Chemotaxis in Methanospirillum hungatei

Methanospirillum hungatei gave a positive chemotactic response to acetate.     

Mercaptan and dicarboxylate inhibitors of hamster dihydroorotase

In mammals, dihydroorotase is part of a trifunctional protein, dihydroorotate synthetase, which catalyzes the first three reactions of de novo pyrimidine biosynthesis. Dihydroorotase catalyzes the formation of a peptide-like bond between the terminal ureido nitrogen and the beta-carboxyl group of N-carbamyl-L-aspartate to yield heterocyclic L-dihydroorotate. A variety of evidence suggests that dihydroorotase may have a catalytic mechanism similar to that of a zinc protease [Christopherson, R. I., & Jones, M. E. (1980) J. Biol. Chem. 255, 3358-3370]. Tight-binding inhibitors of the zinc proteases, carboxypeptidase A, thermolysin, and angiotensin-converting enzyme have been synthesized that combine structural features of the substrates with a thiol or carboxyl group in an appropriate position to coordinate a zinc atom bound at the catalytic site. We have synthesized (4R)-2-oxo-6-thioxohexahydropyrimidine-4-carboxylate (L-6-thiodihydroorotate) and have found that this analogue is a potent competitive inhibitor of dihydroorotase with a dissociation constant (Ki) in the presence of excess Zn2+ ion of 0.17 +/- 0.02 microM at pH 7.4. The potency of inhibition by L-6-thiodihydroorotate in the presence of divalent metal ions decreases in the order Zn2+ greater than Ca2+ greater than Co2+ greater than Mn2+ greater than Ni2+; L-6-thiodihydroorotate alone is less inhibitory and has a Ki of 0.85 +/- 0.14 microM. 6-Thioorotate has a Ki of 82 +/- 8 microM which decreases to 3.8 +/- 1.4 microM in the presence of Zn2+. Zn2+ alone is a moderate inhibitor of dihydroorotase and does not enhance the potency of other inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cooperative binding of Agrobacterium tumefaciens VirE2 protein to single-stranded DNA.

The VirE2 protein of Agrobacterium tumefaciens Ti plasmid pTiA6 is a single-stranded-DNA-binding protein. Density gradient centrifugation studies showed that it exists as a tetramer in solution. Monomeric VirE2 active in DNA binding could also be obtained by using a different protein isolation procedure. VirE2 was found to be thermolabile; brief incubation at 37 degrees C abolished its DNA-binding activity. It was insensitive to the sulfhydryl-specific reagent N-ethylmaleimide. Removal of the carboxy-terminal 37 residues of the 533-residue VirE2 polypeptide led to complete loss of DNA-binding activity; however, chimeric fusion proteins containing up to 125 residues of the VirE2 C terminus were inactive in DNA binding. In nuclease protection studies, VirE2 protected single-stranded DNA against degradation by DNase I. Analysis of the DNA-VirE2 complex by electron microscopy demonstrated that VirE2 coats a single-stranded DNA molecule and that the binding of VirE2 to its substrate is cooperative.     

Nucleotide sequence of the Methylobacterium extorquens AM1 moxF and moxJ genes involved in methanol oxidation

The nucleotide sequence has been determined for two genes involved in methanol oxidation in the facultative methylotroph, Methylobacterium extorquens AM1. The two genes are moxF, encoding the 66-kDa subunit of the methanol dehydrogenase and moxJ, located immediately downstream from moxF, which encodes a 30-kDa protein with unknown function. This information completes the sequence of the 5.86-kb XhoI-SalI fragment containing the moxFJGI region in M. extorquens AM1, and the structure of this gene cluster is presented. Evidence is presented that moxJ is also present in Paracoccus denitrificans. The aa sequence of MoxJ has provided little information concerning its function, but it does appear to contain a signal sequence suggesting a periplasmic location.     

Amylase gene expression in intraspecific and interspecific somatic transformants of Drosophila.

The Amylase locus in Drosophila melanogaster normally contains two copies of the structural gene for alpha-amylase, a centromere-proximal copy, Amy-p, and a distal copy, Amy-d. Products of the two genes may display discrete electrophoretic mobilities, but many strains known to carry the Amy duplication are characterized by a single amylase electromorph, e.g., Oregon-R, which produces the mobility variant AMY-1. A transient expression assay was used in somatic transformation experiments to test the functional status of the Amy genes from an Oregon-R strain. Plasmid constructs containing either the proximal or distal copy were tested in amylase-null hosts. Both genes produced a functional AMY-1 isozyme. Constructs were tested against an AMY-3 reference activity produced by a coinjected plasmid that contains the Amy-d3 allele from a Canton-S strain. With reference to the internal control, the Amy-p and Amy-d genes from Oregon-R expressed different relative activity levels for AMY-1 in transient assays. The transient expression assay was successfully used to test the functional status of Amy-homologous sequences from strains of other species of Drosophila characterized by a single amylase elctromorph, namely, Drosophila pseudoobscura ST and Drosophila miranda S 204. The amylase-null strain of D. melanogaster provided the hosts for these interspecific somatic transformation experiments.     

