CSF, pASLB3-3, was constructed and introduced into Namalwa KJM-1 cells (Hosoi et al., 1988), and cells resistant to 100 nM of methotrexate (MTX) were obtained. Among them, the highest producer, clone SC57, was selected and the productivity of this clone was further characterized. The maximal production of G-CSF was at the most 1.8 g/ml/day using a 25 cm² tissue culture flask, even though the cell number was above 7×10⁵ cells/ml. The limiting factors at high density were analyzed as the deficiency of nutrients, such as glucose, cysteine and serine, and pH control. The depression of specific G-CSF productivity per cell under the batch culture conditions was overcome by using a perfusion culture system, Biofermenter^TM (Sato, 1983) with modifications of nutrients supplementation by a dialysis membrane and/or dissolved oxygen (DO) supplementation by microsilicone fibers. ITPSGF medium was modified to elevate concentrations of amino acids and glucose by 2.0- and 2.5-times, respectively. Under the control of pH at 7.4 and DO at 3 ppm, the specific G-CSF productivity was not depressed even at high cell density (above 1×10⁷ cells/ml), and the amount of G-CSF reached 41 g/ml. These results indicated the possibility of finding the optimum culture conditions for the production of recombinant proteins by Namalwa KJM-1 cells.Abbreviations ABTS 2,2-Azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid - BSA Bovine Serum Albumin - BSA-PBS Phosphate-buffered Saline without Ca²⁺ and Mg²⁺ containing Bovine Serum Albumin - dhfr Dihydrofolate Reductase - DO Dissolved Oxygen - G-CSF Granulocyte Colony-stimulating Factor - HEPES 4-(2-Hydroxyethyl)-1-piperazineethansulfonic Acid - IFN Interferon - MTX Methotrexate - PBS(-) Phosphate-buffered saline without Ca²⁺ and Mg²⁺ - Tween-PBS Phosphate-buffered saline without Ca²⁺ and Mg²⁺ containing 0.05% of Tween 20     

Neonatal hyperthyroidism: alterations in behavioural activity and the metabolism of brain norepinephrine and dopamine.

Daily administration of triiodothyronine (10 μg/100 g) to newborn rats for 30 days produced signs of hyperthyroidism which included accelerated development of physical and behavioural characteristics accompanying maturation. The hyperthyroid rats displayed progressive increases in spontaneous locomotor activity between 14–35 days, which remained elevated well above control levels even at 105 days. Exposure of developing rats to triiodothyronine increased the endogenous levels of striatal tyrosine and tyrosine hydroxylase as well as the concentration of dopamine in hypothalamus, pons-medulla, mid-brain, striatum and hippocampus. The concentration of striatal homovanillic acid and 3,4-dihydroxyphenylacetic acid was also increased in hyperthyroid rats. In contrast, the steady-state levels of norepinephrine remained unaltered resulting in a significant increase in dopamine to norepinephrine ratio in several regions of the brain examined. The elevated levels of dopamine metabolites (homovanillic acid and 3,4-dihydroxyphenylacetic acid) may be due to an increased turnover of dopamine. Our data suggest that increased thyroid hormone levels may lead to an enhanced synthesis as well as utilization of brain catecholamines which in turn may underlie the observed increases in spontaneous locomotor activity.     

Effects of isoproterenol on cyclic-AMP metabolism in rat ventral prostate.

Beta-Adrenergic stimulation of the ventral prostate cyclic-AMP system was investigated by examining the influence of isoproterenol on endogenous cyclic-AMP levels as well as on the activities of adenylate cyclase CEC 4.6.1.1) and cyclic-AMP-dependent and independent protein kinases (EC 2.7.1.37). Administration of isoproterenol (1 mg/kg, ip) resulted in rapid elevation of adenylate cyclase activity (119%) and cyclic-AMP levels (593%). The observed isoproterenol-stimulated changes in cyclic-AMP metabolism of the ventral prostate were time-dependent and maximal stimulation was seen 5 min after treatment with this beta-adrenergic agonist. The increases in prostatic adenylate cyclase and cyclic-AMP also were related to the dose of isoproterenol administered and maximal enhancement of these parameters was seen with 1 mg/kg dose of the agonist. Whereas pretreatment of rats with propranolol (3mg/kg, ip) partially reversed these alterations, administration of an alpha-adrenergic antagonist, phentolamine, even at a dose of 5 mg/kg, failed to elicit any appreciable effect. Stimulation of prostatic soluble protein kinase by isoproterenol was associated with a decrease (33%) in the activity of the cyclic-AMP-dependent protein kinase with a concomitant increase (25%) in that of the independent enzyme. Whereas the ability of the enzyme to bind cyclic-(3H) AMP in vitro was decreased (54%) following isoproterenol treatment, the protein kinase activity ratio (-cyclic-AMP/+cyclic AMP) was significantly elevated from 0.51+/0.05 to 0.95+/0.08. Although propranolol alone had little or no effect on these parameters, it inhibited partially the isoproterenol-induced alterations in cyclic-AMP-dependent protein kinase and the cyclic-AMP binding capacity. Treatment with propranolol also blocked the increases in the kinase activity ratio and in the activity of cyclic-AMP-independent enzyme seen with isoproterenol. Data suggest that the concentration of ventral prostate cyclic-AMP as well as the activities of adenylate cyclase and cyclic-AMP-dependent and independent form of protein kinases are subject to modulation by beta-adrenergic stimulation.     

Influence of clay minerals on sorption of bacteriolytic enzymes

Myxobacteria presumably produce extracellular bacteriolytic enzymes when they are growing in soil. In order to study their ecological significance, adsorption experiments were performed with lytic enzymes produced byMyxococcus virescens in casitone media. Different soils as well as montmorillonite and kaolinite can rapidly adsorb the bacteriolytic but not the proteolytic enzymes. About 1 gm of montmorillonite per liter of cell-free culture solution is enough for the adsorption of 97% of the bacteriolytic enzymes. The adsorption per unit weight is about 100 times greater on montmorillonite than on kaolinite. About 40% of the adsorbed enzymes can be eluted with solutions of high pH or high ionic strength. The only desorbed bacteriolytic enzyme is the alanyl-∈-N-lysine endopeptidase.     

Staining of Some Specific Regions of Human Chromosomes,particularly the Secondary Constriction of No. 9

SEVERAL procedures have been described recently which produce specific patterns of differential staining in human chromosomes^1–9. Techniques which involve DNA denaturation and reannealing reveal deeply stained areas on centromere and secondary constriction regions which have been equated with constitutive heterochromatin⁹.