Ultrastructure of Weddellomyces epicallopisma (Dothideales, Dacampiaceae, Ascomycota), especially of its ascospores]

An ultrastructural study of Weddellomyces epicallopisma (ascomata wall, asci, ascospores and vegetative hyphae), the first done on the family Dacampiaceae, confirms most of the observations made in light microscopy. Moreover it shows that ascospores are provided with an endospore (not visible in light microscope) and that the structure of the ascospore septum is more complex. The similarity of the wall structure between the ascospore and the hyphoid appendages, developed on the upper part of the ascoma, is emphasized.     

Low responsiveness of six-rowed genotypes to androgenesis in barley does not have a pleiotropic basis.

A heterozygous mutant for the two- and six-rowed character was isolated in the barley cultivar Igri through application of sodium azide to isolated microspore cultures and posterior regeneration. Six-rowed and two-rowed homozygotic plants were subsequently identified in the self-pollinated M2 progenies of the original heterozygous M1. Detailed molecular markers confirmed the isogenic nature of this recovered mutant and the original cultivar Igri. A comparative study of the anther culture response of this six-rowed induced mutant vs. diploid 'Igri' was performed to assess whether the two- or six-rowed gene influences anther culture response in barley through a pleiotropic effect or via linkage disequilibrium. No significant differences for any of the recorded variables throughout the in vitro regeneration process were detected between the 'Igri' six-rowed mutant and any of their two-rowed isogenic lines. This suggests that row-type association with anther culture response in barley cultivars is due to the effect of a tight linkage with other genes directly responsible for androgenic response.     

Distribution of alcohol dehydrogenase mRNA in the rat central nervous system. Consequences for brain ethanol and retinoid metabolism.

The localization of alcohol dehydrogenase (ADH) in brain regions would demonstrate active ethanol metabolism in brain during alcohol consumption, which would be a new basis to explain the effects of ethanol in the central nervous system. Tissue sections from several regions of adult rat brain were examined by in situ hybridization to detect the expression of genes encoding ADH1 and ADH4, enzymes highly active with ethanol and retinol. ADH1 mRNA was found in the granular and Purkinje cell layers of cerebellum, in the pyramidal and granule cells of the hippocampal formation and in some cell types of cerebral cortex. ADH4 expression was detected in the Purkinje cells, in the pyramidal and granule cells of the hippocampal formation and in the pyramidal cells of cerebral cortex. High levels of ADH1 and ADH4 mRNAs were detected in the CNS epithelial and vascular tissues: leptomeninges, choroid plexus, ependymocytes of ventricle walls, and endothelium of brain vessels. Histochemical methods detected ADH activity in rodent cerebellar slices, while Western-blot analysis showed ADH4 protein in homogenates from several brain regions. In consequence, small but significant levels of ethanol metabolism can take place in distinct areas of the CNS following alcohol consumption, which could be related to brain damage caused by a local accumulation of acetaldehyde. Moreover, the involvement of ADH in the synthesis of retinoic acid suggests a role for the enzyme in the regulation of adult brain functions. The impairment of retinol oxidation by competitive inhibition of ADH in the presence of ethanol may be an additional origin of CNS abnormalities caused by ethanol.     

Polyvalent inhibitory action of fusidic acid in isolated rat liver cells

The steroidic antibiotic fusidic acid showed a polyvalent action on isolated rat liver cells. It displayed a strong inhibitory capability on protein synthesis in intact cells even stronger than that previously reported in cell-free extracts. Also, it inhibited basal gluconeogenesis and promoted an increase of the membrane permeability to trypan blue. However, the effects on both protein synthesis and basal gluconeogenesis were observed at doses smaller than those required to reduce the cell viability.     

