Hemorheological abnormalities in experimental cerebral ischemia and effects of protein kinase inhibitor on blood fluidity.

The aim of this study was to investigate the mechanisms of the pathogenesis of hyperviscosity following cerebral ischemia. Focal ischemia was produced by embolic occlusion of the right middle cerebral artery (MCA) in rats for 1 hour, followed by recirculation. Twenty-four hours after MCA occlusion, fasudil, a protein kinase inhibitor, was administered intraperitoneally. Blood samples were taken from the abdominal aorta, and viscosity was measured using a cone-plate viscometer. The viscosity of whole blood in the ischemic attack group was significantly increased compared with the sham operated group 24 hours after MCA occlusion. Fasudil dose-dependently and significantly decreased the blood viscosity, and reduced to the normal range after administration of 10 mg/kg of fasudil (sham-operated rats, 5.17+/-0.05 cP; pre dose/ischemic rats, 6.05+/-0.08 cP; post dose/ischemic rats, 5.23+/-0.14 cP; 37.5 sec(-1)). Our findings suggest that cerebral ischemia induces a potent, systemic and long-lasting hyperviscosity, and that the inhibition of protein kinases, especially rho kinase, is efficacious in preventing this hyperviscosity.

Inhibition of neutrophil migration by a protein kinase inhibitor for the treatment of ischemic brain infarction.

This study investigated the therapeutic potential of agents that inhibited neutrophil infiltration in cerebral ischemic infarction. The migration of neutrophils elicited by N-formyl-methionyl-leucyl-phenylalanine, tumor necrosis factor, C5a or platelet-activating factor was potently inhibited by fasudil, an inhibitor of protein kinases including rho kinase, protein kinase C and myosin light chain kinase, and hydroxy fasudil, a metabolite of fasudil, in vitro. In a microembolism model in rats, myeloperoxidase-quantified neutrophil accumulation in the ischemic brain was observed 24 hr after embolization. Intravenous administration of fasudil prevented the accumulation of neutrophils. In rats given fasudil, myeloperoxidase activity in the ipsilateral hemisphere (0.04+/-0.01 unit/g wet tissue) was significantly lower than that in ischemic rats (0.11+/-0.02 unit/g wet tissue). Fasudil also significantly reduced the size of the infarct area and improved neurological functions. These results suggest that neutrophil infiltration into the ischemic brain is involved in the pathogenesis of ischemic injury and that inhibiting neutrophil infiltration may provide an effective therapeutic intervention to reduce ischemic injury.

Correlation between polyploidy and auxotrophic segregation in the imperfect yeast Candida albicans.

In order to clarify the relationship between polyploidization and the capability of phenotypic switching in the imperfect yeast Candida albicans, two types of variants were isolated as segregants from a fusant, which produced a proportion of the cell population with a higher ploidy than the rest, either in a temperature-dependent or -independent manner, when incubated at low (28 degrees C) and high (37 degrees C) temperatures. In the case of the temperature-dependent type of variants, high-ploidy cells appeared at 37 degrees C but rarely at 28 degrees C. This phenotype was named Pldts (temperature-sensitive polyploidization), and the temperature-independent phenotype was called Pld-. The appearance of high-ploidy cells in the culture of the Pldts strain at 37 degrees C was accompanied by a significant increase in the frequency of auxotrophic variants; these variants probably occur as a result of segregation of auxotrophic markers from the heterozygous to the homozygous state. Both Pldts and Pld- phenotypes were recessive in a fusion with a Pld+ parent. An adenine auxotrophic marker (ade1) was introduced into a Pldts strain in a heterozygous state, and the individual high-ploidy cells of this strain, grown at 37 degrees C, were micromanipulated to form colonies, which consisted of red and white sectors appearing at high frequency on a pink background. When the ade1 auxotrophy was introduced into Pld- strains, frequently sectored colonies were produced. These results suggested an increased level of chromosome missegregation in both types of Pld mutants. Analyses by pulsed-field gel electrophoresis of Ade-segregants, derived from a micromanipulated high-ploidy cell of a Pld(ts) strain, suggested the occurrence of nonreciprocal recombination, some of which includes chromosome loss.

