Cloning of multiple genes involved with cobalamin (Vitamin B12) biosynthesis in Bacillus megaterium.

An effective shotgun cloning procedure was developed for Bacillus megaterium by amplifying gene libraries in Bacillus subtilis. This technique was useful in isolating at least 11 genes from B. megaterium which are involved with cobalamin (vitamin B12) biosynthesis. Amplified plasmid banks were transformed into protoplasts of both a series of Cob mutants blocked before the biosynthesis of cobinamide and Cbl mutants blocked in the conversion of cobinamide into cobalamin. Amplification of gene libraries overcame the cloning barriers inherent in the relatively low protoplast transformation frequency of B. megaterium. A family of plasmids was isolated by complementation of seven different Cob and Cbl mutants. Each plasmid capable of complementing a Cob or Cbl mutant was transformed into each one of the series of Cob and Cbl mutants; many of the plasmids isolated by complementation of one mutation carried genetic activity for complementation of other mutations. By these criteria, four different complementation groups were resolved. At least six genes involved in the biosynthesis of cobinamide are carried on a fragment of DNA approximately 2.7 kilobase pairs in length; other genes involved in the biosynthesis of cobinamide were located in two other complementation groups. The physical and genetic data permitted an ordering of genes within several of the complementation groups. The presence of complementing plasmids in mutants blocked in cobalamin synthesis resulted in restoration of cobalamin biosynthesis.     

“Unblocking” Serum Activity <Emphasis Type="Italic">in vitro</Emphasis> in the Polyoma System may Correlate with Antitumour Effects of Antiserum <Emphasis Type="Italic">in vivo</Emphasis>

In a variety of tumour systems, individuals carrying progressively growing neoplasms have lymphoid cells with a specific cytotoxic effect on cultured tumour cells from the same individual^1–4. Since the sera of tumour-bearing individuals have been shown to prevent tumour cell destruction by immune lymphocytes in vitro^2,5–8 and since this serum blocking activity appears early in primary and transplant tumour development^5,7, it has been suggested that the appearance of this serum blocking activity might be responsible for the progressive growth of tumours in individuals having cytotoxic lymphocytes. Counteraction of this blocking activity would thus be of primary importance in facilitating the function of an already existing or bolstered cell-mediated immunity. The serum blocking activity might be inhibited in various ways, by preventing the formation of blocking antibody or by interfering with its action (“unblocking”), as demonstrated in Moloney sarcoma regressor sera⁹. This type of serum also has a therapeutic effect on Moloney sarcomas in vivo^10,11, which has been tentatively attributed to its unblocking activity^8,9 or, possibly, to a complement-dependent cytotoxicity¹⁰. Tumour growth in the Moloney sarcoma system, however, might be due in part to continuous recruitment of neoplastic cells by virus-induced transformation and so the therapeutic effect could be due to a virus-neutralizing serum activity^9,10.     

Analogs of arachidonic acid methylated at C-7 and C-10 as inhibitors of leukotriene biosynthesis

The syntheses and biological activity of (all )-7,7-dimethyl-5-8,- 11,14-eicosatetraenoic acid, (all )-7,7,-dimethyl-5,8,11-eicosatrienoic acid, ( , -7,7-dimethyl-5,8-eicosadienoic acid, (all )-10,10-dimetyl- 5,8,11,14-eicosatetraenoic acid, (all -10,10-dimethyl-5,8,11-eicosatrienoic acid, and .-( , -15-hydroxy-7,7-dimethyl-5,8-eicosadienoic acid are described. These arachidonic acid analogs are all inhibitors of ionophore-induced SRS-A biosynthesis in rat peritoneal cells. Their mode of action may involve inhibition of phospholipase A₂ rather than Δ⁵-lipoxygenase. These compounds failed to exhibit significant activity in an model designed to detect inhibitors of antigen-induced, leukotriene-mediated bronchoconstriction is sensitized guinea pigs.     

