Enzyme activities in Chrysosporium strains

390 strains of Chrysosporium were screened for their ability to produce enzymes. All strains produced: catalase, phosphatase, lipase, amylase, DNAse and phosphoamidase. No strains showed: valine arylamidase, oxidase, -galactosidase, urease, pectolase, protease nor RNAse.     

Structure and expression of the human XPBC/ERCC-3 gene involved in DNA repair disorders xeroderma pigmentosum and Cockayne's syndrome

The human XPBC/ERCC-3 was cloned by virtue of its ability to correct the excision repair defect of UV-sensitive rodent mutants of complementation group 3. The gene appeared to be in addition implicated in the human, cancer prone repair disorder xeroderma pigmentosum group B, which is also associated with Cockayne's syndrome. Here we present the genomic architecture of the gene and its expression. The XPBC/ERCC-3 gene consists of at least 14 exons spread over approximately 45 kb. Notably, the donor splice site of the third exon contains a GC instead of the canonical GT dinucleotide. The promoter region, first exon and intron comprise a CpG island with several putative GC boxes. The promoter was confined to a region of 260 bp upstream of the presumed cap site and acts bidirectionally. Like the promoter of another excision repair gene, ERCC-1, it lacks classical promoter elements such as CAAT and TATA boxes, but it shares with ERCC-1 a hitherto unknown 12 nucleotide sequence element, preceding a polypyrimidine track. Despite the presence of (AU)-rich elements in the 3'-untranslated region, which are thought to be associated with short mRNA half-life actinomycin-D experiments indicate that the mRNA is very stable (t 1/2 greater than 3h). Southern blot analysis revealed the presence of XPBC/ERCC-3 cross-hybridizing fragments elsewhere in the genome, which may belong to a related gene.     

Distribution of salmon gonadotrophin releasing-hormone in the brain and pituitary of the sea bass (Dicentrarchus labrax)

Summary The distribution of salmon gonadotrophin-releasing hormone (sGnRH) was studied in the brain and pituitary of two-year-old immature sea bass (Dicentrarchus labrax) by means of an enzymoimmunoassay (EIA) for sGnRH and immunocytochemistry. The EIA for sGnRH is a competitive assay using a tracer made of sGnRH coupled to acetylcholinesterase from an electric eel. The separation of free and bound tracer is achieved by coating the plates with mouse anti-rabbit IgG monoclonal antibodies. Displacement curves generated by sGnRH and extracts from pituitary and different brain regions showed a good parallelism allowing the assay to be used for sGnRH measurements in this species. Although all parts of the brain contained measurable levels of sGnRH, the highest concentrations were found in the pituitary, the olfactory bulbs and the telencephalon. These data were confirmed by immunocytochemistry. Cell bodies were found in the olfactory bulbs, ventral telencephalon, preoptic region and mediobasal hypothalamus. Immunoreactive fibers could be observed in all parts of the brain including the optic tectum, the cerebellum (corpus and valvula), the vagal lobe, the medulla oblongata and the rostral spinal cord. In most cases, these fibers do not form well defined bundles; however, there was clearly a continuum of immunoreactive fibers, extending from the olfactory bulbs to the pituitary, and along which all the cell bodies described above were located. In the ventral telencephalon and the preoptic region, clear pictures of varicose positive fibers contacting immunoreactive perikarya could be observed. These data indicate that sGnRH is most likely an endogenous peptide in the brain of the sea bass, although the presence of other forms of GnRH cannot be excluded at this point. This study also demonstrates that the general organization of the GnRH systems in the sea bass is highly similar to what has been described in most freshwater teleost species, and provides basis for further studies on the neuroendocrine control of gonadotrophin release in this commercially important species.     

Variation of geosmin content in Anabaena cells and its relation to nitrogen utilization

The addition of the proper amount of ammonium to the culture medium containing nitrate as nitrogen source enhanced the growth rate of Anabaena viguieri. The amount of geosmin produced by these cells varied with the concentrations of ammonium added. A negative correlation between the amount of geosmin produced and of the growth rate of cells was revealed. This was also found in cells grown on various forms of nitrogen sources. Without supply of any nitrogen compound, this organism is capable of fixing gaseous nitrogen, and under these conditions the cells grew relatively slowly. However, they produced more geosmin (per unit protein mass) than cells grown in the presence of combined nitrogen. The isolation of heterocysts, in which nitrogen was fixed, showed that these cells produced higher amounts of geosmin than vegetative cells. The possible relation of nitrogen assimilation to the production of geosmin in the cells was discussed.     