A high-resolution cytogenetic map of human chromosome 9: localization of 203 new cosmid markers by direct R-banding fluorescence in situ hybridization.

A high-resolution cytogenetic map of human chromosome 9 was constructed with 203 newly isolated cosmid markers by direct R-banding fluorescence in situ hybridization. The clones were localized preferentially to R-positive bands throughout chromosome 9. Although the number of clones was roughly proportional to the R-band size, many more clones (ca. 80) were mapped at the q34 region compared to its physical size. Based on these cytogenetic mapping data, one can establish physical contig maps with cosmids and yeast artificial chromosomes and a genetic linkage map essential for positional cloning of responsible genes.

A high-resolution cytogenetic map of human chromosome 12: localization of 195 new cosmid markers by direct R-banding fluorescence in situ hybridization.

We have constructed a high-resolution cytogenetic map of human chromosome 12 with 195 newly isolated cosmids by direct R-banding fluorescence in situ hybridization. The fluorescent signals of 195 clones were evenly distributed throughout chromosome 12, but sublocalized preferentially to R-positive bands. This high-resolution cytogenetic map with an average map distance of 0.73 Mb on bands can, in conjunction with a genetic linkage map, facilitate the analysis of chromosomal and molecular aberrations in genetic diseases and cancers. Moreover, the cytogenetic mapping data provide starting points for establishing contig maps with cosmid clones and yeast artificial chromosomes.

A high-resolution cytogenetic map of human chromosome 5: localization of 206 new cosmid markers by direct R-banding fluorescence in situ hybridization.

We have constructed a high-resolution cytogenetic map of human chromosome 5 with 206 new cosmid clones by direct R-banding fluorescence in situ hybridization. The fluorescent signals of 206 clones were evenly distributed throughout chromosome 5, although they were sublocalized preferentially to R-positive bands. This high-resolution cytogenetic map with an average distance of nearly 1 Mb will serve as important resource for construction of a genetic linkage map, which is essential for positional cloning of responsible genes. Moreover, these mapping data provide many useful landmarks for the construction of contig maps of chromosome 5, as required in the Human Genome Project.

Chromosome mapping of the human cytidine-5'-triphosphate synthetase (CTPS) gene to band 1p34.1-p34.3 by fluorescence in situ hybridization.

The human cytidine-5'-triphosphate synthetase (CTPS) gene was mapped by a direct mapping system combined with fluorescence in situ hybridization and replicated prometaphase R-bands. By high-resolution banding analysis, the signals were localized to band 34.1-34.3 of the short arm of chromosome 1; 1p34.1-p34.3. Simple procedures for the detection of R-bands are described.

Induction of distamycin A-inducible rare fragile sites and increased sister chromatid exchanges at the fragile site.

Expression of distamycin A-inducible rare fragile sites by AT-specific DNA-ligands was examined in lymphoblastoid cell lines derived from heterozygous carriers for the fra(8)(q24), fra(16)(p12), and fra(16)(q22) sites. The sensitivity of fragile site expression to the inducers was different at these fragile sites. The expression of fra(8)(q24) was induced markedly by Hoechst 33258, but not by distamycin A or berenil. An increased expression of fra(16)(p12) was found following treatment with Hoechst 33258 or berenil, but not with distamycin A. At fra(16)(q22), distamycin A markedly induced the fragile site, but Hoechst 33258 and berenil did not. Since their response to the different inducers was similar to that found in cultured lymphocytes, lymphoblastoid cell lines appear to retain their inherent properties. Although BrdUrd alone did not induce any fragile sites, concomitant treatment with BrdUrd plus the inducer was synergistically effective in inducing all the fragile sites. An increased frequency of sister chromatid exchanges was observed at fra(16)(p12) following simultaneous treatment with BrdUrd and berenil, mainly when the site was expressed as an isochromatid gap. Thus, the induced fra(16)(p12) site is a hot spot for the formation of sister chromatid exchanges, as found in other reported fragile sites.