Acetate production from lactate and glucose fermentation by Gluconobacter oxydans

Summary The production of acetate from the fermentation of lactate by Gluconobacter oxydans was studied. Batch experiments showed that glucose was the preferred substrate compared to lactate. A fed-batch culture was fed with a mixture of glucose and lactate followed by periodic addition of lactate. The maximum productivity of acetate was 0.16 g/l h but this value decreased during the fedbatch culture due to growth inhibition by acetate.     

Effects of delta 9-tetrahydrocannabinol on testosterone production in vitro: influence of Ca++, Mg++ or glucose

The major psychoactive component of marihuana, delta 9-tetrahydrocannabinol (THC), influences testicular function. In the present experiments, the addition of THC to incubations of whole decapsulated mouse testes altered testosterone (T) production differentially, depending on the specific gonadotropin used, the dose of THC and/or the amount of divalent cation present in the media. In the presence of luteinizing hormone (LH; 10 ng/ml), and a dose of 25 micrograms THC/ml, T production was significantly decreased, compared to that by testes incubated with LH and vehicle at all Ca++ levels, except at 0.127 or 1.0 mM Ca++. The production of T by these paired testes exposed to either THC or vehicle (ethanol; ETOH), increased as Ca++ concentration approached physiological levels. In contrast, in the presence of follicle-stimulating hormone (FSH; 1 microgram/ml), THC-induced suppression of T production was significant in the absence of Ca++ from the media, and at 12.7 mM Ca++. However, it appeared that the levels of Ca++ did not differentially affect T production in the presence of FSH, whether or not THC was also added. In the presence of human chorionic gonadotropin (hCG; 12.5 mIU/ml), a lower dose of THC (25 ng/ml), stimulated T production at 0.25 to 1 mM Ca++, but had no effect as Ca++ reached 2.5 mM. Without additional Ca++ in the media, this dose of THC significantly reduced T secretion. In contrast, in the presence of hCG, a higher THC dose (25 micrograms/ml), suppressed T accumulation at 0.127, and from 1.0 to 12.7, but had no effect at 0.25 mM, or in the absence of Ca++. In the presence of hCG, the high 25 micrograms/ml dose of THC stimulated T production, in the absence of additional Mg++, and at 0.01 mM Mg++, but THC had no effect at 0.1 mM Mg++, but inhibited T production at 1.1 mM Mg++. In the presence of hCG, 25 micrograms THC/ml produced a consistent suppression of T production across glucose concentrations examined. These findings suggest that the mechanisms by which THC effects testicular steroidogenesis may involve Ca++- and/or Mg++-dependent processes. Differential requirements for these divalent cations by the gonadotropins may explain the interactive effects of THC with LH, hCG or FSH.     

Changes in protein phosphorylation during the cell cycle of Chinese hamster ovary cells

The phosphorylation patterns of proteins were examined during the cell cycle of Chinese hamster ovary cells. This was accomplished by labeling synchronized cells at various times with [32P]orthophosphate and separating the proteins by both isoelectric focusing and nonequilibrium pH gradient two-dimensional gel electrophoresis. The most dramatic changes occurred during late G2/M when approximately eight proteins (including vimentin, lamin B, and histones 1 and 3) showed increased phosphorylation. Ten other proteins appeared to be uniquely phosphorylated during late G2/M. Of these 10 proteins, seven were no longer phosphorylated shortly after mitosis. There is also at least one protein which showed a relative decrease in phosphorylation during late G2/M.     

Characteristics of different cytoplasmic and nuclear estrogen receptors appearing with continuous hormonal exposure

Biochemical properties of cytosol estrogen receptor (ERC) and nuclear estrogen receptor (ERN) from rat uteri continuously exposed in vivo to 17 beta-[2,4,6,7-3H] estradiol ( [3H]E2) for 6 h have been studied on the basis of immunological recognition and chromatographic elution patterns. Overall concentrations of ERC and ERN did not change during this time period when receptor-saturating concentrations of [3H]E2 were maintained (Jakesz, R., Kasid, A., and Lippman, M. E. (1983) J. Biol. Chem. 258, 11798-11806); however, biochemical characteristics were different in ERC and ERN after short or long term hormonal exposure. When ERC from rats treated with estradiol for 30 min was applied to HAP or DEAE columns, two different ER binding components were seen. DNA binding in a cell-free system revealed that these binding components represented an activated and a nonactivated ERC population. After long term hormonal exposure (6 h), only one component of ERC with low DNA binding could be shown despite the preservation of an equivalent quantity of cytoplasmic binding activity. This binder does not react with a monoclonal antibody directed against extranuclear estrogen receptor species. These data suggest disappearance of the activated ERC population, with appearance of a new, immunologically nonrecognizable ERC species with 6 h of continuous hormonal exposure. Elution profiles of ERN on HAP chromatography reveal 2 different binding components at 30 min and at 6 h of continuous [3H]E2 exposure. There is an increase of the population eluted at higher molarity after 6 h of in vivo treatment. This later eluting binding component is the major DNA binder in vitro. ERN from both time points are recognized immunologically by monoclonal antibody. After reaction with the antibody, the sedimentation coefficient shifted to 8-9 S on sucrose gradients, but the previously described faster sedimentation of ERN extracted 6 h after injection persisted. We conclude that ER in both cellular compartments undergoes time-dependent alterations, which may be involved in the initiation of hormone action.     

