Biomedical subjects

Mari Shimura

Publications and source records attributed to Mari Shimura.

8 recordsLinked to original sources

PubMed2006

HIV-1 Vpr induces DNA double-strand breaks.

Recent observations imply that HIV-1 infection induces chromosomal DNA damage responses. However, the precise molecular mechanism and biological relevance are not fully understood. Here, we report that HIV-1 infection causes double-strand breaks in chromosomal DNA. We further found that Vpr, an accessory gene product of HIV-1, is a major factor responsible for HIV-1-induced double-strand breaks. The purified Vpr protein promotes double-strand breaks when incubated with isolated nuclei, although it does not exhibit endonuclease activity in vitro. A carboxyl-terminally truncated Vpr mutant that is defective in DNA-binding activity is less capable of Vpr-dependent double-strand break formation in isolated nuclei. The data suggest that double-strand breaks induced by Vpr depend on its DNA-binding activity and that Vpr may recruit unknown nuclear factor(s) with positive endonuclease activity to chromosomal DNA. This is the first direct evidence that Vpr induces double-strand breaks in HIV-1-infected cells. We discuss the possible roles of Vpr-induced DNA damage in HIV-1 infection and the involvement of Vpr in further acquired immunodeficiency syndrome-related tumor development.

Cell Culture Techniques↗

PubMed2005

Improved gene expression in resting macrophages using an oligopeptide derived from Vpr of human immunodeficiency virus type-1.

Vpr, an accessory gene product of human immunodeficiency virus type-1, is thought to transport a viral DNA from the cytoplasm to the nucleus in resting macrophages. Previously, we reported that a peptide encompassing amino acids 52-78 of Vpr (C45D18) promotes the nuclear trafficking of recombinant proteins that are conjugated with C45D18. Here, we present evidence that C45D18, when conjugated with a six-branched cationic polymer of poly(N,N-dimethylaminopropylacrylamide)-block-oligo(4-aminostyrene) (SV: star vector), facilitates gene expression in resting macrophages. Although there was no difference between SV alone and C45D18-SV with respect to gene transduction into growing cells, C45D18-SV resulted in more than 40-fold greater expression of the exogenous gene upon transduction into chemically differentiated macrophages and human quiescent monocyte-derived macrophages. The data suggest that C45D18 contributes to improving the ability of a non-viral vector to transduce macrophages with exogenous genes and we discuss its further application.

Amino Acid Sequence↗

PubMed2005

Premature sister chromatid separation in HIV-1-infected peripheral blood lymphocytes.

To investigate the mechanism of aneuploidy that is frequently observed in AIDS, we examined premature sister chromatid separation (PCS), a sign of genomic instability, in peripheral blood cells of HIV-1-infected individuals. PCS was found in all six HIV-1 individuals at a high incidence. When peripheral blood cells from healthy volunteers were infected with HIV-1 in vitro, the incidence of PCS increased. This suggests that HIV-1 infection causes PCS and has the potential to induce aneuploidy.

Aneuploidy↗

PubMed2005

Element array by scanning X-ray fluorescence microscopy after cis-diamminedichloro-platinum(II) treatment.

Minerals are important for cellular functions, such as transcription and enzyme activity, and are also involved in the metabolism of anticancer chemotherapeutic compounds. Profiling of intracellular elements in individual cells could help in understanding the mechanism of drug resistance in tumors and possibly provide a new strategy of anticancer chemotherapy. Using a recently developed technique of scanning X-ray fluorescence microscopy (SXFM), we analyzed intracellular elements after treatment with cis-diamminedichloro-platinum(II) (CDDP), a platinum-based anticancer agent. The images obtained by SXFM (element array) revealed that the average Pt content of CDDP-resistant cells was 2.6 times less than that of sensitive cells, and the zinc content was inversely correlated with the intracellular Pt content. Data suggested that Zn-related detoxification is responsible for resistance to CDDP. Of Zn-related excretion factors, glutathione was highly correlated with the amount of Zn. The combined treatment of CDDP and a Zn(II) chelator resulted in the incorporation of thrice more Pt with the concomitant down-regulation of glutathione. We propose that the generation of an element array by SXFM opens up new avenues in cancer biology and treatment.

Antineoplastic Agents↗

PubMed2004

Nuclear trafficking of macromolecules by an oligopeptide derived from Vpr of human immunodeficiency virus type-1.

