Differential distribution of alpha-tubulin isotypes in Euplotes eurystomus determined by confocal immunofluorescence microscopy

Detergent permeabilized Euplotes eurystomus (a fresh water hypotrichous ciliate) was reacted with monoclonal and polyclonal antibodies specific for either detyrosinated or tyrosinated alpha-tubulin (Glu- or Tyr-tubulin). The isolated cytoskeleton-nuclear complex was examined by Western immunoblotting and by immunofluorescent and electron microscopic methods. Both Glu- and Tyr-tubulins were detected by immunoblot analysis. Immunofluorescent microscopy indicated that the alpha-tubulin isotypes are concentrated in different regions of permeabilized cells: Glu-tubulin is located primarily in cirri, membranelles, and surrounding the macro- and micronuclei. Tyr-tubulin is principally at the bases of cirri and membranelles. This differential distribution of alpha-tubulin isotypes is discussed in terms of current concepts concerning the correlation of tubulin post-translational modifications to microtubule stability. Confocal immunofluorescent imaging was of critical importance in clearly differentiating the Glu-tubulin isotype surrounding the macro- and micronuclei from a brilliantly fluorescent environment originating from cytoskeletal structures. In conjunction with conventional and stereo-electron microscopy, confocal optical microscopy provided convincing evidence for a "basket" of microtubules surrounding both nuclei.     

Comparison of antibiotic complexes formed by cultures of Streptomyces griseoruber

Physico-chemical properties of antibiotic complexes formed by various strains of Streptomyces griseoruber: VNIIA 1195, VNIIA 1193-INA 2022/55 and IMiV 1618 were compared. It was shown that the antibiotic complex 1195 differed by the electron absorption spectrum, chromatographic mobility and indicator properties from lateriomycins A and B produced by the type culture of S. griseoruber, Jamaguchi et Saburi, 1955 and strain 70717, as well as from prodigiozin produced by S. griseoruber, VNIIA 1193. By the absorption spectra and chromatographic mobility the components of the antibiotic complex 1195 were identical with cinerubins A and B, pyrromycin and eta- and xi-pyrromycinones included in the composition of antibiotic 1618 produced by S. griseoruber 1618 and differed in their quantitative contents and absence of epsilon-pyrromycinones.     

Relation of bacitracin synthesis and manganese content in Bacillus licheniformis: antibiotic production in colonies forming on media with high levels of Mn2+

Population of B. licheniformis is not homogeneous with respect to its sensitivity to Mn2+. Active bacitracin producing cultures with high and low sensitivity to exogenic manganese are described. In media containing high sublethal concentrations of Mn2+ there were detected two types of bacitracin-producing colonies i.e. light and dark differing likely in the mechanisms of their cell protection from the metal excess. Since bacitracin production did not always correlate with sensitivity to Mn2+ at the level of the colony formation the use of Mn2+ as a selective factor for isolating inactive strains of B. licheniformis is of low efficiency.     

A physical genome map of Pseudomonas aeruginosa PAO.

A complete macrorestriction map of the 5.9 Mb genome of Pseudomonas aeruginosa PAO (DSM 1707) was constructed by the combination of various one- and two-dimensional pulsed field gel electrophoresis techniques. A total of 51 restriction sites (36 SpeI sites, 15 DpnI sites) were placed on the physical map yielding an average resolution of 110 kb. Several genes encoding virulence factors and enzymes of metabolic pathways were located on the anonymous map by Southern hybridization. Distances between the gene loci were similar on the genetic and physical maps, suggesting an even distribution of genome mobility throughout the bacterial chromosome. The four rRNA operons were organized in pairs of inverted repeats. The two-dimensional macro-restriction techniques described herein are generally applicable for the genome mapping of any prokaryote and lower eukaryote which yields resolvable fragment patterns on two-dimensional pulsed field gels.     

Biology of gut anaerobic fungi

The obligately anaerobic nature of the gut indigenous fungi distinguishes them from other fungi. They are distributed widely in large herbivores, both in the foregut of ruminant-like animals and in the hindgut of hindgut fermenters. Comparative studies indicate that a capacious organ of fermentative digestion is required for their development. These fungi have been assigned to the Neocallimasticaceae, within the chytridiomycete order Spizellomycetales. The anaerobic fungi of domestic ruminants have been studied most extensively. Plant material entering the rumen is rapidly colonized by zoospores that attach and develop into thalli. The anaerobic rumen fungi have been shown to produce active cellulases and xylanases and specifically colonise and grow on plant vascular tissues. Large populations of anaerobic fungi colonise plant fragment in the rumens of cattle and sheep on high-fibre diets. The fungi actively ferment cellulose which results in formation of a mixture of products including acetate, lactate, ethanol, formate, succinate, CO2 and H2. The properties of the anaerobic fungi together with the extent of their populations on plant fragments in animals on high-fibre diets indicates a significant role for the fungi in fibre digestion.     

