Kunihiko Fukuchi

Inhibition of herpes simplex virus infection by tannins and related compounds

Several chemically defined plant extracts were investigated for their antiviral action on herpes simplex virus (HSV-1, HSV-2)-infected African green monkey kidney cells and human adenocarcinoma cells, using a plaque formation assay. Among them, the monomeric hydrolyzable tannins, oligomeric ellagitannins and condensed tannins, having galloyl groups or hexahydroxydiphenoyl groups, had the most potent anti-HSV activity. Their 50% effective doses (0.03-0.1 microgram/ml) were by two-three orders of magnitude lower than their 50% cytotoxic doses (greater than 10 micrograms/ml). On the other hand, gallic acid, neutral polysaccharides, chemically modified (N,N-dimethylaminoethyl-, carboxymethyl-, and sulfated-) glucans, sialic acid-rich glycoproteins, and uronic acid-rich pine cone polysaccharide showed little or no activity. Using radiolabeled virus particles, we demonstrated that the anti-HSV effect of the tannins is due to inhibition of virus adsorption to the cells.

Iron deprivation‐induced apoptosis in HL‐60 cells

Iron deprivation of HL‐60 cells with deferoxamine B mesylate (DFO) induced apoptosis. DNA fragmentation became apparent with 10−6 M DFO after 48 h treatment. The apoptosis peak according to the DNA histogram on flow cytometory and typical nuclear collapse and were observed microscopically after 48 h treatment with 10−4 M DFO. Cells treated with 10−4 M DFO for as little as 24 h were shown to be committed to apoptosis, as chromatin condensation progressed gradually thereafter.

Molecular analysis of the Cip1/Waf1 (p21) gene in diverse types of human tumors.

We have screened for mutations in the Cip1/Waf1 gene using Southern blot analysis and the polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) method in diverse human tumors. Seven of 102 (7%) human tumor samples were identified to have point mutations within the coding region of the Cip1/Waf1 gene. Two of the seven mutated cases showed gene rearrangements. These results suggest that the frequency of genetic alterations in the Cip1/Waf1 gene is relatively low in comparison with several known tumor suppressor genes.

Phosphatidylinositol 3-kinase inhibitors, Wortmannin or LY294002, inhibited accumulation of p21 protein after γ-irradiation by stabilization of the protein

Expression of the cyclin kinase inhibitor, p21, is regulated both transcriptionally and posttranscriptionally by the ubiquitin-proteasome degradation pathway. Recently, we reported that DNA damage is required for efficient p21 expression by demonstrating that enhanced p21 mRNA expression induced by DNA damage results in increased p21 protein, but enhanced p21 mRNA without DNA damage does not. In addition, we demonstrated that DNA damage suppressed the ubiquitination of p21. In this study, we analyze the link between p21 stabilization and DNA damage. Enhanced p21 protein expression in ML-1 cells resulting from 15 Gy gamma-irradiation was diminished by Wortmannin or LY294002 pretreatment of cells. However, the levels of p21 mRNA were not affected by inhibitor pretreatment. Wortmannin or LY294002 pretreatment reduces p53 expression after gamma-irradiation to a lesser degree than that of p21. In addition, we examined the involvement of DNA-PK, whose activity is inhibited by Wortmannin or LY294002, in p21 stabilization using the SCID fibroblast cell line and a DNA-PK targeting ML-1 cell line. Accumulation of p21 protein by gamma-irradiation was similar to that of DNA-PK intact cells and was reduced by Wortmannin or LY294002 pretreatment. Involvement of another DNA damage detecting enzyme, the ATM gene product, whose activity is also inhibited by Wortmannin or LY294002, was evaluated. ATM deficient cells induced p21 after gamma-irradiation, gamma-irradiation-induced p21 protein was diminished by pretreatment of cells with Wortmannin or LY294002. We conclude that the p21 stabilization mechanism functions after gamma-irradiation, was sensitive to Wortmannin or LY294002, and required neither DNA-PK nor ATM gene product for activity.

Linezolid-resistant methicillin-resistant Staphylococcus aureus isolated after long-term, repeated use of linezolid

We report a 54-year-old male patient with an infection caused by linezolid-resistant methicillin-resistant Staphylococcus aureus (MRSA), isolated after long-term, repeated use of linezolid. Five MRSA strains isolated from our patient were preserved and submitted to bacteriological analysis. All five of these strains were found to have identical genotypes by pulsed-field gel electrophoresis. Two strains isolated in the early hospital period were sensitive to linezolid, while three isolated in the late hospital period were resistant. These three strains that had acquired resistance to linezolid were found to have a G2576T point mutation in the 23SrRNA domain V. Linezolid-resistant MRSA is rare, but may occur with the long-term, repeated administration of linezolid.

DNA damage induces p21 protein expression by inhibiting ubiquitination in ML-1 cells

We previously reported that deferoxamine, an iron chelating agent, induced p53 and cell accumulation in the G1 phase of ML-1 cells in the same way as the DNA damaging agent, etoposide. Etoposide treatment increased expression of the p21 gene, a cyclin kinase inhibitor, at both the mRNA and protein levels. However, deferoxamine treatment only increased the p21 mRNA level without the appearance of a detectable protein product. A substrate for cyclin kinase, pRB, was unphosphorylated by etoposide treatment, but remained unaffected by deferoxamine, indicating that p21 was functional after etoposide, but not after deferoxamine treatment. Therefore, in the present study, we investigated the involvement of the ubiquitin proteasome pathway in post-transcriptional regulation of p21. By the addition of lactacystin, a proteasome inhibitor, to deferoxamine treatment, the level of unubiquitinated p21 protein product was similar to that induced by etoposide treatment, and the ubiquitinated p21 bands became apparent. After etoposide treatment, the level of ubiquitinated p21 was diminished and a high level of unubiquitinated p21 expression was observed. We concluded that (1) efficient expression of p21 protein requires inhibition of the ubiquitin-proteasome pathway, and (2) DNA damage inhibits the ubiquitination of p21.

