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Masashi Tatsumi

Publications and source records attributed to Masashi Tatsumi.

16 recordsLinked to original sources
PubMed2006

Development of a real-time PCR assay for detection and quantification of Francisella tularensis.

The facultative intracellular bacterium, Francisella tularensis, is an etiological agent of tularemia and is also considered to be a potential biological threat agent due to its extreme infectivity. We established a real-time PCR assay using the LightCycler (LC) system to detect a Francisella-specific sequence of the outer membrane protein (fopA) gene. Twenty-five F. tularensis strains including 16 Japanese isolates were subjected to this LC-PCR assay, and were tested positive, whereas Francisella philomiragia and other bacteria species did not show any specific fluorescent signal. A linear response was observed using F. tularensis genomic DNAs of between 20 fg and 2 ng, corresponding to 1.2 to 1.2 x 10(5) bacteria. The newly established real-time PCR allows the detection of the F. tularensis genome specifically, sensitively, and rapidly. This assay may contribute to the standardization of the laboratory diagnosis of tularemia.

Bacterial Outer Membrane Proteins↗
PubMed2005

Construction and characterization of an infectious molecular clone of HIV-1 subtype A of Indian origin.

India has the second highest number of HIV-1 infected people next to South Africa. The predominant proportion of HIV-1 circulating in India is of subtype C origin, with a small fraction made up of subtypes A and B. In this report, we describe the construction and characterization of the first full-length infectious molecular clone p1579A-1 HIV-1, from an HIV-1 subtype A infected person from India, using long PCR and successive ligation of the amplimers. Phylogenetic analysis of the sequence of the entire proviral DNA and LTR confirmed p1579A-1 to be an HIV-1 subtype A. Analysis of the env gene of p1579A-1 showed a conserved GPGQ motif and the absence of basic amino acids at positions 11 and 25 suggesting CCR5 coreceptor usage. Analysis of env N-linked glycosylation sites revealed fewer sites in the V1 region of envelope compared to other subtype A. Transcription factor binding site analysis of the LTR sequences identified conserved as well as unique transcription factor binding sites (TFBS) in p1579A-1. This infectious clone of HIV-1 can be useful to study the molecular mechanism of dominance of subtype C in India.

Adolescent↗
PubMed2005

A novel small molecular weight compound with a carbazole structure that demonstrates potent human immunodeficiency virus type-1 integrase inhibitory activity.

The integration of reverse transcribed proviral DNA into a host genome is an essential event in the human immunodeficiency virus type 1 (HIV-1) replication life cycle. Therefore, the viral enzyme integrase (IN), which plays a crucial role in the integration event, has been an attractive target of anti-retroviral drugs. Several IN inhibitory compounds have been reported previously, yet none has been successful in clinical use. To find a new, more successful IN inhibitor, we screened a diverse library of 12 000 small molecular weight compounds randomly by in vitro strand-transfer assay. We identified a series of substituted carbazoles that exhibit strand-transfer inhibitory activity at low micromolar concentrations. Of these, the most potent compound exhibited an IC50 of 5.00+/-3.31 microM (CA-0). To analyse the structural determinants of strand-transfer inhibitory activity of the carbazole derivatives, we selected 23 such derivatives from our compound library and performed further analyses. Of these 23 compounds, six showed strong strand-transfer inhibition. The inhibition kinetics analyses and ethidium bromide displacement assays indicated that the carbazole derivatives are competitive inhibitors and not intercalators. An HeLa4.5/LTR-nEGFP cell line was employed to evaluate in vitro virus replication inhibition of the carbazole derivatives, and IC50 levels ranged from 0.48-1.52 microM. Thus, it is possible that carbazole derivatives, which possess structures different from previously-reported IN inhibitors, may become novel lead compounds in the development of IN inhibitors.

Anti-HIV Agents↗
PubMed2004

Identification of a novel isoform of ZAP-70, truncated ZAP kinase.

We identified a novel cDNA encoding truncated ZAP-70, which lacked the SH2 domain and a part of interdomain B, and named it truncated ZAP kinase (TZK). TZK was expressed in the thymus, spleen, and lymph nodes with ZAP-70. TZK was expressed in CD44+CD25- thymocytes up to mature T cells, but ZAP-70 was not expressed in CD44+CD25- or CD44+CD25+ thymocytes. ZAP-70 or TZK was transfected into P116 cells derived from a Jurkat T-cell line deficient in ZAP-70. The P116 cells with ZAP-70 induced the T-cell receptor-mediated signal transduction, but the cells expressing TZK did not. While ZAP-70 was accumulated at the immune synapse, TZK was not. Meanwhile, impaired phosphorylation of SLP-76, one of the substrates of ZAP-70, in P116 cells upon pervanadate stimulation was rescued in the cells expressing TZK. These findings show that TZK is a novel isoform of ZAP-70, which is expressed in pre-T-cell receptor-minus thymocytes and functions as a kinase not associated with T-cell receptor.

