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Biomedical subjects

Shigeru Morikawa

Publications and source records attributed to Shigeru Morikawa.

At least 19 recordsLinked to original sources
PubMed2003

The intracellular association of the nucleocapsid protein (NP) of hantaan virus (HTNV) with small ubiquitin-like modifier-1 (SUMO-1) conjugating enzyme 9 (Ubc9).

Small ubiquitin-like modifier-1 (SUMO-1) conjugating enzyme 9 (Ubc9) conjugates SUMO-1 to target proteins and modulates cellular processes such as signal transduction, transcription regulation, and cell growth regulation. We demonstrated here that the nucleocapsid protein (NP) of Hantaan virus (HTNV) was associated with Ubc9 and SUMO-1 in vivo. Analysis of the interaction between the truncated NPs and Ubc9 revealed that the amino acid residues at the positions between 101 and 238 in the NP were responsible for the interaction. Furthermore, a consensus binding motif of Ubc9 and SUMO-1, MKAE, within this region, especially the second amino acid of the motif, K residue, was crucial for the interaction, and the interaction was essential for the NP to be localized in the perinuclear region. These results indicate that the assembly of the HTNV-NP is regulated by the interaction between the NP and Ubc9. This is the first report to demonstrate the interaction of Ubc9 with a structural protein of negative-strand RNA viruses.

Animals↗
PubMed2003

Establishment of a human preadipose cell line, HPB-AML-I: refractory to PPARgamma-mediated adipogenic stimulation.

In this study, we established a unique cell line, HPB-AML-I (AML-I), from peripheral blood mononuclear cells collected from a patient with acute myeloid leukemia (AML: M1). Morphological and phenotypical analyses of the established AML-I cells demonstrated that they belong to a preadipocyte cell line as indicated by their storage of lipid droplets and expression of surface antigens similar to those found on bone marrow stromal cells (MSC). Through cell culture under adipogenic conditions, effective differentiation of AML-I cells into adipocytes was induced by an adipogenesis inducing cocktail (INC) made up of a mixture of methylisobutylxanthine, hydrocortisone, and indomethacin. By contrast, activation of peroxisome proliferator-activated receptor (PPARgamma), which plays a key role in lipids metabolism and is highly expressed in AML-I cells, decreased the number of lipid droplets in AML-I cells. Here we report the establishment of a unique human derived-preadipocyte cell line, AML-I, and its bi-directional adipogenic response to different type stimulation, i.e., one is a refractory response to troglitazone, a well-known adipogenic stimulator, and a positive response to INC, an adipogenesis induction cocktail. These results suggest that, based on the adipogenic response, there might be some distinct lineages in human adipocytes and that the unique differentiation of AML-I cells should be useful for analyzing both the differentiation and regulation of human preadipocytes.

Adipocytes↗
PubMed2003

Capsaicin inhibits growth of adult T-cell leukemia cells.

Human T-cell leukemia virus type 1 (HTLV-1)-associated adult T-cell leukemia (ATL) is resistant to conventional chemotherapy. We examined the in vitro effects of capsaicin, the principal ingredients of red pepper, on three ATL cell lines. Capsaicin treatment inhibited the growth of ATL cells both in dose- and time-dependent manner. The inhibitory effect was mainly due to the induction of cell cycle arrest and apoptosis. Capsaicin treatment also induced the degradation of Tax and up-regulation of I kappa-B alpha, resulting in the decrease of nuclear factor (NF)-kappa B/p65 DNA binding activity. In addition, the Bcl-2 level was found to be decreased. Based on these findings, capsaicin may be considered for chemoprevention of ATL.

Apoptosis↗
PubMed2003

Detection of immunoglobulin G to Crimean-Congo hemorrhagic fever virus in sheep sera by recombinant nucleoprotein-based enzyme-linked immunosorbent and immunofluorescence assays.

