Enzymatic desulfurization of dibenzothiophene by a cell-free system of Rhodococcus erythropolis D-1

Abstract Pseudomonas syringae cells were exposed to Cu²⁺ alone or in the precence of acetate, proline or cysteine, at concentrations that reduced free Cu²⁺ to 1/10 of the total copper. Ligand concentrations (designated as isoeffective) were determined experimentally using a Cu²⁺-selective electrode and confirmed by computer calculations using published stability constants. Exposure of P. syringae cells to Cu²⁺ alone resulted in rapid and pronounced cell death, and binding of most of the copper in solution. The addition of acetate, proline or cysteine, a few minutes after Cu²⁺ treatment, resulted in a significant reduction in cell death, and in the amount of copper bound to the cells. For short exposures to Cu²⁺, cysteine was more effective than acetate or proline, but after 60 min of treatment, similar results were observed with these ligands. The addition of ligands before Cu²⁺ resulted in even more reduced copper toxicity. The results showed that, at isoeffective concentrations, weak and moderate copper-ligands can effectively antagonize copper toxicity, and that this protective effect does not require previously equilibrated copper-ligand solutions and is not very dependent of the nature of the ligand.     

Precise identification of gene products in hearts after in vivo gene transfection, using Sendai virus-coated proteoliposomes.

Both efficient gene transfer and the exact identification of gene product are required for gene therapy. Gene transfection of green fluorescence protein (GFP) might be useful for the reporter. After in vivo cotransfection of GFP and beta-galactosidase (beta-Gal) genes in Sendai virus-coated proteoliposomes to rat hearts, we compared the sensitivity and specificity of three methods: GFP detection, histochemical staining (HC) of beta-Gal activity, and immunostaining (IS) of the beta-Gal protein. Fluorescence microscopy and double staining of HC and IS revealed that both GFP and IS were equally sensitive and fourfold superior to HC at the peak of gene expression. However, different from skeletal muscle, the GFP of transfected cardiomyocytes showed two demerits: the fluorescence quenching due to the intense staining of beta-Gal activity, and nonspecific autofluorescence from myocardium. Thus, specific IS would be so far the most reliable to identify the gene product in heart.     

Single-locus control of the mast cell population in mouse skin

The number of mast cells in connective tissue from dorsal skin varied markedly among mouse strains. Inbred strains of mice were typed into three groups, high (NC and NZB mice), low (B6, B10, and BALB/c mice), and intermediate (C3H/He and DBA/2 mice), by their mast cell content in the skin. However, the strain differences in the number of mast cells was marginal at the age of weaning but became distinct with age. This could be explained mainly by the frequently observed clustering of mast cells in adult NC and NZB mice and the rarely observed clustering in younger mice as well as in adult B10 and BALB/c mice. The breeding experiment revealed that the difference in the number of mast cells between NC and B10 mice was controlled by a single autosomal dominant locus, for which we propose the designation Mcr (mast cell regulator). The role of the Mcr locus with regard to the frequency of the mast cell population in connective tissue is discussed.     

Sulfhydryl modification and activation of phenylalanine hydroxylase by dinitrophenyl alkyl disulfide

A new family of asymmetric thiol-disulfide exchange reagents, the dinitrophenyl alkyl disulfides (DNPSSR), was used to modify rat liver phenylalanine hydroxylase. The results indicate that the enzyme has two different types of reactive sulfhydryl (SH) residues per subunit. One SH residue was modified selectively by a DNPSSR having a neutral and hydrophilic alkyl group, and this modification was accompanied by appreciable activation of enzyme; the other SH residue was modified only by an anionic DNPSSR, and this modification did not result in activation. The catalytic properties of phenylalanine hydroxylase activated by DNPSSR were similar to those of the N-ethylmaleimide- (NEM-) modified enzyme, but the process of activation by DNPSSR was quite different from modification with NEM. An analysis of the reaction kinetics of the modification and of catalysis by the modified enzyme suggests that DNPSSR modification causes a change in the subunit interaction leading to a loss of the negative cooperativity normally seen with phenylalanine hydroxylase.     

Postglacial environmental change of the Pacific Ocean off the coasts of central Japan

Downcore changes in microfossil assemblages and oxygen isotope ratios in three piston cores recovered from the Northwestern Pacific, off central Japan, show that the subtropical Kuroshio front was located to the south of C-4 core site (Lat. 33° N) during the last glacial. The front then advanced northward, passing over the C-4 site and the C-6 site (34.6° N) at about 13 ka and 10 ka, respectively, and reached the C-1 core site (36° N) at about 7 ka. After 5.5 ka it retreated to the area between the C-1 and C-6 sites. A brief but significant cold event, the readvance of the cold Oyashio Current, is recognized between 11 and 10 ka in the two northern cores, but the current did not reach the southern C-4 site. A contemporaneous cold event is known in the North Atlantic, and the cooling was probably a global phenomenon likely to be associated with lowering of sea level. Contamination of isotopically light water is apparent between 14 and 11 ka in the marked change in isotopic composition of benthic foraminifers. Oxygen isotope ratios of planktonic foraminifers show that prior to the advance of the Kuroshio front, the surface water at these core sites was isotopically lighter than the Kuroshio water at that time.     

