Isolation and Characterization of Mutants of Clostridium acetobutylicum ATCC 824 Deficient in Acetoacetyl-Coenzyme A:Acetate/Butyrate:Coenzyme A-Transferase (EC 2.8.3.9) and in Other Solvent Pathway Enzymes

Mutants of Clostridium acetobutylicum ATCC 824 exhibiting resistance to 2-bromobutyrate or rifampin were isolated after nitrosoguanidine treatment. Mutants were screened for solvent production by using an automated alcohol test system. Isolates were analyzed for levels of butanol, ethanol, acetone, butyrate, acetate, and acetoin in stationary-phase batch cultures. The specific activities of NADH- and NADPH-dependent butanol dehydrogenase and butyraldehyde dehydrogenase as well as those of acetoacetyl-coenzyme A:acetate/butyrate:coenzyme A-transferase (butyrate-acetoacetate coenzyme A-transferase [EC 2.8.3.9]) (CoA-transferase), butyrate kinase, and phosphotransbutyrylase were measured at the onset of stationary phase. Rifampin-resistant strain D10 and 2-bromobutyrate mutant R were found to be deficient in only CoA-transferase, while several other mutants exhibited reduced butyraldehyde dehydrogenase and butanol dehydrogenase activities as well. The colony morphology of 2-bromobutyrate mutant R was similar to that of the parent on RCM medium; however, it had about 1/10 the level of CoA-transferase and increased levels of butanol dehydrogenase and butyraldehyde dehydrogenase. A nonsporulating, spontaneously derived degenerated strain exhibited reduced levels of butyraldehyde dehydrogenase, butanol, dehydrogenase, and CoA-transferase compared with those of the original strain. When C. acetobutylicum ATCC 824 was grown on medium containing low levels of 2-bromobutyrate, an altered colony morphology was observed. Not all strains resistant to 2-bromobutyrate (12 mM) were non-solvent-producing strains.     

Acute and chronic effects of an anionic surfactant on some freshwater tubificid species

We report the results of research on acute and chronic effects of linear alkylbenzensulfonate (LAS) on two tubificid species. 96 h LC₅₀ assay values were estimated at 10° for Limnodrilus hoffmeisteri and Branchiura sowerbyi exposed to different concentrations of LAS dissolved in water, both with and without sediment. The presence of sediments modified LAS toxicity and increased values: NOEC and LOEC resulted in values 2.5 times higher for Branchiura sowerbyi and 4–4.5 times for Limnodrilus hoffmeisteri, when the sediments were present. The chronic effects induced by a long exposure to LAS were evaluated for different stages of the biological cycle of Branchiura sowerbyi. Using concentrations between the NOEC and LOEC (0.5, 2.5, and 5 ppm), with control, we could observe that: 1) at 5 ppm the cocoons were laid precociously compared to controls, 2) in all treated series the number of cocoons was lower than in controls, 3) the mean number of oocytes per cocoon was lower for the worms submitted to LAS, compared to the control, 4) the period of embryonic development was similar for all used concentrations and for control, and 5) the number of degenerated cocoons was unchanged by the LAS treatment.     

Induction of vancomycin resistance in Enterococcus faecium by inhibition of transglycosylation

Vancomycin resistance has recently been recognized among clinical isolates of enterococci. Resistance is inducible, and associated with production of a novel 39 kDa membrane protein. The mechanism by which exposure to vancomycin, which does not penetrate the cell membrane, induces resistance is unknown. In the vancomycin resistant strain Enterococcus faecium 228, resistance was also inducible by moenomycin, suggesting that inhibition of the transglycosylation step in peptidoglycan synthesis may be required for induction of resistance. Cytoplasmic pools of peptidoglycan precursors were increased after exposure to vancomycin or moenomycin, representing a potential means for regulation of induction.     