Crimean-Congo hemorrhagic fever virus is a tick-borne virus that causes severe hemorrhagic symptoms with an up to 50% mortality rate in humans. Wild and domestic animals, such as sheep, cattle and goats, are the reservoirs. The recombinant nucleoprotein-based Crimean-Congo hemorrhagic fever virus antibody detection systems for sheep sera were developed by enzyme-linked immunosorbent assay (ELISA) and an indirect immunofluorescence assay techniques. The samples used for evaluation were 80 sera collected from sheep in a Crimean-Congo hemorrhagic fever-endemic area (western part of the Xinjiang Uygur Autonomous Region) and 39 sera collected from sheep in a disease-free region (Shandong province, eastern China). The ELISA and indirect immunofluorescence assay using recombinant nucleoprotein of the virus proved to have high sensitivity and specificity for detecting the immunoglobulin G antibodies to the virus in sheep sera. Within this limited number of samples, the recombinant nucleoprotein-based ELISA and indirect immunofluorescence assay are considered to be useful tools for seroepidemiological study of virus infections in sheep sera.

Animals↗
PubMed2003

Analysis of linear B-cell epitopes of the nucleoprotein of ebola virus that distinguish ebola virus subtypes.

Ebola virus consists of four genetically distinguishable subtypes. We developed monoclonal antibodies (MAbs) to the nucleoprotein (NP) of Ebola virus Zaire subtype and analyzed their cross-reactivities to the Reston and Sudan subtypes. We further determined the epitopes recognized by these MAbs. Three MAbs reacted with the three major subtypes and recognized a fragment containing 110 amino acids (aa) at the C-terminal extremity. They did not show specific reactivities to any 10-aa short peptides in Pepscan analyses, suggesting that these MAbs recognize conformational epitope(s) located within this region. Six MAbs recognized a fragment corresponding to aa 361 to 461 of the NP. Five of these six MAbs showed specific reactivities in Pepscan analyses, and the epitopes were identified in two regions, aa 424 to 430 and aa 451 to 455. Three MAbs that recognized the former epitope region cross-reacted with all three subtypes, and one that recognized the same epitope region was Zaire specific. One MAb, which recognized the latter epitope region, was reactive with Zaire and Sudan subtypes but not with the Reston subtype. These results suggest that Ebola virus NP has at least two linear epitope regions and that the recognition of the epitope by MAbs can vary even within the same epitope region. These MAbs showing different subtype specificities might be useful reagents for developing an immunological system to identify Ebola virus subtypes.

Amino Acid Sequence↗
PubMed2003

A patient with Crimean-Congo hemorrhagic fever serologically diagnosed by recombinant nucleoprotein-based antibody detection systems.

We treated a male patient with Crimean-Congo hemorrhagic fever (CCHF). The diagnosis of CCHF was confirmed by reverse transcription-PCR and recombinant nucleoprotein (rNP)-based immunoglobulin G (IgG) and IgM capture enzyme-linked immunosorbent assays of serially collected serum samples. The patient was treated with intravenous ribavirin and recovered with no consequences. The study indicates that rNP-based CCHF virus antibody detection systems are useful for confirming CCHF virus infections. This case also suggests that intravenous ribavirin therapy may be promising for the treatment of CCHF patients.

Adult↗
PubMed2003

Antigen capture enzyme-linked immunosorbent assay for specific detection of Reston Ebola virus nucleoprotein.

Antigen capture enzyme-linked immunosorbent assay (ELISA) is one of the most useful methods to detect Ebola virus rapidly. We previously developed an antigen capture ELISA using a monoclonal antibody (MAb), 3-3D, which reacted not only to the nucleoprotein (NP) of Zaire Ebola virus (EBO-Z) but also to the NPs of Sudan (EBO-S) and Reston Ebola (EBO-R) viruses. In this study, we developed antigen capture ELISAs using two novel MAbs, Res2-6C8 and Res2-1D8, specific to the NP of EBO-R. Res2-6C8 and Res2-1D8 recognized epitopes consisting of 4 and 8 amino acid residues, respectively, near the C-terminal region of the EBO-R NP. The antigen capture ELISAs using these two MAbs detected the EBO-R NP in the tissues from EBO-R-infected cynomolgus macaques. The antigen capture ELISAs using Res2-6C8 and Res2-1D8 are useful for the rapid detection of the NP in EBO-R-infected cynomolgus macaques.

Animals↗
PubMed2003

[Lassa virus].

Explore the source record for details and available documents.

Animals↗
PubMed2002

Genetic diversity of the M RNA segment among Crimean-Congo hemorrhagic fever virus isolates in China.