Molecular analysis of the cyclin-dependent kinase inhibitor genes, p15, p16, p18 and p19 in the myelodysplastic syndromes

The myelodysplastic syndromes (MDS) are a heterogeneous group of clonal blood disorders characterized by dyshematopoiesis with a frequent evolution to acute leukemia. Chromosomal deletions rather than translocations are the predominant karyotypic abnormalities in MDS, suggesting a recessive mechanism in the pathogenesis of MDS, such as inactivation of tumor suppressor genes. A group of cyclin-dependent kinase inhibitors, p15 (INK4B), p16 (INK4A), p18 (INK4C) and p19 (INK4D), are candidate tumor suppressor genes. To determine whether genetic alterations of these genes play an important role in the development and/or progression of MDS, we examined 46 samples from MDS patients by Southern blotting, single-strand-conformation polymorphism (SSCP) using polymerase chain reaction (PCR) and sequencing of DNA. These samples included 13 refractory anemias (RA), four refractory anemias with ringed sideroblasts (RARS), 16 refractory anemias with an excess of blasts (RAEB), eight refractory anemias with an excess of blasts in transformation (RAEB-T) and five chronic myelomonocytic leukemia (CMMoL) samples. Except for allelic polymorphisms or silent point mutations, no alterations of coding regions of these four CDKI genes were identified. In summary, genetic abnormalities of the p15, p16, p18 and p19 genes are rare events in the development and/or progression of MDS.

Antibiotic susceptibility survey of blood-borne MRSA isolates in Japan from 2008 through 2011

We conducted an antibiotic susceptibility survey of 830 blood-borne methicillin resistant Staphylococcus aureus collected from nationwide hospitals in Japan over a three-year period from January 2008 through May 2011. Antibiotic susceptibility was judged according to the criteria recommended by the Clinical Laboratory Standard Institute. Over 99% of the MRSA showed to be susceptible to teicoplanin, linezolid, sulfamethoxazole/trimethoprim and vancomycin, and over 97% of them were susceptible to daptomycin, arbekacin and rifampin. The majority of the MRSA strains showed resistant to minocycline, meropenem, imipenem, clindamycin, ciprofloxacin, cefoxitin, and oxacillin in the rates of 56.6, 72.9, 73.7, 78.7, 89.0, 99.5, and 99.9%, respectively. Among the MRSA strains, 72 showed reduced susceptibility to vancomycin, including 8 strains (0.96%) of vancomycin-intermediate S. aureus (VISA), 54 (6.51%) of heterogeneous vancomycin-intermediate S. aureus (hVISA), and 55 (5.63%) of β-lactam antibiotics-induced vancomycin resistant S. aureus (BIVR). Unexpectedly, among the 54 hVISA and 55 BIVR, 45 isolates (83.3% and 81.8%, respectively) showed both hVISA and BIVR phenotypes. A new trend of vancomycin resistance found in this study was that VISA strains were still prevalent among the bacteremic specimens. The high rates of the hVISA/BIVR two-phenotypic vancomycin resistance, and the prevalence of VISA in the bloodborne MRSA call attention in the MRSA epidemiology in Japan.

Identification of the regulatory region required for ubiquitination of the cyclin kinase inhibitor, p21.

The expression of cyclin kinase inhibitor p21 is regulated by the ubiquitin-proteasome protein degradation system, as well as by transcriptional regulation. Generally, ubiquitination is regulated by the phosphorylation of the substrate. In this study, we identified the region of p21 responsible for the regulation of ubiquitination. Since the phosphorylation sites of p21 are distributed in the C-terminal region, we constructed sequential C-terminal truncated fragments and examined their ubiquitination in eukaryotic cells. The ubiquitination was observed in the 1-164 (full length) and 1-157 fragments with the same efficiency, but not in the 1-147 fragment. The lack of ubiquitination in the 1-147 fragment was unlikely due to the removal of a Lys residue at position 154, since the p21 K154R mutant was ubiquitinated as efficiently as the full-length p21. Furthermore, the 148-157 deleted form of p21 was not ubiquitinated, just like the 1-147 fragment. Thus, the C-terminal 148-157 region, not a ubiquitination site by itself, should contain an essential regulatory region for the efficient ubiquitination of p21.

Direct proteasome inhibition by clasto-lactacystin β-lactone permits the detection of ubiquitinated p21waf1 in ML-1 cells

The ubiquitin proteasome pathway regulates the expression of major cellular regulatory proteins. The ubiquitin proteasome system has been demonstrated to be involved in the expression of the cyclin kinase inhibitor, p21. Ubiquitinated p21 is degraded immediately by 26S proteasome, therefore, the detection of p21 is difficult. We report here an improvement for the detection of ubiquitinated p21 using a proteasome inhibitor, clasto-lactacystin beta-lactone. A p21-enriched cell lysate is obtained by pretreating the cells with deferoxamine to induce p21 mRNA expression followed by treatment with 1x10(-6) M beta-lactone. The concentration of p21 from the cell lysate was performed using an anti-p21 antibody crosslinked to protein G Sepharose. Ubiquitinated p21 was detected on Western blots of the concentrated sample using an anti-ubiquitin antibody. This detection system will be used for further analysis of the regulation of p21 ubiquitination.