Animals↗
PubMed2004

Regulation of osteoclastogenesis by three human RANKL isoforms expressed in NIH3T3 cells.

Receptor activator of nuclear factor-kappaB ligand (RANKL) induces osteoclastogenesis by binding with the receptor, receptor activator of nuclear factor-kappaB in the presence of macrophage colony-stimulating factor. Three human RANKL isoforms, hRANKL1, hRANKL2, and hRANKL3, were identified. hRANKL1 was identical to previously reported RANKL and possessed intracellular, transmembrane, and extracellular domains, hRANKL2 did not have the intracellular domain, and hRANKL3 did not have the intracellular and transmembrane domains. When bone marrow macrophages were cultured with NIH3T3 cells expressing hRANKL1, osteoclasts were formed, but when cultured with NIH3T3 cells expressing hRANKL2 or hRANKL3, no tartrate resistant acid phosphatase-positive cell was observed. In the coculture system, coexpression of hRANKL3 with hRANKL1 significantly inhibited the formation of osteoclasts by hRANKL1, but coexpression of hRANKL2 with hRANKL1 did not affect the osteoclastogenesis by hRANKL1 significantly. These results suggest that the activity of osteoclastogenesis by hRANKL1 is regulated by the attenuator, hRANKL3.

Amino Acid Sequence↗
PubMed2004

Specific reactions between purified HIV-1 particles and CD4+ cell membrane fragments in a cell-free system of virus fusion or entry.

The initial step of human immunodeficiency virus type 1 (HIV-1) infection has been studied by Env-mediated fusion or entry assays with appropriate cells expressing CD4 or CXCR4/CCR5 receptors in cultures, where many factors underlying cellular activities likely regulate the fusion/entry efficiency. Here we attempted to develop a more simplified in vitro cell-free fusion/entry reaction that mimics HIV-1 infection in cultures. Membrane fragments of target cells and intact infectious HIV-1 particles were purified, mixed and incubated. The core p24 protein was released from the purified virions and detected by ELISA without detergents in the supernatant of the reaction mixtures. This release reaction proceeded temperature-dependently and in a dose-dependent manner between the virion and membrane fractions, and was specific for HIV-1 Env and CD4. Env-deleted or VSV-G-pseudotyped HIV-1 released little p24, if any. Pretreatment of the membrane fragments with anti-CD4 antibodies inhibited the p24 induction from both X4-tropic and R5-tropic HIV-1. Furthermore, X4 but not R5 HIV-1 reacted with the membrane prepared from intrinsically CXCR4-positive HeLa-CD4 cells, whereas both viruses reacted with that prepared from CCR5-transduced HeLa-CD4 cells, indicating that this cell-free reaction mimics coreceptor usage of HIV-1 infection. Therefore, a potent entry inhibitor of X4 HIV-1, SDF-1alpha, blocked the release from X4 but not R5 HIV-1. Inversely, a weak entry inhibitor of R5 HIV-1, MIP-1beta, partially affected only the release from R5 HIV-1. These results suggest that this cell-free reaction system provides a useful tool to study biochemical fusion/entry mechanisms of HIV-1 and its inhibitors.

Antiviral Agents↗
PubMed2003

High prevalence of diverse forms of HIV-1 intersubtype recombinants in Central Myanmar: geographical hot spot of extensive recombination.

OBJECTIVES: To investigate the molecular epidemiology and genetic structure of HIV-1s causing the epidemic in Central Myanmar and to explore the genesis of HIV epidemic in this area. DESIGN: A molecular epidemiological investigation was conducted in 1999-2000 in the city of Mandalay among high-risk populations and the structural features of circulating HIV-1s were analyzed. METHODS: HIV-1 genotypes of 59 specimens were screened based on gag (p17) and env (C2/V3) regions. Near full-length nucleotide sequences of HIV-1 isolates with subtype discordance were determined and their recombinant structures were characterized. RESULTS: Three lineages of HIV-1 strains, including CRF01_AE (27, 45.8%), subtype B' (Thailand variant of subtype B) (15, 25.4%) and subtype C (8, 13.6%), were distributed in Mandalay, while substantial portions (9, 15.3%) of specimens showed various patterns of subtype discordance in different regions of HIV-1 genomes. The study on six HIV-1 isolates with subtype discordance revealed that they were highly diverse types of unique recombinant forms (URFs) comprised of various combinations of three circulating subtypes. One URF was a particularly complex mosaic that contained 13 recombination breakpoints between three HIV-1 subtypes. Approximately half of recombinants showed 'pseudotype' virion structures, in which the external portions of envelope glycoproteins were exchanged with different lineages of HIV-1 strains, suggesting the potential selective advantage of 'pseudotype' viruses over parental strains. CONCLUSION: The study revealed the unique geographical hot spot in Central Myanmar where extensive recombination events appeared to be taking place continually. This reflects the presence of highly exposed individuals and social networks of HIV-1 transmission.