Regulation of Myelin P0 Glycoprotein Synthesis in Cultured Rat Schwann Cells and Continuous Rat PNS Cell Lines

We studied the effects of agents that raise intracellular cyclic AMP on synthesis of myelin components by cultured neonatal rat sciatic nerve Schwann cells and by continuous PNS cell lines derived from the fusion of neonatal rat sciatic nerve Schwann cells with rat RN22 Schwannoma. Treatment with N6,2'-O-dibutyryl cyclic AMP (dibutyryl cyclic AMP) caused a fourfold increase in Schwann cell incorporation of 35SO4 into sulfogalactosylceramide (sulfatide), and elicited a 10- to 20-fold increase in such incorporation by the continuous PNS cell lines; a similar effect on PNS cell line sulfatide radiolabelling was obtained with forskolin. Cultured Schwann cells expressed barely detectable levels of myelin P0 glycoprotein (P0) mRNA and myelin basic protein (MBP) mRNA. Treatment of the Schwann cells with axolemmal fragments or with dibutyryl cyclic AMP did not elicit a detectable increase in the levels of these mRNAs. The PNS cell lines constitutively expressed much higher levels of P0 mRNA than did the Schwann cells, and synthesized immunochemically demonstrable P0 glycoprotein, but did not express MBP. Treatment of the PNS cell lines with dibutyryl cyclic AMP markedly reduced expression of P0 mRNA and also diminished immunoreactive P0 glycoprotein. These PNS cell lines should prove useful for further studies of the control of Schwann cell differentiation.     

Enhancement and inhibition of benzo[a]pyrene-induced SOS function in E. coli by synthetic antioxidants

8 antioxidants were tested in the SOS chromotest for induction of SOS function and for modulation of benzo[a]pyrene-induced SOS function. None of the antioxidants leads to increased beta-galactosidase activity by itself. Butylated hydroxytoluene at concentrations between 10(-5) M and 3 X 10(-4) M enhances benzo[a]pyrene-induced SOS function at benzo[a]pyrene concentrations between 10(-6) M and 3 X 10(-5) M. Butylated hydroxyanisole, ethoxyquin, propyl gallate and octyl gallate also slightly enhance benzo[a]pyrene-induced SOS function at concentrations up to 3 X 10(-4) M though to a lesser degree than butylated hydroxytoluene. Dodecyl gallate, vitamin C and alpha-tocopherol do not increase benzo[a]pyrene action. In concentrations exceeding 3 X 10(-4) M all synthetic antioxidants tested but not vitamin C and alpha-tocopherol decrease beta-galactosidase activity both in the absence and, more extensively, in the presence of benzo[a]pyrene. Preliminary data suggest that the apparent suppression of benzo[a]pyrene-induced SOS function is not due to an effect on the formation of benzo[a]pyrene metabolites by the metabolizing system used.     

Dinitrogenase reductase (Fe-protein) of nitrogenase in the cyanobacterial symbionts of three Azolla species: Localization and sequence of appearance during heterocyst differentiation

Transmission electron microscopy and immunocytological labeling were used to study the distribution and ontological occurrence of dinitrogenase reductase (Fe-protein) of nitrogenase in cyanobacterial symbionts within young leaves of the water-ferns Azolla filiculoides Lamarck, A. caroliniana Willdenow, and A. pinnata R. Brown. Rabbit anti-dinitrogenase reductase antisera and goat anti-rabbit-immunoglobulin G antibody conjugated to colloidal gold were used as probes. Western blot analyses showed that a polypeptide of approx. 36 kDa (kdalton) was recognized in the symbionts of all three Azolla species and that the polyclonal sera used were monospecific. In all symbionts, nitrogenase was immunologically recognizable within heterocysts. It was absent from vegetative cells, and also from the akinetes of the A. caroliniana and A. pinnata symbionts. The differentiation of vegetative cells into heterocysts in all three symbionts was initiated by formation of additional external cell-wall layers and narrowing of the neck followed by loss of glycogen, mild vesiculation of thylakoid membranes, and the appearance of polar nodules. No nitrogenase was detected at these early stages, but it appeared in the intermediate proheterocyst stage concomitantly with the formation of contorted membranes, and reached the strongest labeling in mature heterocysts, containing extensive tightly packed membranes. Nitrogenase was evenly distributed throughout heterocysts except at the polar regions, which contained honey-comb configurations and large polar nodules. With increased age of the A. caroliniana and A. pinnata symbionts, heterocysts became highly vesiculated, with a concomitant decrease in the amount of nitrogenase detected.Abbreviations IgG Immunoglobulin G - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate - TEM transmission electron micrograph     