Utilization of alpha-ketoglutarate as a precursor for transmitter glutamate in cultured cerebellar granule cells

Alpha-ketoglutarate together with an amino group donor (alanine) was shown to be able to serve as a precursor for the glutamate pool which is released by potassium-induced depolarization (i.e., transmitter glutamate) in cerebellar granule cells. However, these compounds could not be utilized as precursors for intracellular glutamate or for release of transmitter aspartate. The formation of transmitter glutamate was inhibited by the transamination inhibitor aminooxyacetic acid but not by phenylsuccinate, an inhibitor of the dicarboxylate carrier in the mitochondrial membrane. Both of these inhibitors have previously been found to inhibit synthesis of transmitter glutamate from glutamine. The results support the hypothesis that alpha-ketoglutarate and alanine undergo transamination in the cytosol to form pyruvate and glutamate, and that this glutamate pool is available for transmitter release of glutamate but does not constitute the major intracellular pool of glutamate.     

Streptomycin and N-Methyl-N’-nitro-N-nitroso-guanidine inhibit incorporation of14C-precursors of macromolecule synthesis inEuglena gracillis

Streptomycin and N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) cause a 100% hereditary bleaching ofEuglena gracilis. In spite of the outcomes being the same, the corresponding mechanisms differ. By investigating the incorporation of the¹⁴C-labelled precursors of macromolecule synthesis we showed streptomycin to inhibit the synthesis of nucleic acids and proteins to the same extent (56%), whereas in the case of MNNG a 66% inhibition of nucleic acid synthesis and a 24% one of protein synthesis were observed.     

Microflora participating in the decomposition of carboxymethyl cellulose continuously added to the soil

The microbial community in the soil was analyzed during four weeks of a continuous enrichment of structural chernozem soil samples with a 0.1% solution of carboxymethyl cellulose (CMC) under aerobic and semianaerobic conditions. During the first 14 d, the total amount of the aerobic and anaerobic, cellulose-degrading microorganisms increased significantly. Various metabolic pathways were u‘ed te decompose the substrate: diverse metabolic systems were activated and different groups of microorganisms preferred in dependence on the presence of oxygen or the source of mineral nitrogen. In the later phases of cultivation, a decrease in the concentration of zymogenous microflora and in the level of substrate mineralization was observed ovon though CM-cellulase activity remained high. During the fourth week of cultivation, a conspicuous increase in the numbers of oligothropic bacteria occurring in the colcnies of the microorganisms degrading cellulose was found. The representatives of prosthecobacteria (Caulobacter, Hyphomicrobium, Prosthecomicrobium spp.) andSeliberia sp. were thus identified. This “microflora of dispersion” attends the zymogenous microbes degrading CMC and indicates later phases of the process of decomposition.     

Synthesis and secretion of mucous glycoprotein by the gill of Mytilus edulis. I. Histochemical and chromatographic analysis of [14C]glucosamine bioincorporation

The ability of the isolated gill epithelium of Mytilus edulis to incorporate [14C]glucosamine as a precursor in the biosynthesis and secretion of mucous glycoproteins was investigated. Localization of mucous cells in the gill filament was achieved using histochemical staining techniques. Mucus cells containing neutral and acidic mucins were found in the lateral region, whereas mucus cells containing primarily neutral or sulfated mucins were found in the abfrontal region. Autoradiographic results showed that in both regions, the mucous cells were rich in content of the incorporated radiolabel. The secreted glycoproteins containing the incorporated radiolabel were analyzed by column chromatography using Bio-Gel P-2 and P-6. Two populations of the glycoproteins differing in molecular size were isolated. Upon alkaline reductive borohydride cleavage of the O-glycosidic linkages of the high molecular weight protein, about 70% of the radiolabel and 85% of the carbohydrate content were removed from the protein. The alkaline borohydride cleavage resulted in the formation of at least six oligosaccharide chains of various lengths of sugar units. Gas chromatographic analysis of the carbohydrate composition shows that the glycoproteins contain N-acetylglucosamine, N-acetylgalactosamine, and galactose, fucose, and mannose as the neutral monosaccharides. The above results indicate that the isolated gill epithelium of M. edulis is capable of incorporating [14C]glucosamine in the synthesis of secretable mucin-type glycoproteins.     

Membrane-buffer partition coefficients of tetracaine for liquid-crystal and solid-gel membranes estimated by direct ultraviolet spectrophotometry

The membrane-buffer partition coefficient of tetracaine was measured by direct ultraviolet spectrophotometry in dimyristoylphosphatidylcholine unilamellar liposomes at temperatures above and below the main phase transition. The partition coefficients of uncharged tetracaine to solid-gel (18 degrees C) and liquid-crystal (30 degrees C) membranes were 6.9 x 10(4) and 1.2 x 10(5), respectively. Despite the general assumption that local anesthetic binding to the solid membrane is negligible, this study showed that the solid membrane binding amounts to 57.5% of the liquid membrane binding. Binding of the charged form to the liquid or solid membrane was not detectable under the present experimental condition of 0.03 mM tetracaine bulk concentration. The present method measures metachromasia of local anesthetics when bound to lipid membranes. Its advantage is that the separation of the vesicles from the solution is not required. A linearized equation is presented that estimates the partition coefficient or binding constant graphically from a linear plot of the absorbance data. The method is applicable for estimation of drug partition when a measurable spectral change occurs due to complex formation.