Analysis of ligand-binding to the kringle 4 fragment from human plasminogen

The interaction of the isolated human plasminogen kringle 4 with the four -amino acid ligands -aminocaproic acid (ACA), N-acetyl-l-lysine (AcLys), trans-aminomethyl(cyclohexane)carboxylic acid (AMCHA) and p-benzylaminesulfonic acid (BASA) has been further characterized by ¹H-NMR spectroscopy at 300 and 600 MHz. Pronounced high-field shifts, reaching 3 ppm, are observed for AMCHA resonances upon binding to kringle 4, which underscores the relevance of ligand lipophilic interactions with aromatic side chains at the binding site. Ligand titration curves for the nine His and Trp singlets found in the kringle 4 aromatic spectrum reveal a striking uniformity in the kringle response to the various ligands. The average binding curves exhibit a clear Langmuir absorption isotherm saturation profile and the data were analyzed under the assumption of one (high affinity) binding site per kringle. Equilibrium association constants (K _a ) and first order dissociation rate constants (k _off) were derived from linearized expressions of the Langmuir isotherm and of the spectral line-shapes, respectively. The results for the four ligands, at 295 K, pH^* 7.2, indicate that: (a) AMCHA exhibits the strongest binding (K _a =159 mM ^-1) and ACA the weakest (K _a =21 mM ^–1) with AcLys and BASA falling in between; (b) ACA dissociates readily (k _off = 5.3 × 10³ s^–1) and AMCHA associates the fastest (k _off = 2.0 × 10⁸ M ^–1 s^–1) while the kinetics for BASA exchange is relatively slow (k _off = 0.8 × 10³ s^–1, k _on = 0.6 × 10⁸ M ^–1s^–1); (c) the ligand-binding kinetics is close to diffussion-controlled.Abbreviations ACA -aminocaproic acid - AcLys N-acetyl-l-lysine - AMCHA t-aminomethyl(cyclohexane)carboxylic acid - BASA p-benzylaminesulfonic acid - K4 kringle 4 - NOE nuclear Overhauser effect - ppm parts-per-million - pH^* glass electrode pH reading uncorrected for deuterium isotope effects - K _a ligand-kringle 4 equilibrium association constant - k _off ligand-kringle 4 dissociation rate constant - k _on ligand-kringle 4 association rate constant     

Osmotic and cryoprotective effects of glycerol-sucrose solutions on day-3 mouse embryos

The relative volume of Day-3 mouse embryos changed as a linear function of the reciprocal of osmolality [corrected] of non-permeating solutes after 10 min exposure to sucrose and glycerol-sucrose solutions at 20 degrees C. The slope of the linear regression line was less in glycerol-sucrose than in sucrose solutions because glycerol permeation caused re-expansion. Before freezing by direct transfer to -180 degrees C the embryos were placed into glycerol-sucrose in 1-step (1-step equilibration) or first into glycerol and then into glycerol-sucrose (2-step equilibration). Using 2-step equilibration the post-thaw survival rate was substantially higher at 3.0 and 4.0 M-glycerol levels and less dependent on changes in the sucrose concentration within the range of 0.125 to 1.0 M than with 1-step equilibration. Under optimal conditions 90-95% of rapidly frozen embryos developed to blastocysts in vitro and 30% into live young in vivo. It is suggested that the cryoprotective role of glycerol is due to its ability to reduce osmotic pressure differences between the extra and intracellular spaces during rapid freezing of embryos.     

Covalent labeling of opioid receptors with 3H-D-Ala2-Leu5-enkephalin chloromethyl ketone. I. Binding characteristics in rat brain membranes

The chloromethyl ketone derivative of D-Ala2-Leu5-enkephalin was synthesized in a radioactive form, and the resulting compound (3H-DALECK) was used to label opioid receptors. 3H-DALECK binds with high affinity, specificity and saturability to rat brain membranes. The number of sites labeled is 130 fmoles/mg protein. Unlabeled opioids inhibited the binding of 3H-DALECK; etorphine and DAGO being most potent. A 10-fold preference for mu sites over delta was seen in site-specific competition experiments; while DALECK displayed low affinity for kappa sites of rat brain. DALECK irreversibly blocked a certain population of sites. Approximately 40% of 3H-DALECK binding at 15 min, and 60% at 60 min association time did not dissociate in the presence of a large excess of unlabeled DALECK and was resistant to washing. Autoradiography performed after SDS-PAGE revealed specific alkylation of proteins with molecular weight of 74, 65, 56, 43 and 34 kD. These results demonstrate the applicability of using 3H-DALECK to covalently label opioid receptors.     

Tn10 mutagenesis in Azotobacter vinelandii

Summary The tetracycline-resistant transposon Tn10 and its high-hopper derivative Tn10HH104 were introduced into the Azotobacter vinelandii genome using suicide conjugative plasmids derived from pRK2013. Several types of mutants induced by either of these elements are described. Nif^- mutants (deficient in nitrogen fixation) were easily isolated, whereas the isolation of other mutant types (auxotrophs, sugar non-users) required special selection conditions. The characterization of the mutations as transposon insertions was often complicated and sometimes required a combination of genetic and physical tests. A common source of complication, the existence of double inserts, was found among the mutants induced by Tn10HH104 but not among those induced by Tn10. Both the high-hopper and the wild-type element proved to undergo secondary transpositions, albeit at different frequencies. Another type of complication, the existence of heterozygotes, occurred because of the high level of redundancy of the A. vinelandii genome.