Construction and properties of K1 type killer wine yeasts

Summary With the use of a protoplast fusion technique the killer character of K1 type was transferred into four industrial Saccharomyces wine yeasts. The prototrophic yeast strains active against standard sensitive and K2 killer Saccharomyces strains, resistant to K1 killer toxin were constructed with no changes in technological properties.     

Divergence between GLUT4 mRNA and protein abundance in skeletal muscle of insulin resistant rats

Hyperglycemia and skeletal muscle insulin resistance coexist in uncontrolled type 2 diabetes mellitus. Similar defects in insulin action were observed in glucose-infused, normal rats, a model of glucose toxicity. In these rats insulin-stimulated glucose uptake by skeletal muscle was decreased due to a post-receptor defect. We investigated whether the impaired glucose uptake resulted from a decrease in the abundance of the predominant muscle glucose transporter (GLUT4) mRNA and/or protein. GLUT4 protein abundance in the hyperglycemic rats was not different from the control group despite a 50% decrease in muscle glucose uptake. GLUT4 mRNA abundance was 2.5-fold greater in the hyperglycemic rats as compared to the control animals. We conclude that the coexistence of hyperglycemia and hyperinsulinemia results in (1) a defect in GLUT4 compartmentalization and/or functional activity and (2) a divergence between GLUT4 mRNA levels and translation.     

Lysyl oxidase-mediated crosslinking in granulation tissue collagen in two models of hyperglycemia.

Enzymatically mediated crosslinks and nonenzymatic glycation were quantified in granulation tissue collagen in two models of hyperglycemia, diabetes and galactosemia, that have opposite effects on collagen solubility. The effects of castration, which alters collagen solubility, was also investigated. Collagen from both diabetic and galactosemic rats had significantly increased levels of dihydroxylysinonorleucine (DHLNL), a difunctional reducible crosslink. Galactosemic rats had significantly decreased levels of hydroxypyridinium, a trifunctional product of DHLNL and hydroxylysine, relative to control values, while diabetic rats had normal levels. Values for all other detectable crosslinks in collagen from hyperglycemic rats were indistinguishable from control values. Nonenzymatic glycation was increased in both groups of hyperglycemic rats. In diabetic rats, but not in galactosemic rats, nonenzymatic glycation was strongly correlated with DHLNL content. Castration had no effect on crosslink content of collagen from diabetic or galactosemic rats. This study demonstrates that (1) collagen crosslinking is abnormal in granulation tissue collagen in both experimental diabetes and galactosemia, (2) these changes are similar to those observed in skin collagen from insulin-dependent diabetic subjects and (3) the crosslinking abnormalities are not correlated with alterations in collagen solubility. We conclude that hyperglycemia-associated increases in immature crosslinks cannot account for altered collagen solubility, although impaired maturation of such crosslinks may be partially responsible for the lathyrogenic effect of galactosemia.     

Mechanisms for activation and subsequent removal of cytosolic Ca2+ in bradykinin-stimulated neuronal and glial cell lines

Mechanisms for activation and for removal of cytosolic Ca2+ after stimulation with bradykinin were investigated in two neural cell lines by measuring cytosolic Ca2+ activity and 45Ca2+ fluxes. In the neuronal (neuroblastoma x glioma hybrid) and in the glial (rat glioma) cell lines, the transient, bradykinin-induced rise in cytosolic Ca2+ activity (determined by fura-2 or indo-1 fluorescence) was blocked by a bradykinin B2 receptor antagonist. Ca2+ ionophores (ionomycin and 4-Br-A23187) caused a comparable transient rise in cytosolic Ca2+ activity. After addition of ionophores, the Ca2+ response to bradykinin was reduced or completely blocked in both cell lines. At the concentrations used, the ionophores primarily depleted intracellular Ca2+ stores and prevented refilling of the stores. Thus, the bradykinin-induced rise of cytosolic Ca2+ activity seems to be mostly due to Ca2+ release from internal stores. In the neuronal but not in the glial cell line, a brief stimulation by bradykinin of 45Ca2+ uptake was followed by a long-lasting inhibition below control values. Thus, in the neuronal cells bradykinin presumably blocks Ca2+ channels by a readily reversible, pertussis toxin-insensitive mechanism. Excess cytosolic Ca2+ of the bradykinin-stimulated cells is mostly not resequestered into the internal Ca2+ pool accessible to bradykinin, but is mainly extruded through the plasma membrane, as indicated by (i) stimulation of 45Ca2+ release by bradykinin, (ii) quick reduction by bradykinin of cellular 45Ca2+ content of cells preequilibrated with 45Ca2+, and (iii) diminution of the ionophore-inducible Ca2+ response after the addition of bradykinin.     

