Virulence-associated genetic regions comprising 31 kilobases of the 230-kilobase plasmid in Shigella flexneri 2a.

By random transposon Tn5 insertions, we previously identified six virulence-associated SalI fragments, B, D, F, G, H, and P, in the 230-kilobase plasmid pMYSH6000 of Shigella flexneri 2a. In this study, we analyzed the sites of 134 independent Tn5 insertions on four contiguous SalI fragments, B, P, H, and D, of pMYSH6000 and identified five virulence-associated regions; four were associated with inducing a positive Sereny test (Ser), invasion into epithelial cells (Inv), binding to Congo red (Pcr), and inhibition of bacterial growth (Igr), and one was associated with the Ser and Inv but not with the Pcr or Igr phenotypes. Hybridization studies revealed that these virulence-associated DNA regions were highly conserved among 15 other virulence plasmids of four species of Shigella and enteroinvasive Escherichia coli. These data indicate that at least seven separate genetic determinants on the virulence plasmid are required for full expression of the virulence phenotype of shigellae.     

Enhancement of inositol phospholipid metabolism and activation of protein kinase C in ras-transformed rat fibroblasts

The inositol phospholipid metabolism is one of the main pathways of signal transduction in cells. We measured the activities of its key enzymes in v-Ha-ras-transformed 208F rat fibroblasts. In the ras-transformed clones, incorporation of [32P]Pi into intermediates of the inositol phospholipid metabolism was stimulated. The activities of phosphatidylinositol and phosphatidylinositol-4-phosphate kinases in the transformed clones were about 35-50% more than in untransformed cells, indicating increased inositol phospholipid metabolism. However, the activity of diacylglycerol kinase in their membrane fraction was 25-35% less than that of untransformed cells, although the total diacylglycerol kinase activity did not change. The imbalance of these kinases could constitute one of the main reasons leading to the increased level of inositol phosphates and the accumulation of diacylglycerol to 2-2.2 times that in control 208F cells. Phosphatidylinositol-4,5-bisphosphate-phospholipase C activity did not change on the transformation when assayed under various conditions. The increased level of diacylglycerol caused intracellular translocation, activation, and down-regulation of protein kinase C changes which may be one of the essential events in transformation by the v-Ha-ras gene.     

Evidence for direct binding of Clostridium botulinum type E derivative toxin and its fragments to gangliosides and free fatty acids

Clostridium botulinum type E derivative toxin and its heavy chain bound to gangliosides GT1b, GD1a and GQ1b and saturated and unsaturated free fatty acids with chain lengths of 14-20 carbons. The L-H-1 fragment lacking the carboxyl-terminal portion of the heavy chain bound to free fatty acids but not to gangliosides. These observations led us to a new hypothesis on the mechanism of binding between botulinum toxin and gangliosides; the carboxyl-terminal portion (H-2 fragment) of the heavy chain binds to an oligosaccharide residue of gangliosides and then the amino-terminal portion (H-1 fragment) interacts with the hydrophobic portion of gangliosides consisting of fatty acids.     

Enhanced Phosphorylation of Tyrosine Hydroxylase at More Than One Site Is Induced by 56 mM K+ in Rat Pheochromocytoma PC 12 Cells in Culture

Incubation of rat pheochromocytoma PC12 cells with dibutyryl cyclic AMP or 56 mM K+ is associated with increased activity and enhanced phosphorylation of tyrosine hydroxylase in situ. Following incubation of the PC12 cells with 32Pi, rapid isolation of the tyrosine hydroxylase, and tryptic digestion of the enzyme, two distinct 32P-peptides can be identified after paper electrophoresis. 56 mM K+ increases 32Pi incorporation into both of these peptides, whereas dibutyryl cyclic AMP increases 32Pi incorporation into only one of these peptides. The rate of increase in the incorporation of 32Pi into these two peptides in cells treated with 56 mM K+ is similar. The phosphorylation of tyrosine hydroxylase in PC12 cells occurs exclusively on serine residues. These results suggest that tyrosine hydroxylase in PC12 cells is phosphorylated on serine residues at two or more distinct sites after 56 mM K+ -induced depolarization. Since only one of these sites is phosphorylated by cyclic AMP-dependent protein kinase, activation of tyrosine hydroxylase by 56 mM K+ may involve phosphorylation by multiple protein kinases in rat pheochromocytoma PC12 cells.     

Fine structural and immunohistochemical studies of goat adenohypophysial cells

Summary The fine structure of each type of anterior pituitary cell in the male goat was studied through the application of a superimposition technique in which adjacent thick sections were used to identify individual cells beforehand by light-microscopic immunohistochemistry. A cone of the pars intermedia protrudes into the pars anterior, being surrounded by the narrow pituitary cleft; the immunohistochemical appearances of the cells forming the cone resemble those of the pars anterior. Several follicles appear in the pars anterior. Ultrastructurally GH cells resemble prolactin cells. The secretory granules of both types are spherical; the diameter of the former is about 340 nm, whereas that of the latter is about 440 nm. ACTH cells are polygonal in shape with secretory granules, about 180 nm in diameter, scattered throughout the cytoplasm. TSH cells, which are spherical in shape, contain the smallest secretory granules, 150 nm in diameter. The highly electron-dense LH cells contain numerous secretory granules about 210 nm in diameter. Their nuclei are irregular with incisures. Thus, the anterior pituitary cells of the goat are ultrastructurally characteristic and species-specific.     

Production of monoclonal antibodies against Clostridium botulinum type E derivative toxin

Abstract Five monoclonal antibodies (MCA; E–8–2, 9–1, 11–2, 12–4, and 13–1) against Clostridium botulinum type E derivative toxin were prepared. Their ELISA titers were higher than or equivalent to that of conventional polyclonal antibody. Three of them (E-8–2, 12–4, and 13–1) possessed the neutralizing activity comparable to that of polyclonal antibody. The results of binding-competition experiments indicated that the monoclonal antibodies bound to different sites on the type E toxin molecule. Immunoblotting analyses demonstrated that E-8–2, 9–1, and 11–2 react to fragment I (heavy chain) of the toxin. By use of these monoclonal antibodies, it may be possible to scrutinize the structure-function relationship of botulinum toxins and cross reactions between type E and F toxins.     

False positive reaction for carcinoembryonic antigen in paget cells

Virchows Archiv B Cell Pathology - Paget cells from cases of mammary and extramammary Paget’s disease were examined for carcinoembryonic antigen (CEA) and CEA-related antigens by the...     

Use of monoclonal antibodies in enzyme linked immunosorbent assay (ELISA) for detection of botulinum type B toxins

Use of polyclonal antibodies failed to correlate mouse assay with enzyme linked immunosorbent assay (ELISA) in titration of culture fluid of different strains of Clostridium botulinum type B. If ELISA is performed with such a monoclonal antibody that is capable of neutralizing the toxin, however, the lethal toxicity can be determined quantitatively.     

Theophylline delays human sleep phase

Twelve healthy male volunteers were given theophylline 250 mg in order to test effects on 24-hr rhythms. Rhythms of sleep/wake and subjective sleepiness were delayed. Ingestion of xanthines such as theophylline in coffee, tea, colas and chocolate may contribute to some sleep disorders. Theophylline might likewise be useful in treating disorders of circadian oscillators.