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.     

Differentiation of Langerhans Cells from Interdigitating Cells Using CD1a and S-100 Protein Antibodies

The present study shows that Langerhans cells can be differentiated from Interdigitating cells at the light microscopic level. Superficial lymph nodes and skin taken from necropsies and the lymph nodes of dermatopathic lymphadenopathy (DPL) were used for this experiment. Sections of lymph node and skin were embedded using the acetone, methyl benzoate and xylene (AMeX) method and dendritic cells were immunostained with anti S-100 protein antibody (S-100, and OKT-6 (CD1a) using the restaining method. Langerhans cells in the skin were positive for both CD1a and S-100. Dendritic cells positive for both CD1a and S-100, and dendritic cells positive for S-100, but not for CD1a were observed in superficial lymph nodes. In normal superficial lymph nodes, there were more interdigitating cells than Langerhans cells. The majority of the dendritic cells in the DPL were Langerhans cells. We conclude that the S-100 and CD1a positive cells are Langerhans cells, and the S-100 positive-CD1a negative cells are interdigitating cells.     

Hydrops fetalis caused by intrauterine human parvovirus infection

During a large outbreak of erythema infectiosum in 1987 in Toyama prefecture, Japan, a 32-year-old woman acquired a mild rash on her arms and legs at 18 weeks of gestation. At 26 weeks and 4 days of gestation, the fetus died by hydrops fetalis and pregnancy was terminated. Histological studies of the fetus revealed degeneration of erythroblastic cells in the liver and bone marrow. Extensive extramedullary hematopoiesis and hemosiderin deposits were observed in the liver. Antibody response to human parvovirus B19 virus was demonstrated in maternal sera by ELISA. Furthermore, dot hybridization with the molecularly cloned DNA probe revealed the presence of human parvovirus DNA sequence in the fetal liver, spleen, lung, kidney and placenta. This report describes the first case in Japan of hydrops fetalis caused by human parvovirus B19 infection.     

Insulin-like growth factor I rapidly stimulates tyrosine phosphorylation of a Mr 185,000 protein in intact cells

The type I insulin-like growth factor (IGF) receptor, like the insulin receptor, contains a ligand-stimulated protein-tyrosine kinase activity in its beta-subunit. However, in vivo, no substrates have been identified. We used anti-phosphotyrosine antibodies to identify phosphotyrosine-containing proteins which occur during IGF-I stimulation of normal rat kidney and Madin-Darby canine kidney cells labeled with ortho[32P]phosphate. Both cells provide a good system to study the function of the type I IGF receptors because they contain high concentrations of these receptors but no insulin receptors. In addition, physiological levels of IGF-I, but not insulin, stimulated DNA synthesis in growth-arrested cells. IGF-I stimulated within 1 min of tyrosine phosphorylation of two proteins. One of them, with a molecular mass between 97 and 102 kDa, was supposed to be the beta-subunit of the type I IGF receptor previously identified. The other protein had an approximate molecular mass of 185 kDa, which resembled, by several criteria, pp 185, originally identified during the initial response of Fao cells to insulin binding (White, M. F., Maron, R., and Kahn, C. R. (1985) Nature 318, 183-186). These data suggest that tyrosine phosphorylation of pp 185 may occur during activation of both the type I IGF receptor and the insulin receptor, and it could be a common substrate that transmits important metabolic signals during ligand binding.     

Biosynthesis of apolipophorin-III by the fat body in locusts

Biosynthesis of locust apolipophorin-III (apo-III) was studied in vitro. Gel electrophoresis and immunoblotting analyses of the locust hemolymph demonstrated that apo-III first appears in the hemolymph on the day 3 of the adult stage after the final molt and its hemolymph concentration increases thereafter. When incubated in vitro in a medium containing radioactive amino acid, the fat body cells synthesized the radiolabeled apo-III and released it into the medium. The developmental change in the apo-III synthesizing activity in the fat body reflected that of the apo-III concentration in the hemolymph. RNA isolated from the adult fat body directed the synthesis of apo-III as a major translation product in a cell-free system. These results indicate that the fat body is the tissue responsible for the synthesis of the locust apo-III, and biosynthesis of apo-III is developmentally regulated at the level of mRNA in accordance with the flight activity of the locust.     

Enzymic Synthesis of Leukotriene B4 in Guinea Pig Brain

Leukotriene B4 [5(S), 12(R)-dihydroxy-6, 14-cis-8,10-trans-eicosatetraenoic acid] was obtained from endogenous arachidonic acid when slices of the guinea pig brain cortex were incubated with the calcium ionophore A 23187. Enzymes involved in its synthesis, arachidonate 5-lipoxygenase [arachidonic acid to 5(S)-hydroperoxy-6-trans-8,11,14-cis-eicosatetraenoic acid and subsequently to leukotriene A4] and leukotriene A4 hydrolase (leukotriene A4 to B4), were present in the cytosol fraction. Arachidonate 5-lipoxygenase was Ca2+-dependent, and was stimulated by ATP and the microsomal membrane, as was noted for the enzyme from mast cells. The lipid hydroperoxides stimulated 5-lipoxygenase by four- to sixfold. The leukotriene A4 hydrolase activity was rich in brain, and the specific activity (0.4 nmol/min/mg of protein) was much the same as that of guinea pig leukocytes. High activities of these enzymes were detected in the olfactory bulb, pituitary gland, hypothalamus, and cerebral cortex. Since leukotriene B4 is enzymically synthesized in the brain, possible roles related to neuronal functions or dysfunctions deserve to be examined.     

The different effects of recombinant human tumor necrosis factor on rat fibrosarcoma sublines

Summary The antitumor effect of recombinant human tumor necrosis factor (rH-TNF) on two clones of rat fibrosarcoma with different metastatic potential to lymph nodes was examined. The colony formation of clone A, which has high metastatic potential, was completely inhibited by continuous exposure to rH-TNF at 50 U/ml. In contrast, colony formation of clone G, which has low metastatic potential, was not inhibited by high concentrations of rH-TNF (10,000 U/ml). The inhibitory effect of rH-TNF on colony formation by clone A was also observed with a 1-h exposure to rH-TNF. This effect was time and concentration dependent, as determined by the colony assay, ³H-thymidine uptake assay, and ⁵¹Cr-release assay. ³H-thymidine and ³H-uridine uptake per cell of clone A exposed to rH-TNF was not decreased. This suggests that the mechanisms of the antitumor effect of rH-TNF were not due to inhibition of DNA and RNA synthesis of tumor cells. In vivo growth and lymph node metastases of clone A inoculated i.p. to Donryu strain rats were completely suppressed by 14 consecutive i.p. injections of 10⁵ or 10⁶ U/kg per day of rH-TNF. On the other hand the growth of clone G was not influenced by rH-TNF administration.     

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.