Structural difference of the insulin receptors from circulating monocytes and erythrocytes

We compared insulin receptors obtained from cells widely used in human studies, the circulating monocytes and erythrocytes. Biochemically, these receptors possess both binding (alpha-subunit) and tyrosine kinase (beta-subunit) activities similar to insulin receptors from other sources. Subtle differences in molecular weight, however, were detected between the alpha-subunits of these two cell types when analyzed by NaDodSO4-PAGE. Crosslinked [125I]insulin-labeled alpha-subunit of the monocyte insulin receptor was of higher apparent molecular weight than the alpha-subunit derived from red cells. Neuraminidase treatment of the alpha-subunits from each cell type indicated more sialic acid residues were present on the monocyte than the red cell alpha-subunit. The structural properties of the insulin receptors of human circulating cells are similar but not identical to insulin receptors of other characterized systems.     

The Role of Surface Water and Light on Differentiation in the Cellular Slime Molds

A key environmental factor in inducing final fruiting body differentiation in the migrating slugs of cellular slime molds is loss of contact with a surface film of water. If the slug tip reaches above the water film, fruiting tends to occur, but if it stays in contact with the surface water, continued migration is favored. Light is effective in promoting fruiting only if the phototactic slugs orient away from surface (as with overhead light) and therefore, only indirectly affects differentiation.     

Low level transport of IgA to bile via the asialoglycoprotein receptor

The rat and rabbit transport IgA from blood to bile by a highly efficient transcellular pathway mediated by secretory component (SC). Other mammals do not express SC on liver hepatocytes, but they do transport a small amount of IgA to bile. In the first part of this study, human polymeric IgA was radiolabeled and depleted of SC binding activity by successive affinity adsorption. Transport of this preparation intact to rat bile was 4%, but was reduced to 2% when 50 mg unlabeled asialoglycoprotein was preadministered. The 2% decline corresponds to the percent of asialo-orosomucoid diverted to bile from the lysosomal pathway. In guinea-pigs, missorting of asialo-orosomucoid intact to bile was 10% of the injected dose. Transport of normal human IgA to bile was 1-2%, even though guinea-pigs do not express SC in the liver. Excess unlabeled asialofetuin reduced the transport of asialo-orosomucoid by 10-fold and IgA by 6-fold. This demonstrates that the asialoglycoprotein receptor can mediate transport of IgA to bile in small amounts, but that this transport may be only a biological artifact resulting from limited fidelity of intracellular protein sorting.     

Affinity labeling of dihydrofolate reductase with an antifolate glyoxal

Dihydrofolate reductases from different species contain several highly conserved arginines, some of which have been shown by x-ray crystallography to have their guanido groups near the p-aminobenzoyl glutamate moiety of enzyme-bound methotrexate. The orientation of one of these (Arg-52) appears to be completely reversed in comparing the crystal structures of Escherichia coli with Lactobacillus casei enzyme (Bolin, J. T., Filman, D. J., Matthews, D. A., Hamlin, R. C., and Kraut, J. (1982). J. Biol. Chem. 257, 13650-13662). We synthesized a novel antifolate containing a glyoxal group designed to react specifically with active-site guanido groups which are able to approach the p-aminobenzoyl carbonyl of methotrexate. The binding of this compound to the enzyme was competitive with dihydrofolate (DHF) in ordinary buffers. In borate buffer at pH 8.0 it inactivated dihydrofolate reductases from both E. coli and L. casei at similar maximum rates, while the chicken liver enzyme was more slowly inactivated. The inactivation was stoichiometric, paralleled the loss of the glyoxal chromophore, and showed saturation kinetics. Inhibitor binding and thus inactivation was enhanced by NADPH, while DHF protected the enzyme. This allowed calculation of the Kd for DHF which was found to be identical with its Km. The stoichiometrically inactivated enzyme displayed the 340-nm chromophore characteristic of 4-aminopteridines bound to dihydrofolate reductase confirming active-site labeling with normal orientation of the ligand. The ligand remained covalently bound to inactivated enzyme upon denaturation at low pH but dissociated at neutral pH. Computer graphic modeling of the crystal structures predicted reaction of Arg-31 but not Arg-52 in L. casei dihydrofolate reductase and of only Arg-52 in the E. coli enzyme. Purification of the CNBr fragments from the inactivated enzymes gave a single labeled peptide for each species. The particular peptide tagged in each case was unaffected by the presence of NADPH and was in excellent agreement with the crystallographic predictions.