Comparative Analysis of argK-tox Clusters and Their Flanking Regions in Phaseolotoxin-Producing Pseudomonas syringae Pathovars

DNA fragments containing argK-tox clusters and their flanking regions were cloned from the chromosomes of Pseudomonas syringae pathovar (pv.) actinidiae strain KW-11 (ACT) and P. syringae pv. phaseolicola strain MAFF 302282 (PHA), and then their sequences were determined. Comparative analysis of these sequences and the sequences of P. syringae pv. tomato DC3000 (TOM) (Buell et al., Proc Natl Acad Sci USA 100:10181–10186, 2003) and pv. syringae B728a (SYR) (Feil et al., Proc Natl Acad Sci USA 102:11064–11069, 2005) revealed that the chromosomal backbone regions of ACT and TOM shared a high similarity to each other but presented a low similarity to those of PHA and SYR. Nevertheless, almost-identical DNA regions of about 38 kb were confirmed to be present on the chromosomes of both ACT and PHA, which we named “tox islands.” The facts that the GC content of such tox islands was 6% lower than that of the chromosomal backbone regions of P. syringae, and that argK-tox clusters, which are considered to be of exogenous origin based on our previous studies (Sawada et al., J Mol Evol 54:437–457, 2002), were confirmed to be contained within the tox islands, suggested that the tox islands were an exogenous, mobile genetic element inserted into the chromosomes of P. syringae strains. It was also predicted that the tox islands integrated site-specifically into the homologous sites of the chromosomes of ACT and PHA in the same direction, respectively, wherein 34 common gene coding sequences (CDSs) existed. Furthermore, at the left end of the tox islands were three CDSs, which encoded polypeptides and had similarities to the members of the tyrosine recombinase family, suggesting that these putative site-specific recombinases were involved in the recent horizontal transfer of tox islands. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users.     

Mitochondrial DNA analysis of Sporothrix schenckii clinical isolates from China

Mitochondrial DNA (mtDNA) types based on restriction fragment length polymorphism (RFLP)patterns with HaeIII were investigated in clinical isolates of Sporothrix schenckii in China. In addition to 23 mtDNA types (Types 1–23) so far reported, a new mtDNA type (Type 24) was found in this study. Type 24 was divided into two subtypes, Subtype 24A and 24B based on RFLP with EcoRV. Sixty-seven isolates in China consisted of 58 isolates of Type 4, 5 of Type 6, 1 of Type 5, 1 of Type 20 and 2 of Type 24. Based on the phylogeny of the mtDNA types (Types 1–24) constructed by estimating sequence divergences of mtDNA, mtDNA types clustered into two groups: Group A (Types 1–3, Type 11, Types 14–19 and Types 22–23) and Group B (Types 4–10, Types 12–13,Types 20–21 and Type 24). These results suggest that mostS. schenckii isolates in China belong to Group B.This revised version was published online in October 2005 with corrections to the Cover Date.     

Stress fibers in the mesenteric mesothelial cells of the large intestine of the bullfrog,Rana catesbeiana

Summary Actin-containing cytoplasmic fibers were visualized in the mesenteric mesothelial cells of the large intestine of bullfrog tadpoles by rhodamine-phalloidin staining of en face preparations of mesothelial cells. These fibers were concurrently stained by immunofluorescence using antibodies to myosin or -actinin. Electron microscopy showed the presence of bundles of microfilaments in the basal cytoplasm of the cells. Such fibers in the mesothelial cells may be comparable to the stress fibers present in cultured cells. The mesothelial cells initially formed axially oriented stress fibers when they changed from a rhombic to a slender spindle-like shape. On the other hand, stress fibers disappeared as cells transformed from elongated to polygonal shapes during the period of metamorphic climax. Expression of stress fibers in these cells appears to be related to the degree of tension loaded on the mesentery, which may be generated by mesenteric winding. These stress fibers in the mesothelial cells may serve to regulate cellular transformation. They may also help to maintain cellular integrity by strengthening the cellular attachment to subepithelial tissue against tensile stress exerted on the mesentery.     

Effect of catalase-specific inhibitor 3-amino-1,2,4-triazole on yeast peroxisomal catalase in vivo

3-Amino-1,2,4-triazole (3-AT) is known as an inhibitor of catalase to whose active center it specifically and covalently binds. Subcellular fractionation and immunoelectronmicroscopic observation of the yeast Candida tropicalis revealed that, in 3-AT-treated cells in which the 3-AT was added to the n-alkane medium from the beginning of cultivation, catalase transported into peroxisomes was inactivated and was present as insoluble aggregated forms in the organelle. The aggregation of catalase in peroxisomes occurred only in these 3-AT-treated cells and not in cells in which 3-AT was added at the late exponential growth phase. Furthermore, 3-AT did not affect the transportation of catalase into peroxisomes. The appearance of aggregation only in cells to which 3-AT was added from the beginning of cultivation suggests that, in the process of catalase transportation into yeast peroxisomes, some conformational change may take place and that correct folding may be inhibited by the binding of 3-AT to the active center of catalase. Accordingly, 3-AT will be an interesting compound for investigation of the transport machinery of the peroxisomal tetrameric catalase.     