Protection against cisplatin toxicity by administration of glutathione ester

The role of cellular glutathione in the prevention of toxicity due to the anti-cancer drug cisplatin (cis-diamminedichloroplatinum) was explored in mice treated with buthionine sulfoximine (BSO), a selective inhibitor of gamma-glutamylcysteine synthetase (and therefore of glutathione synthesis), and with glutathione and glutathione monoisopropyl ester. Pretreatment of mice with BSO enhanced the lethal toxicity of cisplatin by about twofold. Administration of glutathione ester (dose, 2.5-7.5 mmol/kg) protected against lethal cisplatin toxicity; glutathione was also effective, but much less so. Glutathione ester, in contrast to glutathione, is effectively transported into cells and split to glutathione intracellularly. The previous findings that administered glutathione does not protect against lethal toxicity due to cadmium ions and mercuric ions, whereas glutathione ester does, suggest that intracellular glutathione is required for protection against these heavy metal ions. That administration of glutathione has a protective effect on cisplatin toxicity suggests that the toxic effects of cisplatin may be exerted both intracellularly and extracellularly, and that extracellular glutathione (or its degradation products) may form a complex with cisplatin extracellularly. The finding that glutathione ester is more effective than glutathione in protecting against the toxicity of cisplatin suggests that use of glutathione ester may be therapeutically advantageous.     

3H]prostaglandin uptake in vivo by rabbit uterine tissues and blastocysts

Day-6 pregnant rabbits were anesthetized and subjected to a mid-ventral laparotomy. [3H] Prostaglandin F2alpha) (PGF2alpha) [3H]PGE2, [14C]Urea or [14C]Sucrose were instilled into the uterine lumen via the uterotubal junction. The amounts instilled/uterine horn were respectively 3.7 +/- 0.3, 3.5 +/- 0.3, 5.7 +/- 1.3 and 2.7 +/- 1.6 muCi in 20mul of buffer. Animals were killed at 1, 2, 9, 19 or 21 h after radioactive instillation, and the amounts of radioactivity in blastocysts, uterine tissue, peritoneal cavity washings and urine evaluated by liquid scintillation spectrometry. A gradient of radioactivity was observed from the uterotubal junction to the cervical end of the uterus. Large amounts of [3H]PG were found in the injected horn and associated blastocysts with a considerable crossover to the non-injected horn, but little in the associated blastocysts. Much of the blastocysts associated- [3H]PG remained unmetabolized. Large amounts of metabolized [3 H] were found in urine. [14C]Urea was taken up by uterine tissue in the injected horn, but there was little cross over to the non-injected horn. Urea was also found in urine. Much of the [14C]Sucrose remained in the injected horn, and little was recovered from the urine. It was found that at 9 h, but not at 19 h, after [3 H]PG instillation, the PG was localized at the site of the blastocysts in the injected but not in the contralateral horn. Significantly more [3H]PGF2alpha than [3H]PGE2 was localized in this situation. [14C]Urea was not localized at the site of the blastocysts in urea injected horns. (ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis of eicosanoids by gamma-interferon-differentiated U937 cells

Interferons induce morphological, biochemical and functional alterations in monocyte macrophage and myeloid cell lines. We studied the effect of 3 days incubation with gamma-interferon from human buffy coats on the global synthesis of arachidonic acid metabolites by U937 cells. Interferon-induced morphologic changes including cytoplasmic and nuclear changes and the appearance of multiple lysosomal-like granules consistent with cellular differentiation were observed by electron microscopy. The labeling of phosphatidylserine, phosphatidylcholine and phosphatidylethanolamine was increased and that of phosphatidylinositol, free fatty acids as 3H-arachidonic acid and neutral lipids reduced, when interferon-treated cells were incubated with 3H-arachidonic acid. Interferon caused qualitative and quantitative changes in the synthesis of cyclooxygenase and lipoxygenase products. A23187, a calcium ionophore, and the tumor promotor, phorbol myristate acetate, greatly increased the synthesis by interferon-differentiated cells of 2 cyclooxygenase products; synthesis of lipoxygenase products was reduced. In the presence of indomethacin, 'shunting' into putative lipoxygenase products occurred. The relationship between interferon-induced morphologic and functional changes, the development of altered phospholipid and eicosanoid metabolism and the identity of these metabolites are yet to be established.