The complete nucleotide sequences of the medium (M) segment of seven Chinese isolates of Crimean-Congo hemorrhagic fever (CCHF) virus were determined. The M-segment RNA of CCHF virus comprises 5356-5377 nucleotides depending on the isolate and encodes a protein comprising 1689-1697 amino acids in a viral complementary sense. Phylogenetic analysis of the M segment showed that the Chinese CCHF virus isolates were clustered into three groups, one of which was more closely related to a Nigerian isolate. Pairwise comparison of a precursor protein showed that amino-terminal regions comprising 250 amino acids were extraordinary heterogeneous, with a 22.4% identity in amino acids being observed between the most distantly related isolates. Since all the viruses were isolated from 1966 to 1988 within a restricted area in the Xinjiang Autonomous Region in western China, the results indicate that a multisource virus population is endemic in this region.

Animals↗
PubMed2002

Chimeric recombinant hepatitis E virus-like particles as an oral vaccine vehicle presenting foreign epitopes.

Many viral and bacterial pathogens establish infections through mucosal surfaces in their initial stage. However, only a few nonreplicating molecules successfully induce strong mucosal immune reaction without the addition of adjuvants by oral administration. To overcome this difficulty, we investigated whether hepatitis E virus-like particles (HEV-VLPs) could be utilized as a carrier molecule for foreign antigenic epitopes and to stimulate mucosal immunity without the need for adjuvants. To accomplish this goal, we incorporated a B cell epitope tag, consisting of 11 amino acids at the C-terminal of HEV-VLP. The chimeric VLP showed morphology similar to that of the mature HEV virion and VLP. The inserted epitope was reactive with a specific monoclonal antibody in the VLP form, suggesting that it was exposed on the surface of the VLP. After oral administration without adjuvant, this chimeric HEV induced significant levels of specific IgG and IgA to both the inserted epitope and HEV-VLP in intestinal secretions. These humoral immune responses were observed as early as 2 weeks after the first immunization. These results suggest the potential of HEV-VLP as a mucosal vaccine carrier vehicle for the presentation of antigenic epitopes through oral administration.

Administration, Oral↗
PubMed2002

Human T-cell leukemia virus type I Tax transactivates the matrix metalloproteinase-9 gene: potential role in mediating adult T-cell leukemia invasiveness.

Human T-cell leukemia virus type I (HTLV-I) is the etiologic agent of adult T-cell leukemia (ATL) and of tropical spastic paraparesis/HTLV-I-associated myelopathy. Infiltration of various tissues by circulating leukemic cells is a characteristic of ATL. Matrix metalloproteinases (MMPs), which mediate the degradation of the basement membrane and extracellular matrix, play an important role in metastasis and tumor cell dissemination. The aim of this study was to explore whether expression of MMP-2 and MMP-9 was deregulated by HTLV-I infection. The data showed that HTLV-I-infected T-cell lines expressed high levels of MMP-9 compared with uninfected T-cell lines. In contrast, the levels of the related MMP-2 were not significantly altered by HTLV-I infection. In addition, the elevated expression of MMP-9 in HTLV-I-infected cells was attributable to the action of the viral transactivator protein Tax. The results show that Tax can activate the MMP-9 promoter and induce MMP-9 expression in T cells, indicating that the constitutive expression of MMP-9 in virus-infected cell lines is at least in part mediated by Tax. Activation of the MMP-9 promoter by Tax occurs mainly through the action of NF-kappaB and SP-1. The biologic significance of these observations was validated by the following 2 findings: MMP-9 expression was increased in primary ATL cells, and plasma MMP-9 levels were elevated in ATL patients. In addition, plasma levels of MMP-9 correlated with organ involvement in ATL patients. Together these data suggest that overexpression of MMP-9 in HTLV-I- infected cells may be in part responsible for the invasiveness of ATL cells.

Enzyme Induction↗
PubMed2002

Bone marrow transplantation in a child with Wiskott-Aldrich syndrome latently infected with acyclovir-resistant (ACV(r)) herpes simplex virus type 1: emergence of foscarnet-resistant virus originating from the ACV(r) virus.