Melanocyte development: with a message of encouragement to young women scientists

This is a semi-biographical review describing my research on melanocyte development and related personal experiences. Having been educated and trained as a dermatologist, I have been involved in many clinically-oriented studies, however, what has always interested me the most is pigment cell biology. Since I started working at St Marianna University in 1991, I have been undertaking research on melanocyte development and relevant growth factors using mice as models. My research in this field was inspired by my collaborations with various scientists, mostly from the field of biology. Many of these specialists I have met at meetings of the Societies of Pigment Cell Research (PCR). Stem cell factor (SCF, Kitl) and endothelin 3 (EDN3) have been identified as indispensable factors regulating the development of melanocytes. Mice mutant at loci encoding those factors (or their receptors) such as Sl/Sl (receptors W/W) and ls/ls (receptors s/s) have white coat colors and white patches, respectively. Our murine neural crest cell (NCC) primary cultures derived from Sl/Sl embryos showed that EDN3 cannot develop melanocyte precursors without SCF and that EDN3 can elicit proliferation and differentiation in the presence of SCF. These results suggest that without EDN3 and the endothelin type B receptor (EDNRB), melanocytes can not fully increase in number, which could well be the cause of the partial white coat color of ls/ls and s/s mice. Contamination with factors derived from the serum in medium or in feeder cells sometimes causes experimental errors, and therefore we established three immortal cell lines derived from NCC in different developmental stages and designated them as NCCmelb4, NCCmelb4M5 and NCCmelan5, all of which can survive without feeder cells. Using these cell lines and NCC primary cultures, we studied the effect of many factors related to melanocyte development. From the results, it has become evident that Vitamin D3 induces EDNRB expression by NCCmelb4 cells. In addition to the International Pigment Cell Conference (IPCC), I have also taken part in many annual meetings of the Japanese Society for Pigment Cell Research (JSPCR), Pan American Society for Pigment Cell Research (PASPCR) and European Society for Pigment Cell Research (ESPCR). Not only have I learned a great deal, I have enjoyed myself immensely at those meetings. Moreover, I have made many good friends there, some of whom I have collaborated with in my research. To conclude, I would like to give my message 'be ambitious' to young scientists, especially young women.     

Lack of mutagenic and toxic effects of low dose potassium bromate on kidneys in the Big Blue rat

Potassium bromate (KBrO3) has been classified as a genotoxic carcinogen based on positive results in the Ames test, and chromosome aberration and micronucleus tests. The purpose of the present study was to investigate the dose-response relationship for in vivo mutagenic and toxic effects of KBrO3 in the kidneys of Big Blue rats. In experiment 1, male Big Blue rats were divided into 8 groups. KBrO3 was dissolved in tap water and administered to groups 1-8 at concentrations of 0, 0.02, 0.2, 2, 8, 30, 125 and 500 ppm, respectively, for 16 weeks. Experiment 2 was performed to investigate the effects of KBrO3 at the 0.002 ppm dose approximately contained in the tap water on rat kidneys. Ten Big Blue rats were divided into 2 groups and given distilled water and tap water, respectively, for 16 weeks. In experiment 1, treatment with 500 ppm KBrO3 significantly increased the mutant and total mutation frequencies and frequency of GC to TA transversion of the lacI gene in the kidney compared to non-treatment control group, but 125 ppm and lower doses of KBrO3 had no effects. Histopathologically, renal toxic changes were observed in groups administered KBrO3 at 30 ppm or higher in a dose-dependent manner. PCNA positive cell indices in renal tubular cells were significantly increased in the kidney at doses of 125 and 500 ppm, but not at 30 ppm or lower doses, as compared to the control group. Furthermore, 8-hydroxy-2'-deoxyguanosine formation, a marker of oxidative stress, was significantly increased at 500 ppm. In experiment 2, there were no differences in any parameter between the distilled water and tap water groups. These results suggest the existence of no-effect levels for in vivo mutagenic and toxic effects, proliferation stimulus, and oxidative stress of KBrO3 in rat kidneys.     

Development of multiple-locus variable-number tandem-repeat analysis for rapid genotyping of Ehrlichia ruminantium and its application to infected Amblyomma variegatum collected in heartwater endemic areas in Uganda

The rickettsial bacterium Ehrlichia ruminantium is the causative agent of heartwater, a serious tick-borne disease in ruminants. The genetic diversity of organisms in the field will have implications for cross-protective capacities of any vaccine developed, and for an effective vaccine design strategy proper genotyping and understanding of existing genetic diversity in the field is necessary. We searched for variable-number tandem-repeat (VNTR) loci for use in a multi-locus VNTR analysis (MLVA). Sequencing analysis of 30 potential VNTRs using a panel of 17 reference strains from geographically diverse origins identified 12 VNTRs with allelic profiles differing between strains. Application of MLVA to 38 E. ruminantium-infected Amblyomma variegatum collected from indigenous cattle in 6 different districts of Uganda identified 21 MLVA types. The discriminatory power of MLVA was greater than that of map1 PCR-restriction fragment length polymorphism analysis, with which only 6 genotypes were obtained. The high discriminatory power as well as cost- effective performance of MLVA provide the potential for this technique to be applied in the future with respect to optimizing vaccine trials by identifying local strain diversity, and also raise the possibility of exploring the association between E. ruminantium genotypes and phenotypes such as pathological outcome in the ruminant host.