Disappearance of terminal deoxynucleotidyltransferase in human malignant T-lymphoblasts treated with a human alpha interferon preparation

Human T-lymphoblastoid cell lines RPMI 8402, MOLT-3, and CCRF-CEM were treated with interferon (IFN) to determine if the treatment would result in the disappearance of cellular terminaldeoxynucleotidyltransferase (TdT), a possible differentiation marker for T-lymphocytes. Incubation of RPMI 8402 cells in the presence of IFN preparation caused a decrease in the number of TdT-positive cells and in TdT activity of the cell extract. The inhibition of cell multiplication was dose dependent. The anticellular effect of IFN preparation was cytostatic, not cytocidal. The IFN preparation modified neither the TdT content nor proliferation of MOLT-3 and CCRF-CEM cell lines. The effects of IFN preparation thus varied with the cell line.     

Distribution of Clostridium botulinum type C in Ishikawa prefecture, and applicability of agglutination to identification of nontoxigenic isolates of C. botulinum type C.

Since deaths of waterfowls have frequently been observed in Lake Kahoku near Kanazawa city, Japan, we attempted an ecological study on Clostridium botulinum type C in four other lakes as well as Lake Kahoku. One hundred and twenty-nine (56%) of 230 soil samples collected gave rise to lethal toxicity in mice with the characteristic “wasp-waist” symptom. All of the 51 samples arbitrarily selected were neutralized by C. botulinum type C antitoxic serum. A further seasonal study throughout the year at a given shore area of Lake Kahoku disclosed that nearly all samples gave rise to toxicity due to C. botulinum type C during the autumn season when the most waterfowls congregate. Toxigenic strains of C. botulinum type C were isolated together with nontoxigenic strains that were culturally and biochemically similar to the toxigenic strains. Both the toxigenic and nontoxigenic strains were equally agglutinable by an antiserum prepared against one of the nontoxigenic strains. Further extensive studies on the specificity of the agglutination method for identification were performed with 112 strains of 46 clostridial species. None of the strains used except some strains of C. novyi type A and a strain of C. botulinum type D was agglutinable. Based on the findings for cultural, biochemical, and agglutinable properties, the nontoxigenic strains were identified as C. botulinum type C. Also, C. novyi type A isolates showing colonies covered with a small pearly layer zone but surrounded by an aberrantly wide lecithinase zone are discussed.     

Impact on energy metabolism of quantitative and functional cyclosporine-induced damage of kidney mitochondria

In this study we have measured, under experimental conditions which maintained efficient coupling, respiratory intensity, respiratory control, oxidative phosphorylation capacity and protonmotive force. Succinate cytochrome-c reductase and cytochrome-c oxidase activities were also studied. These investigations were carried out using kidney mitochondria from cyclosporine-treated rats (in vivo studies) and from untreated rats in the presence of cyclosporine (in vitro studies). Inhibition of respiratory intensity by cyclosporine did not exceed 21.1% in vitro and 15.9% in vivo. Since there was no in vitro inhibition of succinate cytochrome-c reductase and cytochrome-c oxidase activities, the slowing of electron flow observed can be interpreted as a consequence of an effect produced by cyclosporine between cytochromes b and c₁. Cyclosporine had no effect on respiratory control either in vitro or in vivo. Statistically significant inhibition of the oxidative phosphorylation was observed both in vitro (6.6%) and in vivo (12.1%). Moreover, cyclosporine did not induce any change of membrane potential either in vivo or in vitro. Our findings show that cyclosporine is neither a protonophore, nor a potassium ionophore. In cyclosporine-treated rats we noticed a decrease of protein in subcellular fraction, including the mitochondrial fraction. The role of the inhibition respiratory characteristics by cyclosporine in nephrotoxicity in vivo must take account of these two parameters: inhibition of the respiratory characteristics measured in vitro and diminution of mitochondrial protein in cyclosporine-treated rats.     

Prophages integrating into prophages: A mechanism to accumulate type III secretion effector genes and duplicate Shiga toxin-encoding prophages in Escherichia coli

Bacteriophages (or phages) play major roles in the evolution of bacterial pathogens via horizontal gene transfer. Multiple phages are often integrated in a host chromosome as prophages, not only carrying various novel virulence-related genetic determinants into host bacteria but also providing various possibilities for prophage-prophage interactions in bacterial cells. In particular, Escherichia coli strains such as Shiga toxin (Stx)-producing E. coli (STEC) and enteropathogenic E. coli (EPEC) strains have acquired more than 10 prophages (up to 21 prophages), many of which encode type III secretion system (T3SS) effector gene clusters. In these strains, some prophages are present at a single locus in tandem, which is usually interpreted as the integration of phages that use the same attachment (att) sequence. Here, we present phages integrating into T3SS effector gene cluster-associated loci in prophages, which are widely distributed in STEC and EPEC. Some of the phages integrated into prophages are Stx-encoding phages (Stx phages) and have induced the duplication of Stx phages in a single cell. The identified attB sequences in prophage genomes are apparently derived from host chromosomes. In addition, two or three different attB sequences are present in some prophages, which results in the generation of prophage clusters in various complex configurations. These phages integrating into prophages represent a medically and biologically important type of inter-phage interaction that promotes the accumulation of T3SS effector genes in STEC and EPEC, the duplication of Stx phages in STEC, and the conversion of EPEC to STEC and that may be distributed in other types of E. coli strains as well as other prophage-rich bacterial species.     

Cancer Cell Analyses at the Single Cell-Level Using Electroactive Microwell Array Device

Circulating tumor cells (CTCs), shed from primary tumors and disseminated into peripheral blood, are playing a major role in metastasis. Even after isolation of CTCs from blood, the target cells are mixed with a population of other cell types. Here, we propose a new method for analyses of cell mixture at the single-cell level using a microfluidic device that contains arrayed electroactive microwells. Dielectrophoretic (DEP) force, induced by the electrodes patterned on the bottom surface of the microwells, allows efficient trapping and stable positioning of single cells for high-throughput biochemical analyses. We demonstrated that various on-chip analyses including immunostaining, viability/apoptosis assay and fluorescent in situ hybridization (FISH) at the single-cell level could be conducted just by applying specific reagents for each assay. Our simple method should greatly help discrimination and analysis of rare cancer cells among a population of blood cells.