A human leukocyte antigen (HLA)-matched unrelated bone marrow transplantation (BMT) was performed in a 13-year-old patient with the congenital immunodeficiency syndrome, Wiskott-Aldrich syndrome. The patient had a history of acyclovir (ACV)-resistant (ACV(r)) herpes simplex virus type 1 (HSV-1) infections prior to BMT. After BMT, the skin lesions caused by HSV-1 relapsed on the face and genito-anal areas. Ganciclovir (GCV) therapy was initiated, but the mucocutaneous lesions worsened. An HSV-1 isolate recovered from the lesions during this episode was resistant to both ACV and GCV. The ACV(r) isolate was confirmed to have the same mutation in the viral thymidine kinase (TK) gene as that of the previously isolated ACV(r) isolates from the patient. After treatment switch to foscarnet (PFA), there was a satisfactory remission but not a complete recovery. Although the mucocutaneous lesions improved, a PFA-resistant (PFA(r)) HSV-1 was isolated 1 month after the start of PFA therapy. The PFA(r) HSV-1 isolate coded for the same mutation in the viral TK gene as the ACV(r) HSV-1 isolates. Furthermore, the PFA(r) isolate also expressed a mutated viral DNA polymerase (DNA pol) with an amino acid (Gly) substitution for Val at position 715. This is the first report on the clinical course of a BMT-associated ACV(r) HSV-1 infection that subsequently developed resistance to foscarnet as well.

Acyclovir↗
PubMed2002

Importance of C-terminus of herpes simplex virus type 1 thymidine kinase for maintaining thymidine kinase and acyclovir-phosphorylation activities.

We previously isolated an acyclovir (ACV)-resistant herpes simplex virus type 1 (HSV-1), strain TAR, from a child with Wiskott-Aldrich syndrome. An acyclovir-sensitive HSV-1, strain TAS, had been isolated from the same patient before the isolation of HSV-1 TAR. The TK protein of ACV-sensitive HSV-1 TAS was composed of 376 amino acids, while that of HSV-1 TAR was composed of 407 amino acids with altered amino acid residue between positions 355-407. The elongation of TK was caused by a single nucleotide deletion of cytosine from a homopolymer stretch of 4 cytosine residues between positions 1061-1064. There was no viral TK activity in HSV-1 TAR-infected Vero cells, indicating the importance of the C-terminal portion of TK protein from positions 355-376. Recombinant TK polypeptides with amino acid deletions at the C-terminus were prepared, and TK and ACV-phosphorylation activities were examined. Deletion of 5 and 6 amino acids from the C-terminus of the TK polypeptide of HSV-1 TAS resulted in a reduction of TK activity by approximately 75% and 100%, respectively. These mutant TK polypeptides did not phosphorylate ACV. These results indicate that amino acid residues from positions 371-376 in the C-terminal portion of HSV-1 TK protein are essential for keeping TK and ACV-phosphorylation activities.

Acyclovir↗
PubMed2002

Genotypic and phenotypic characterization of the thymidine kinase of ACV-resistant HSV-1 derived from an acyclovir-sensitive herpes simplex virus type 1 strain.

Twenty-four strains of acyclovir (ACV)-resistant (ACV(r)) herpes simplex virus type 1 (HSV-1) were generated from the HSV-1 TAS strain by exposure to ACV, and the genotype and phenotype of the thymidine kinase (TK) from these mutants were analyzed. The TK polypeptide of the ACV(r) HSV-1 strains was examined by Western blot using an anti-HSV-1 TK rabbit serum. The sensitivity of each strain to ACV, foscarnet and cidofovir (CDV) was also determined. A single guanine (G) insertion or a single cytosine (C) deletion was detected in 12 of the 24 ACV(r) strains at the G or C homopolymer stretches within the TK gene. Genotypic analysis predicted that two thirds of the ACV(r) HSV-1 strains expressed truncated TK polypeptides, while one third expressed viral TK polypeptide with a single amino acid substitution at various sites. Western blot abnormalities in the viral TK polypeptides were identified in 21 ACV(r) strains. There was an inverse correlation between the susceptibility of the HSV-1 mutant strains to ACV and that to CDV. Nucleotide sequencing of the TK gene and Western blot analysis of the viral TK polypeptides are considered to be one of the methods for predicting virus sensitivity to ACV and CDV.