Vorkommen des Auerhuhns (Tetrao urogallus major C. L. Brehm, 1831) am Athos (Greichenland)

Summary At the Mount Athos the Capercaillie is spread in the montane high forests at an altitude between 1140 m and 1340 m above sea level. This isolated occurrence at the southern boundary of the species area and 140 km to the south of the breeding place in the Rhodope Mountains is the southernmost occurrence recorded so far. In former times, the Capercaillie presumably was widespread in the mountain forests of Central and Northern Greece.     

Detection of Rotavirus in Sewage

For detection of rotavirus, domestic sewage was concentrated by two different methods: (i) adsorption to and elution from positively charged Seitz filters, followed by ultracentrifugation, and (ii) chemical precipitation. The concentrated fluids were tested by an enzyme-linked immunosorbent assay and electron microscopy. In 6 of 24 (25%) samples, rotavirus was detectable after the combined filtration and ultracentrifugation technique with both an enzyme-linked immunosorbent assay and electron microscopy. No positive results were obtained after chemical precipitation.     

Quantitation of gibberellins and the metabolism of [3H]gibberellin A1 during somatic embryogenesis in carrot and anise cell cultures

In a carrot (Daucus carota L.) cell line lacking the ability to undergo somatic embryogenasis, and in carrot and anise (Pimpinella anisum L.) cell lines in which embryogenesis could be regulated by presence or absence of 2,4-dichlorophen-oxyacetic acid (2,4-D), in the medium (+2,4-D=no embryogenesis,-2,4-D=embryo differentiation and development), the levels of endogenous gibberellin(s) (GA) were determined by the dwarfrice bioassay, and the metabolism of [³H]GA₁ was followed. Embryos harvested after 14 d of subculture in-2,4-D had low levels (0.2–0.3 g g^-1 dry weight) of polar GA (e.g. GA₁-like), but much (3–22 times) higher levels of less-polar GA (GA_4/7-like); GA₁, GA₄ and GA₇ are native to these cultures. Conversely, the undifferentiated cells in a non-embryogenic strain, and proembryos of an embryogenic strain (+2,4-D) showed very high levels of polar GA (2.9–4.4 g g^-1), and somewhat reduced levels of less-polar GA. Cultures of anise undergoing somatic embryo development (-2,4-D) metabolized [³H]GA₁ very quickly, whereas proembryo cultures of anise (+2,4-D) metabolized [³H]GA₁ slowly. The major metabolites of [³H]GA₁ in anise were tentatively identified as GA₈-glucoside (24%), GA₈ (15%), GA₁-glucoside (8%) and the ¹⁽¹⁰⁾GA₁-counterpart (2%). Thus, high levels of a GA₁-like substance and a reduced ability to metabolize GA₁ are correlated with the absence of embryo development, while lowered levels of GA₁-like substance and a rapid metabolism of GA₁ into GA₈ and GA-conjugates are correlated with continued embryo development. Exogenous application of GA₃ is known to reduce somatic embryogenesis in carrot cultures; GA₄ was found to have the same effect in anise cultures. Thus, a role (albeit negative) in somatic embryogenesis for a polar, biologically active GA is implied.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - GA gibberellin(s) or gibberellin-like substances - GC-RC gas chromatography-radiochromatogram counting - HPLC high-presare liquid chromatography - Rt retention time - TLC thinlaver chromatography     

Sensitive immunological detection of translation products in SV40 plaques

A simple and sensitive in situ radioimmunoassay, using simian virus 40 (SV40) proteins as a model, has been developed for the detection of specific translation products of foreign genes cloned in viral vectors. This assay is based on the coupling of all proteins in viral plaques to diazophenylthioether (DPT)-paper. Specific proteins bound to the filter are detected by autoradiography after sequential incubation with (i) unfractionated and unlabeled specific antiserum and (ii) 125I-labeled protein A from Staphylococcus aureus. This assay detects SV40-specific proteins in individual plaques as early as 42 h after infection and its sensitivity limit is below 5 x 10(8) molecules of the major SV40 capsid protein, VP1.     

Immunological detection of specific proteins in total cell extracts by fractionation in gels and transfer to diazophenylthioether paper

We describe a sensitive immunological procedure for the detection of specific proteins in total cell extracts and for the comparison of antigenically related polypeptides. Proteins are fractionated in polyacrylamide gels and transferred electrophoretically to diazophenylthioether paper, to which they bind covalently. Specific proteins are identified by incubation with specific antibody and 125 I-labeled protein A from Staphylococcus aureus, followed by autoradiography. High-resolution separation of proteins prior to transfer is achieved by polyacrylamide gradient gel electrophoresis in the presence of sodium dodecyl sulfate or by nonequilibrium pH gradient electrophoresis, followed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Further information can be obtained by limited enzymatic proteolysis of the proteins in the gel following polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and analysis of the cleavage products by gel electrophoresis at right angles to the first gel. We show the application of this technique to the detection and comparison in extracts from infected cells of proteins related immunologically to the simian virus 40 capsid proteins VP1 and VP3.