Adolescent↗
PubMed2003

"All-in-One Assay", a direct phenotypic anti-human immunodeficiency virus type 1 drug resistance assay for three-drug combination therapies that takes into consideration in vivo drug concentrations.

A novel phenotypic anti-human immunodeficiency virus type 1 (HIV-1) drug resistance assay is described. Three drugs at concentrations equivalent to those determined in in vivo pharmacokinetics, were mixed in a well, serially diluted by 10-folds, and added to incubations of clinical HIV-1 isolates and CCR-5 expressing HeLa/CD4+ cells which was previously reported as the MAGIC-5 cells (Antimicrob. Agents Chemother. 45 (2001) 495) to determine the 95% inhibitory dilution (ID(95)) of the combination regimens. The ID(95) of efavirenz (EFV)-containing regimens was ten-times lower than that of nevirapine (NVP)-containing regimens against HIV-1 isolated from antiviral therapy naive patients. However, the difference was not apparent by the conventional fold resistance measurement based on the 50% inhibitory concentration. Furthermore, the synergistic effects of drug combinations against clinical HIV-1 isolates can be evaluated by our assay. The ID(95)s of EFV- and nelfinavir (NFV)- containing regimens against HIV-1 from naive patients were less than 0.01 whereas those against resistant viruses were over 0.05, although the clinical cut-off values are to be determined in larger clinical studies. Our assay, designated "All-in-One Assay", that can examine resistance to three drugs simultaneously under consideration of in vivo drug concentrations described above might be useful in practice.

Anti-HIV Agents↗
PubMed2002

Processing of the HTLV-II envelope precursor glycoprotein gp63 by furin is essential for cell fusion activity.

To investigate the relationship between the fusogenic properties of HTLV-II and the processing of the envelope precursor glycoprotein gp63, recombinant cowpox virus expressing this protein was used to infect a range of cell lines derived from different species. Syncytium formation and gp63 processing were observed in all cells with the exception of LoVo cells, which are known to have a dysfunctional form of the endoprotease, furin. Furin has been shown to be necessary for the processing of a number of viral envelope glycoproteins, and gp63 contains a consensus sequence (305)Arg-Arg-Arg-Arg, which is a furin substrate motif. Pulse-chase studies demonstrated gp63 processing in Vero but not in LoVo cells. In addition it could be shown that expression of recombinant furin restored the processing of gp63 to gp46 in LoVo cells, and this resulted in syncytium formation. Our findings suggest that furin plays a pivotal role in cleavage of the HTLV-II envelope gp63, which in turn is a prerequisite for the fusogenic properties of the virus.

Animals↗
PubMed2002

Isolation and characterization of an infectious HIV type 1 molecular clone from a patient with primary infection.

We isolated a subtype B infectious DNA clone of human immunodeficiency virus type 1 (HIV-1) from a seronegative patient with acute infection and determined the entire nucleotide sequence. All the reading frames encoding the structural proteins (Gag, Pol, and Env) and nonstructural proteins (Tat, Rev, Vpr, Vif, and Nef) were found. Although moat functional domains in these proteins were conserved, we identified a duplication of the T cell factor lei (TCF-1alpha) element in the long terminal repeat and many variations in the N-linked glycosylation sites in the V4-V5 region but not in the V1-V3 loop of Env, compared with prototype subtype B clones. Furthermore, phylogenetic analysis of the entire nucleotide sequence indicated that this HIV-1 was distinct from the prototype subtype B clones, suggesting that transmitted viruses can be variants. This HIV-1 DNA done will be a useful prototype for investigating the mechanism of HIV-1 transmission.

Amino Acid Sequence↗
PubMed2002

Construction and characterization of an infectious molecular clone derived from the CRF01_AE primary isolate of HIV type 1.

An infectious molecular clone (named p95TNIH022) was constructed using long-range polymerase chain reaction products derived from a clinical isolate (95TNIH022) of HIV-1 CRF01_AE obtained from an asymptomatic Thai carrier in 1995. The virus in the supernatant from p95TNIH022-transfected 293T cells showed infectivity in peripheral blood mononuclear cells (PBMCs) as well as in MAGIC5 cells, which express CD4 and CCR5, but not in the original MAGI cells, indicating that p95TNIH022 is an infectious molecular clone with CCR5 tropism. Interestingly, p95TNIH022-derived virus induced profound cell killing in infected PBMCs, as in cells infected with the parental isolate.

