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.     

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.     

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.     

Growth of the malignant and nonmalignant human squamous cells in a protein-free defined medium

Summary A novel protein-free synthetic medium has been developed for the culture of human squamous cell carcinoma cells. This medium, designated PF86-1, supports the serial subcultivation of six out of nine human squamous cell carcinoma cell lines in a protein-free, chemically defined condition without the adapting culture from serum-containing conditions. These cell lines growing in PF86-1 exhibited nearly equal potency to grow in massive culture without noticeable changes in morphology but presented a significantly decreased level of colony forming efficiency when compared with the cells cultured in serum-containing media, suggesting the implication of some autocrine mechanism. Interestingly, this medium supported the growth of normal human squamous cells of oral mucosa and skin for more than 2 mo. in the primary explant culture in spite of high levels of calcium ion concentration, where the overgrowth of fibroblasts as contaminant was not observed. These results suggest that PF86-1 supports the growth of cells derived from epidermal tissues selectively and provides the same defined condition for growth of malignant and nonmalignant human squamous cells. It seems, therefore, that PF86-1 allows investigations on the products of squamous cell carcinoma cells or on the differences of growth mechanisms between normal and neoplastic human squamous cells.     

Adenovirus interaction with distinct integrins mediates separate events in cell entry and gene delivery to hematopoietic cells.

A major impediment to the effective use of adenovirus vectors for gene therapy is a lack of knowledge of how these vectors interact with diverse cell types in vivo. Adenovirus attachment to most human cell types is mediated by the fiber protein, which binds to an as yet unidentified cell receptor. In contrast to this, we report that adenovirus type 2 (Ad2) attachment to hematopoietic cells is facilitated by interaction of the penton base protein with members of the beta2 integrin family. Adenovirus particles were capable of binding to human monocytic cells, which lack fiber receptors, and virus binding could be blocked by a soluble penton base or by a function-blocking monoclonal antibody to integrin alphaMbeta2. To confirm the role of alphaMbeta2 integrins in Ad2 binding to hematopoietic cells, we analyzed virus attachment and gene delivery to CHO cells expressing recombinant beta2 integrins. alphaMbeta2-expressing CHO cells supported 3- to 5-fold-higher levels of Ad2 binding and 5- to 10-fold-larger amounts of gene delivery than did nontransfected CHO cells, indicating that alphaMbeta2 facilitates adenovirus attachment to and infection of hematopoietic cells. While beta2 integrins promote Ad2 attachment to hematopoietic cells, further studies demonstrated that alphav integrins were required for the next step in infection, virus internalization into cell endosomes. These studies reveal a novel pathway of Ad2 infection of hematopoietic cells mediated by distinct integrins which facilitate separate events in virus entry. They also suggest a possible strategy for selective adenovirus-mediated gene delivery to hematopoietic cells.