Very low osmotic water permeability and membrane fluidity in isolated toad bladder granules

Summary Osmotic water permeability of the apical membrane of toad urinary epithelium is increased greatly by vasopressin (VP) and is associated with exocytic addition of granules and aggrephores at the apical surface. To determine the physiological role of granule exocytosis, we measured the osmotic water permeability and membrane fluidity of isolated granules, surface membranes and microsomes prepared from toad bladder in the presence and absence of VP.P _f was measured by stopped-flow light scattering and membrane fluidity was examined by diphenylhexatriene (DPH) fluorescence anisotropy. In response to a 75mm inward sucrose gradient, granule size decreased with a single exponential time constant of 2.3±0.1 sec (sem, seven preparations, 23°C), corresponding to aP _f of 5×10^–4 cm/sec; the activation energy (E _a ) forP _f was 17.6±0.8 kcal/mole. Under the same conditions, the volume of surface membrane vesicles decreased biexponentially with time constants of 0.13 and 1.9 sec; the fast component comprised 70% of the signal. Granule, surface membrane and microsome time constants were unaffected by VP. However, in surface membranes, there was a small decrease (6±2%) in the fraction of surface membranes with fast time constant. DPH anisotropies were 0.253 (granules), 0.224 (surface membrane fluidity is remarkably lower than that of surface and microsomal membranes, and (4) rapid water transport occurs in surface membrane vesicles. The unique physical properties of the granule suggests that apical exocytic addition of granule membrane may be responsible for the low water permeability of the unstimulated apical membrane.     

Insertional inactivation of a gene which controls expression of vancomycin resistance on plasmid pHKK100

Expression of inducible high level vancomycin resistance (Vmr) in enterococci appears to require other plasmid-encoded genes in addition to the previously described structural genes vanA and vanH. Tn917 mutagenesis was used to identify such a region in the Vmr plasmid pHKK100. Insertional inactivation of a 693-bp open reading frame upstream from vanH resulted in complete loss of Vmr. This putative 26,642-Da protein has been designated VanR.     

A mite survey in the dumpsters for urban solid rubbish

Summary The presence of house dust mites and storage mites in dumpsters was investigated in 3 different areas of Venice hinterland. The aim of this study was to find a relationship between some reported respiratory symptoms among 6 workers who were responsible for moving the dumpsters to the truck and the bio-aerosol released from such operation. These symptoms were closely related to the work and after allergological evaluation we found sensitisation to house dust mites in all 6 employees. The mine exposure in the workplace was assessed by a standardized sampling and analysis of dust obtained after brushing the inside surfaces of dumpsters. In 50% of the specimens (10/20) we found mites belonging to the following families: Pyroglyphidae (Dermatophagoides spp.), Acaridae (Acarus siro) and a lower amount of Cheyletidae (Cheyletus spp.). An indirect test (guanine test) was also performed and gave positivity in 16 dust samples (80%). The insides of dumpsters showed to be an optimal environment for mites survival and reproduction because of cracks and grooves on the walls, the high humidity level and the presence of mould. These mites probably come from dwelling places and other environments (markets, slaughters-houses, farms, etc.). This suggests that mites allergens can be released during rubbish discharging from the dumpsters and represent a possible risk for the employees.