Base Sequence↗
PubMed2002

Isolation and characterization of replication-competent molecular DNA clones of HIV type 1 CRF01_AE with different coreceptor usages.

We have isolated replication-competent molecular clones of HIV-1 circulating recombinant form CRF01_AE with different coreceptor usages. After lambda phage cloning of unintegrated circular proviral DNAs derived from a CRF01_AE strain (HIV-1NH1), isolated in Japan, the infectious molecular clone, designated p93JP-NH1, was reconstituted. 93JP-NH1 showed an X4 and R5 phenotype in NP2 cell-based coreceptor utilization assays and exerted robust replication in human T cell lines, including MT2, M8166, and PM1 cells, whereas it propagated modestly in peripheral blood mononuclear cells. The CRF01_AE molecular clone with R5 phenotype (p93JP-NH2env) was then constructed by replacing the env gene of p93JP-NH1 with that of a nearly isogenic CRF01_AE R5 strain isolated from an epidemiologically linked case. The phylogeny and recombination break-point analysis confirmed that these clones shared an A/E recombinant structure similar to that of the prototype CRF01_AE strain, CM240. These replication-competent CRF01_AE molecular clones with different coreceptor usages would be useful tools for the study of CRF01_AE, one of the most prevalent strains in Asia.

Base Sequence↗
PubMed2002

Interference between D30N and L90M in selection and development of protease inhibitor-resistant human immunodeficiency virus type 1.

We studied the evolutionary relationships between the two protease inhibitor (PI) resistance mutations, D30N and L90M, of human immunodeficiency virus type 1 (HIV-1). The former is highly specific for nelfinavir resistance, while the latter is associated with resistance to several PIs, including nelfinavir. Among patients with nelfinavir treatment failure, we found that D30N acquisition was strongly suppressed when L90M preexisted. Thus, D30N/L90M double mutations not only were detected in a very limited number of patients but also accounted for a minor fraction within each patient. In the disease course, the D30N and L90M clones readily evolved independently of each other, and later the D30N/L90M double mutants emerged. The double mutants appeared to originate from the D30N lineage but not from the L90M lineage, or were strongly associated with the former. However, their evolutionary pathways appeared to be highly complex and to still have something in common, as they always contained several additional polymorphisms, including L63P and N88D, as common signatures. These results suggest that D30N and L90M are mutually exclusive during the evolutionary process. Supporting this notion, the D30N/L90M mutation was also quite rare in a large clinical database. Recombinant viruses with the relevant mutations were generated and compared for the ability to process p55gag and p160pol precursor proteins as well as for their infectivity. L90M caused little impairment of the cleavage activities, but D30N was detrimental, although significant residual activity was observed. In contrast, D30N/L90M demonstrated severe impairment. Thus, the concept of mutual antagonism of the two mutations was substantiated biochemically and functionally.

Cloning, Molecular↗
PubMed2002

Inefficient enhancement of viral infectivity and CD4 downregulation by human immunodeficiency virus type 1 Nef from Japanese long-term nonprogressors.

It has been reported that patients infected with nef-defective human immunodeficiency virus type 1 (HIV-1) do not progress to AIDS; however, mutations that abrogate Nef expression are not common in long-term nonprogressors (LTNPs). We postulated that Nef function might be impaired in LTNPs, irrespective of the presence or absence of detectable amino acid sequence anomalies. To challenge this hypothesis we compared in vitro function of nef alleles that were derived from three groups of Japanese patients: LTNPs, progressors, and asymptomatic carriers (ACs). The patient-derived nef alleles were subcloned into a nef-defective infectious HIV-1 molecular clone and an expression vector. We first examined Nef-dependent enhancement of infection in a single-round infectivity assay by the use of MAGNEF cells, in which Nef is required more strictly for the infection than in the parent MAGI cells. All nef alleles from LTNPs showed reduced enhancement in the infectivity of nef-defective HIV-1 mutants compared to the nef alleles of progressors or ACs. Second, we found that nef alleles from LTNPs were less efficient in CD4 downregulation than those of progressors or ACs. Third, all nef alleles from LTNPs, progressors, and ACs reduced the cell surface expression of major histocompatibility complex class I to a similar level. Last, there was no correlation between Hck-binding activity of Nef and clinical grouping. In conclusion, we detected inefficient enhancement of HIV-1 infectivity and CD4 downregulation by HIV-1 nef alleles of LTNPs. It awaits further study to conclude that these characteristics of nef alleles are the cause or the consequence of the long-term nonprogression after HIV-1 infection.

Alleles↗