PubMed Health⌕ Search

Biomedical subjects

H Shida

Publications and source records attributed to H Shida.

At least 19 recordsLinked to original sources
PubMed2001

Sequence analysis of fibroblast growth factor receptor 2 ( FGFR2 ) in Japanese patients with craniosynostosis.

Recently, mutations of the fibroblast growth factor receptor ( FGFR ) genes have been detected in syndromic craniosynostosis. We examined nucleotide sequences of FGFR2 in Japanese craniosynostosis patients (Crouzon syndrome: 9 cases; Apert syndrome: 6 cases; scaphocephaly: 3 cases as non-syndromic patients) by polymerase chain reaction (PCR) followed by direct sequencing methods. The results demonstrated FGFR2 heterozygous mutations at codons 252, 290 of exon 7, and at codon 342, 354 of exon 9 in Crouzon syndromes. In Apert syndrome patients, Ser252Trp and Pro253Arg were detected in five and one patients, respectively. No mutation was detected in one case of Crouzon, all cases of scaphocephaly and healthy individuals. Thus far sequence analysis of FGFR2 in syndromic craniosynostosis has been reported in many white patients, whereas in Japanese only several cases have been studied. The current study with 18 patients confirmed that a similar series of mutations occur in Japanese patients as in white patients regardless of ethnicity and environment. The frequency of the mutation was 82% (9/11 cases) in Japanese Crouzon patients. The ratio of S252W:P253R was 5 : 1 in Japanese Apert patients. Moreover, in Japanese Apert patients, complication rate of cleft palate was 60% for mutation of Ser252Trp and 0 of 2 patients for Pro253Arg, with their syndactyly score being 4.90 and 5.50, respectively.

Acrocephalosyndactylia↗
PubMed2001

Rat CRM1 is responsible for the poor activity of human T-cell leukemia virus type 1 Rex protein in rat cells.

Rat models of human T-cell leukemia virus type 1 (HTLV-1)-related diseases such as adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis have been reported. However, these models do not completely reproduce human diseases partly because HTLV-1 replicates poorly in rats. We investigated here the possible reason for this. We found that the activity of Rex in rat cells is quite low compared to that in human cells. As Rex function depends largely on the CRM1 protein, whose human type (human CRM1 [hCRM1]) directly binds to Rex and exports it from the nucleus to the cytoplasm, we assessed whether rat CRM1 (rCRM1) could act as well as hCRM1 as a cofactor for Rex activity. We first cloned a cDNA encoding rCRM1 and found that both rCRM1 and hCRM1 could bind to and export Rex protein to the cytoplasm with similar efficiencies. However, unlike hCRM1, rCRM1 could hardly support Rex function because of its poor ability in inducing the Rex-Rex interaction required for RNA export into the cytoplasm. These observations suggest that the poor ability of rCRM1 to act as a cofactor for Rex function may be responsible for the poor replication of HTLV-1 in rats.

Amino Acid Sequence↗
PubMed2000

Dox-dependent SIVmac with tetracycline-inducible promoter in the U3 promoter region.

An attenuated live vaccine is a candidate in developing vaccines against human immunodeficiency virus type 1 (HIV-1). The study using macaques and simian immunodeficiency virus (SIVmac) showed an attenuated virus to be more effective than any other vaccine candidate. However, development of a safer vaccine is required for clinical application. In this study, we constructed pSIVmac Delta nef with tetracycline inducible promoter (pTet) and tried to control viral expression in a drug-dependent manner. Promoter/enhancer motifs in the U3 region of the long terminal repeats (LTRs) were serially deleted and replaced with pTet. In mutant LTRs, which lack NF-kappaB and Sp1 binding sites, TATA box motifs, and the 5' half of the U3 region, promoter activity was stringently controlled by doxycycline (Dox). Their activities were similar to or higher than that of wild-type LTR in the presence of Dox, based on the transient chloramphenicol acetyltransferase reporter assay. Three of these mutant LTRs were introduced into the pSIVmac239 Delta nef genome. Viral protein from these viruses was efficiently expressed in a Dox-dependent manner after transfection to a HeLa cell, which expresses reverse tetracycline transactivator (rtTA). The 2-LTR-form viral DNA of these viruses could be detected in M8166 cells that had been infected with supernatants from the transfected rtTA HeLa cell. These results suggest that pSIVmac Delta nef containing mutant LTRs can proceed through one viral replication cycle consisting of transcription, formation of viral particles, infection to cells, and reverse transcription. Although continuous replication of these Dox-dependent viruses requires a supply of rtTA as a constituent for the pTet-On viral genome, the successful replacement of the original promoter with a drug-dependent promoter suggests a new possibility for developing a safer attenuated live virus.