Acyclovir↗
PubMed2002

Characterization of granulocyte colony-stimulating factor receptor expressed on human lymphocytes.

We have studied the characterization of granulocyte colony-stimulating factor receptor (G-CSFR) in human lymphocytes. About one-third to one-quarter of the B lymphocytes from peripheral B-cell sources displayed G-CSF binding on the two-colour immunofluorescence study. The rate of G-CSFR-expressing (G-CSFR+) B cells was higher in bone marrow and cord blood than in peripheral blood, spleen and tonsil. G-CSFR expression was greater in the surface immunoglobulin D (IgD)-positive (sIgD+) B-cell population, but scarce in the sIgD- B-cell population. In tonsil, G-CSFR+ B cells were present among the cells with naive B and germinal-centre B phenotypes, but those with memory B phenotype were rarely found on triple-colour immunofluorescence analysis. Mitogen-activated, but not resting, T lymphocytes also showed G-CSF binding. Several continuous T- and B-cell lines expressed functional G-CSFR, because the addition of G-CSF enhanced the proliferative response of these cell lines. A sequence analysis of G-CSFR mRNA isoforms obtained from the T and B cells revealed that G-CSFR was derived from class I and class IV mRNA. Our results indicated that G-CSFR was constitutively expressed on the B-cell surface and was inducible in T cells.

B-Lymphocytes↗
PubMed2002

MD41, a novel T helper 0 clone, mediates mast-cell dependent delayed-type hypersensitivity in mice.

In a previous study on mouse, we have shown that delayed-type hypersensitivity (DTH) could be classified into two types according to MC requirement. The first type of DTH could be elicited by sensitization with methylated human serum albumin (MHSA) in complete Freund's adjuvant (CFA) in both wild type and mast-cell deficient (W/Wv) mice. The second type could be elicited by MHSA in incomplete Freund's adjuvant (IFA) sensitization in wild type but not W/Wv mice. While the former was related to classic tuberculin (tbc)-type DTH, the latter appeared to be a novel mast-cell dependent DTH (MD-DTH). In order to investigate the mechanism of MD-DTH, in this study, we generated an effector T-cell clone (MD41) from lymph node cells of MHSA in IFA-sensitized mice and analysed its pattern of cytokine production. Our results from cytokine assays show that following antigen stimulation, MD41 cells produce significant amounts of the T helper 1 (Th1) cytokine interferon-gamma (IFN-gamma) as well as the Th2 cytokines interleukin (IL)-4 and IL-10. In addition, double staining for IL-4 and IFN-gamma revealed that MD41 cells produce both Th1- and Th2-type cytokines simultaneously, which suggest that MD41 represents a Th0 clone rather than a mixture of Th1 and Th2 clones. Adoptive transfer of MD41 cells into wild-type mice resulted in the development of DTH skin reactions similar to those produced by active sensitization, with very similar histological findings. However, DTH skin reactions could not be induced in W/Wv mice unless first reconstituted with normal bone marrow MC (BM-MC). Therefore, our study suggests that in conjunction with tissue MC, MD41, a less-polarized MD-DTH-derived Th0 clone, is capable of developing murine DTH to the same extent as strongly polarized Th1 cells and mediates MD-DTH rather than tbc-type DTH.

Adoptive Transfer↗
PubMed2002

Development of an immunofluorescence method for the detection of antibodies to Ebola virus subtype Reston by the use of recombinant nucleoprotein-expressing HeLa cells.

An indirect immunofluorescent assay (IFA) to detect Ebola virus subtype Reston (EBO-R) antibodies was developed by the use of a HeLa cell line stably expressing EBO-R nucleoprotein (NP). This IFA has a high specificity for the detection of EBO-R IgG antibodies in both hyperimmune rabbit sera and monkey sera collected during an EBO-R outbreak in the Philippines in 1996. Furthermore, this IFA showed a higher sensitivity for the detection of EBO-R antibodies than did the IFA using HeLa cells expressing the NP of Ebola virus subtype Zaire. These results suggest that this new IFA is useful for seroepidemiological studies of EBO-R infection among monkeys.

Animals↗