NADPH production from NADP+ with a glucose dehydrogenase system involving whole cells and immobilized cells of Gluconobacter suboxydans

Summary A convenient and efficient method of NADPH production from NADP⁺ has been established using a glucose dehydrogenase system involving whole cells and immobilized cells of Gluconobacter suboxydans IFO 3172. Using airdried cells of the bacterium, the optimum conditions for NADPH production were examined, including the cell and glucose concentrations, NADP⁺ concentration, pH, buffer and reaction temperature. Under suitable conditions, the conversion ratio and the amount of NADPH accumulated reached about 100% and 73 mg/ml of the reaction mixture, respectively, after 1-h reaction. Intact cells of the bacterium also showed high NADPH production even in the reaction mixture without a surfactant. The addition of Triton X-100 to the reaction mixture and freeze-thawing treatment of intact cells enhanced the production. The NADPH production method was further improved by using cells of the bacterium immobilized by entrapment in a -carrageenan gel lattice. The immobilized cells had almost the same enzymatic properties as the air-dried cells. The conditions for the continuous production of NADPH with an immobilized cell column were also investigated. NADPH was produced in a good yield (about 95%) with this continuous process.     

Structure of human phenylethanolamine n-methyltransferase gene: existence of two types of mRNA with different transcription initiation sites

The chromosomal gene for human phenylethanolamine N-methyltransferase (PNMT; EC 2.1.1.28) was isolated from a human genomic library using a cloned human PNMT cDNA as a probe, and the nucleotide sequence was determined. PNMT is encoded in a single gene which consists of three exons. We observed newly the presence of minor PNMT mRNA (type B) besides the major mRNA (type A) as reported previously (Kaneda et al., J. Biol. Chem. 263, 7672–7677, 1988) by Northern hybridization. Type B mRNA carries an approximately 700 nucleotide-long untranslated region in the 5′ terminus. This suggests that two types of mRNA are produced from a single gene through the use of two alternative promoters. A TATA-like sequence locates 30 base pair upstream from the cap site of type A mRNA. Upstream of the cap site, there are several sequences resembling Spl binding sites and glucocorticoid responsive elements, with the latter also found in the first intron.     

Cadmium-induced synthesis of metallothioneins in human T and B cell purified by a fluorescence activated cell sorter

Human peripheral blood lymphocytes were reacted with fluorescein-conjugated antibodies specific to T or B cell surface antigen and fractionated with a fluorescence activated cell sorter. The isolated T and B cells were examined for their capacity to synthesize metallothioneins (MTs). Analysis by gel electrophoresis indicated that both T and B cells were able to produce MTs in a Cd2+-inducible manner, suggesting that both cells types have a mechanism of protection against Cd2+ toxicity.     

Paradoxical enhancement of interleukin-2-mediated cytotoxicity against K562 cells by addition of a low dose of methotrexate

Summary In vitro effects of methotrexate (MTX) on interleukin-2(IL-2)-mediated cytotoxicity of peripheral blood mononuclear cells (PBMC) were studied. PBMC were incubated with human recombinant IL-2 (25 U/ml) for 72 h; during the last 24 h, various concentrations (10 pM–1 µM) of MTX were added to the culture. Cytotoxicity against k562 cells was measured by a 4-h⁵¹Cr-release assay. The IL-2-mediated cytotoxicity was paradoxically increased at around a concentration (10 nM) MTX. Such a low concentration of MTX showed no anti-proliferative effect on cell growth. This enhancement with 10 nM MTX was shown only in an E-rosette⁺ (E⁺) population, but not in E-rosette^– (E^–). In addition, when E⁺ cells were treated with an anti-CD16 monoclonal antibody plus complement after incubation with IL-2 and MTX, MTX-induced enhancement was lost, suggesting that an E⁺CD16⁺ cell population was mainly involved in this augmentation. Positively sorted E⁺CD16⁺ cells showed similar enhancement of cytotoxicity after treatment with IL-2 plus MTX. On the other hand, MTX treatment did not show the phenotypical changes including of the E⁺CD16⁺ cells, indicating that this treatment did not affect the differentiation and proliferation of the specific cell subset. Our results indicate that a low dose of MTX could have a role in the regulation of immunological anti-cancer surveillance systems through the natural killer and lymphokine-activated cytotoxic cells.This work was supported in part by a Grant-in-Aid for Cancer Research (1–10) from the Ministry of Health and Welfare in Japan