Vpr, an accessory gene product of HIV-1, is incorporated into cells when added to the culture medium. Via such function Vpr has been shown to transduce a protein into cells that is expressed as a chimeric protein with Vpr. The domain required for protein transduction, however, remained to be clarified. Here we identified a sequence encompassing 52-78 amino acids of Vpr (C45D18) that enables nuclear trafficking of proteins. When chemically synthesized C45D18 was added to the culture medium of human cord blood mononuclear (CBMN) cells, most cells became positive for the incorporated C45D18. Furthermore, recombinant proteins conjugated with the C45D18 were efficiently transduced and transported to regions corresponding to the nucleus. Incorporation of C45D18-conjugated protein was observed within a few hours after addition of the protein, independent of cellular growth. Although it is well known that Tat-derived peptide has a transducing activity, C45D18 was more active than Tat peptide for trafficking proteins into cells. Taking together with results from FACS analysis revealing that more than 90% of CBMN cells were positive for X-gal staining after treatment of C45D18-conjugated beta-galactosidase, we propose that C45D18 translocates bioactive macromolecules directly into the nucleus.

Active Transport, Cell Nucleus↗

PubMed2004

Binding of 14-3-3beta but not 14-3-3sigma controls the cytoplasmic localization of CDC25B: binding site preferences of 14-3-3 subtypes and the subcellular localization of CDC25B.

The dual specificity phosphatase CDC25B positively controls the G2-M transition by activating CDK1/cyclin B. The binding of 14-3-3 to CDC25B has been shown to regulate the subcellular redistribution of CDC25B from the nucleus to the cytoplasm and may be correlated with the G2 checkpoint. We used a FLAG-tagged version of CDC25B to study the differences among the binding sites for the 14-3-3 subtypes, 14-3-3beta, 14-3-3epsilon and 14-3-3sigma, and the relationship between subtype binding and the subcellular localization of CDC25B. All three subtypes were found to bind to CDC25B. Site-directed mutagenesis studies revealed that 14-3-3beta bound exclusively near serine-309 of CDC25B1, which is within a potential consensus motif for 14-3-3 binding. By contrast, 14-3-3sigma bound preferentially to a site around serine-216, and the presence of serine-137 and -309 enhanced the binding. In addition to these binding-site differences, we found that the binding of 14-3-3beta drove CDC25B to the cytoplasm and that mutation of serine-309 to alanine completely abolished the cytoplasmic localization of CDC25B. However, co-expression of 14-3-3sigma and CDC25B did not affect the subcellular localization of CDC25B. Furthermore, serine-309 of CDC25B was sufficient to produce its cytoplasmic distribution with co-expression of 14-3-3beta, even when other putative 14-3-3 binding sites were mutated. 14-3-3epsilon resembled 14-3-3beta with regard to its binding to CDC25B and the control of CDC25B subcellular localization. The results of the present study indicate that two 14-3-3 subtypes can control the subcellular localization of CDC25B by binding to a specific site and that 14-3-3sigma has effects on CDC25B other than the control of its subcellular localization.

14-3-3 Proteins↗

PubMed2004

Nuclear export signal in CDC25B.

CDC25B is a dual-specificity phosphatase that activates CDK1/cyclin B. The nuclear exclusion of CDC25B is controlled by the binding of 14-3-3 to the nuclear export signal (NES) of CDC25B, which was reported to be amino acids H28 to L40 in the N-terminal region of CDC25B. In studying the subcellular localization of CDC25B, we found a functional NES at V52 to L65, the sequence of which is VTTLTQTMHDLAGL, where bold letters are leucine or hydrophobic amino acids frequently seen in an NES. The deletion of this NES sequence caused the mutant protein to locate exclusively in nuclei, while NES-fused GFP was detected in the cytoplasm. Moreover, the introduction of point mutations at some of the critical amino acids impaired cytoplasmic localization. Treatment with leptomycin B, a potent inhibitor of CRM1/exportin1, disrupted the cytoplasmic localization of both Flag-tagged CDC25B and NES-fused GFP. From these results, we concluded that the sequence we found is a bona fide NES of CDC25B.

Active Transport, Cell Nucleus↗

PubMed2003

Loss of p53 induces M-phase retardation following G2 DNA damage checkpoint abrogation.

Most cell lines that lack functional p53 protein are arrested in the G2 phase of the cell cycle due to DNA damage. When the G2 checkpoint is abrogated, these cells are forced into mitotic catastrophe. A549 lung adenocarcinoma cells, in which p53 was eliminated with the HPV16 E6 gene, exhibited efficient arrest in the G2 phase when treated with adriamycin. Administration of caffeine to G2-arrested cells induced a drastic change in cell phenotype, the nature of which depended on the status of p53. Flow cytometric and microscopic observations revealed that cells that either contained or lacked p53 resumed their cell cycles and entered mitosis upon caffeine treatment. However, transit to the M phase was slower in p53-negative cells than in p53-positive cells. Consistent with these observations, CDK1 activity was maintained at high levels, along with stable cyclin B1, in p53-negative cells. The addition of butyrolactone I, which is an inhibitor of CDK1 and CDK2, to the p53-negative cells reduced the floating round cell population and induced the disappearance of cyclin B1. These results suggest a relationship between the p53 pathway and the ubiquitin-mediated degradation of mitotic cyclins and possible cross-talk between the G2-DNA damage checkpoint and the mitotic checkpoint.

4-Butyrolactone↗