Direct solid phase sequencing of genomic and plasmid DNA using magnetic beads as solid support.

Approaches to direct solid phase sequencing of genomic and plasmid DNA have been developed using magnetic beads, coated with streptavidin, as solid support. The DNA is immobilized through selective incorporation of biotin into one of the strands. A single stranded template, suitable for sequencing, is obtained through strand-specific elution. Using this concept, in vitro amplified plasmid DNA and chromosomal DNA were sequenced directly from single colonies. The solid phase approach ensures that the amplification and the sequencing reactions can be performed under optimal conditions. The system was found to be suitable for sequencing using both isotope- and fluorescent-labelled primers.     

A Monte Carlo investigation of homogeneity tests of the odds ratio under various sample size configurations

Epidemiologic data for case-control studies are often summarized into K 2 x 2 tables. Given a fixed number of cases and controls, the degree of sparseness in the data depends on the number of strata, K. The effect of increasing stratification on size and power of seven tests of homogeneity of the odds ratio is studied using Monte Carlo methods. In all the designs considered here, the numbers of cases and controls per stratum are the same. Considering both size and power in non-sparse-data settings, we recommend the Breslow-Day statistic (1980, Statistical Methods in Cancer Research, 1. The Analysis of Case-Control Studies, p. 142; Lyon: International Agency for Research on Cancer) for general use. In sparse-data settings the T4 statistic of Liang and Self (1985, Biometrika 72, 353-358) performs the best when all tables, regardless of sample size, have odds ratios generated from the same distribution. In sparse-data settings characterized by a large table with an odds ratio of 1 and many small tables with odds ratios greater than 1, the T5 statistic of Liang and Self (1985) performs the best. One of the most important results of this study is the generally low power for all homogeneity tests especially when the data are sparse.     

Influence of platelet-activating factor on leukotriene D4-induced contractions of the guinea pig parenchymal strip

Platelet-activating factor (PAF) and sulphidopeptide leukotrienes, such as leukotriene D4 (LTD4), are potent constrictors that are probably released simultaneously in a variety of inflammatory respiratory events. The purpose of the present study was to determine whether LTD4-induced contractions of guinea pig parenchymal lung strips (GPPS) are modified in the presence of PAF. The contractile responses of isolated GPPS to cumulative doses of LTD4, acetylcholine, histamine, and potassium chloride in the presence of PAF (0.1 nM, 0.1 microM) were compared with parallel controls. There was no significant alteration of the response to acetylcholine and potassium chloride and the PAF-induced inhibition of the response to histamine, although significant, was not concentration dependent. In contrast, PAF in a concentration range from 0.1 nM to 1.0 microM caused a marked, concentration-dependent reduction of LTD4-induced contractions. Pretreatment with the PAF receptor antagonist, BN52021, prevented the attenuation of LTD4-induced contraction by PAF. The attenuation of LTD4-induced contraction by PAF was also prevented by pretreatment with indomethacin or with the thromboxane synthase inhibitor U63,557A, but not by pretreatment with the lipoxygenase inhibitors BW755c or nordihydroguaiaretic acid. Thus inhibition of LTD4-induced GPPS contraction by PAF is receptor dependent and probably secondary to thromboxane generation. The respiratory smooth muscle response to leukotrienes may be modified significantly by concomitant PAF release.     

Kinetic and morphological evidence for endocytosis of mammalian cell integrin receptors by using an anti-fibronectin receptor beta subunit monoclonal antibody

Monoclonal antibody (mAb) 7E2.2, which recognizes the beta subunit of the hamster fibronectin receptor (FnR) (Brown, P.J., and Juliano, R. L. (1988) Exp. Cell Res. 177. 303), was used to examine the distribution of and to quantify the internalization of the FnR and possibly related integrins on adherent fibroblasts. Purified 7E2.2 IgG was iodinated and used in binding and internalization studies. Binding to Chinese hamster ovary cells was saturable with a Km of 0.3 nM and an estimated total number of cell surface beta subunits at 2 x 10(5) per cell. The FnR colocalized with fibronectin at cell adhesion contact sites and also was distributed evenly over the dorsal cell surface as discrete clusters. By using a direct immunocolloidal gold approach, the FnR was not associated with coated pits at 4 degrees C until internalization followed warming of the labeled cells to 37 degrees C. A proportion of the FnRs were endocytosed with a half-time of 6.5 min and, consistent with clathrin-mediated uptake, this was sensitive to hypertonic conditions. Receptor-immunocomplexes rapidly became localized within coated pits, small diameter tubules, and peripheral endosomes but the majority remained at the cell surface. At subsaturating concentrations of bound 7E2.2, approximately one-fourth of the total cell receptor population resided intracellularly at any one moment following steady-state; however, appreciable degradation of the iodinated mAb was not detected following accumulation for 4 h at 37 degrees C. These data showed that at least a portion of the FnR are endocytosed via a receptor-mediated pathway and suggested that these receptors do not immediately enter a degradative compartment.