On the mechanism of ATP-induced shape changes in the human erythrocyte membranes: the role of ATP

In the preceding paper (Sheetz, M. and S.J. Singer. 1977. J Cell Biol. 73:638-646) it was shown that erythrocyte ghosts undergo pronounced shape changes in the presence of mg-ATP. The biochemical effects of the action of ATP are herein examined. The biochemical effects of the action of ATP are herein examined. Phosphorylation by ATP of spectrin component 2 of the erythrocyte membrane is known to occur. We have shown that it is only membrane protein that is significantly phosphorylated under the conditions where the shape changes are produced. The extent of this phosphorylation rises with increasing ATP concentration, reaching nearly 1 mol phosphoryle group per mole of component 2 at 8mM ATP. Most of this phosphorylation appears to occur at a single site on the protein molecule, according to cyanogen bromide peptide cleavage experiments. The degree of phosphorylation of component 2 is apparently also regulated by a membrane-bound protein phosphatase. This activity can be demonstrated in erythrocyte ghosts prepared from intact cells prelabeled with [(32)P]phosphate. In addition to the phosphorylation of component 2, some phosphorylation of lipids, mainly of phosphatidylinositol, is also known to occur. The ghost shape changes are, however, shown to be correlated with the degree of phosphorylation of component 2. In such experiment, the incorporation of exogenous phosphatases into ghosts reversed the shape changes produced by ATP, or by the membrane-intercalating drug chlorpromazine. The results obtained in this and the preceding paper are consistent with the proposal that the erythrocyte membrane possesses kinase and phosphates activities which produce phosphorylation and dephosphorylation of a specific site on spectrin component 2 molecules; the steady-state level of this phosphorylation regulates the structural state of the spectrin complex on the cytoplasmic surface of the membrane, which in turn exerts an important control on the shape of the cell.     

On the three-dimensional structure and catalytic mechanism of triose phosphate isomerase

Triose phosphate isomerase is a dimeric enzyme of molecular mass 56 000 which catalyses the interconversion of dihydroxyacetone phosphate (DHAP) and D-glyceraldehyde-3-phosphate. The crystal structure of the enzyme from chicken muscle has been determined at a resolution of 2.5 A, and an independent determination of the structure of the yeast enzyme has just been completed at 3 A resolution. The conformation of the polypeptide chain is essentially identical in the two structures, and consists of an inner cylinder of eight strands of parallel beta-pleated sheet, with mostly helical segments connecting each strand. The active site is a pocket containing glutamic acid 165, which is believed to act as a base in the reaction. Crystallographic studies of the binding of DHAP to both the chicken and the yeast enzymes reveal a common mode of binding and suggest a mechanisms for catalysis involving polarization of the substrate carbonyl group.     

The effects of N-alkylmaleimides on the activity of rat liver glucose 6-phosphatase.

A series of N-alkylmaleimides has been synthesized and used to investigate the thiol groups that are essential for the activity of rat liver microsomal glucose 6-phosphatase. All of the N-alkylmaleimides inactivated glucose 6-phosphatase when preincubated with microsomes (microsomal fractions) at pH 6.5 and 30 degrees C. When enzyme activity was assayed in intact microsomes, the inactivation was non-linear with respect time, showing an initial rapid phase followed by a slower secondary phase. During the initial rapid phase the inactivation may apparently be completely reversed by disrupting the microsomal membrane with detergent. However, after longer exposure to N-alkylmaleimides the reversal is no longer complete. This observation was explained by the results obtained from studying the inactivation in detergent-disrupted microsomes. In this case glucose 6-phosphatase was also completely inactivated, but much more slowly than was seen in intact microsomes, and the process was linear with respect to time. When assayed in both intact and detergent-disrupted microsomes, glucose 6-phosphatase inactivation was dependent on the number of carbon atoms in the alkyl side chain of the N-alkylmaleimides; this dependence was much more marked in disrupted microsomes. Analysis of the data showed that in neither case was there a saturating effect at high concentrations of maleimide. The data have been interpreted to suggest that there are are least two thiol groups essential for activity located in two separate non-polar regions of the membrane-enzyme system. The conclusions are discussed in the light of the current model for the microsomal glucose 6-phosphatase system.     

Selective inhibition of prostaglandin biosynthesis by gold salts and phenylbutazone

Gold salts and phenylbutazone selectively inhibit the synthesis of PGF_2α and PGE₂ respectively. Lowered production of one prostaglandin species is accompanied by an increased production of the other. Selective inhibition by these drugs was observed in the presence of adrenaline, reduced glutathione and copper sulphate under conditions when most anti-inflammatory compounds inhibited PGE₂ and PGF_2α syntheses equally. It is postulated that selective inhibitors may have a different mode of action and beneficial effects may be related to the endogenous ratio of PGE to PGF required for normal function.