Doxycycline↗
PubMed2000

Mixed dust fibrosis and tuberculosis in comparison with silicosis and macular pneumoconiosis.

BACKGROUND: To assess the relationship between mixed dust fibrosis (MDF) and tuberculosis. METHODS: We performed a comparative analysis with MDF, silicosis, and macular pneumoconiosis (Mac), using autopsy records from 1975 to 1994. RESULTS: Prevalences of having tuberculosis among MDF, silicosis, and Mac were not significantly different, albeit a tendency of higher prevalence in silicosis. Cure rates of tuberculosis were, in order, silicosis < MDF < Mac (P=0. 085). Death rates associated with tuberculosis were, in order, silicosis > MDF=Mac (P=0.911). With respect to the two types of association with tuberculosis, i.e., combined type (tuberculopneumoconiosis) and complicated one (pneumoconiosis with tuberculosis); the former was significantly dominant in silicosis, the latter was significantly dominant in Mac, and intermediate in MDF. As a whole, the complicated type had a tendency of a higher cure rate than the combined type (P=0.071). Although the differences of profiles between the combined and complicated types were not statistically significant, the combined type had a tendency to have longer duration of exposure to dusts, earlier registration for treatment, higher profusion score, and earlier death compared with the complicated type. CONCLUSIONS: From our findings, MDF takes an intermediate position between silicosis and Mac regarding the relationship with tuberculosis. The type of association with tuberculosis rather than the kind of background pneumoconiosis seemed to be more important in light of responsiveness to the treatment.

Autopsy↗
PubMed2000

Induction of recombinant gene expression in stably transfected cell lines using attenuated vaccinia virus MVA expressing T7 RNA polymerase with a nuclear localisation signal.

There are major drawbacks using vaccinia virus (VV) expressing T7 polymerase for eukaryotic expression. VV is infectious for humans and due to cytosolic replication of Poxviridae, transient transfection of T7 promoter containing plasmids is necessary, which varies in efficiency. Several improvements have been introduced to this system to enhance expression of herpes viral glycoproteins. Stably transfected cell lines were generated with an EBV-based episomal plasmid vector which can be pushed to increasing copy numbers under selective pressure. The avirulent vaccine MVA strain was adopted to generate a safe laboratory vector for inserting the bacteriophage T7 RNA polymerase gene with (+) or without (-) a nuclear localisation signal. Constructs were designed for recombination into the VV haemagglutinin gene as recombinants could not be isolated successfully when inserting into the MVA thymidine kinase locus. Both T7 MVA recombinants induced foreign protein expression in transiently transfected cells but only the T7-/+ MVA induced target protein expression in stably transfected cells. The level of protein expression by this induction mechanism was comparable to, or superior to levels obtained with VV recombinants expressing the gene under control of the VV 11 k IE promoter. The results suggests that the T7+ MVA virus can be used to induce gene expression in stable recombinant cell lines and offers an attractive and safe alternative to other inducible eucaryotic expression systems.

Animals↗
PubMed1999

Protective effects against simian immunodeficiency virus agm (SIVagm) infection in cynomolgus monkeys immunized with a recombinant vaccinia virus expressing the SIVagm envelope gene.

Protective immunity induced by a recombinant vaccinia virus expressing the envelope (Env) protein of a simian immunodeficiency virus strain, SIVagm, (SEN-RVV), was evaluated in cynomolgus monkeys (Macaca fascicularis). Three monkeys were immunized twice with SEN-RVV and boostered with the purified SIVagm Env protein. These monkeys developed high titers of anti-SIVagm Env antibody, especially after boostering. After challenge with polyclonal SIVagm, no virus was recovered from two of the monkeys and no provirus DNA was detected in one of these two. After autopsy, however, proviral DNA was detected in the spleen of this monkey. These results suggest that this immunization regimen could not completely protect the monkeys from SIV infection but that it did reduce the replicability of the challenged virus.

Animals↗
PubMed1999

Determinant in human immunodeficiency virus type 1 for efficient replication under cytokine-induced CD4(+) T-helper 1 (Th1)- and Th2-type conditions.

Cytokines are potent stimuli for CD4(+)-T-cell differentiation. Among them, interleukin-12 (IL-12) and IL-4 induce naive CD4(+) T cells to become T-helper 1 (Th1) or Th2 cells, respectively. In this study we found that macrophage-tropic human immunodeficiency virus type 1 (HIV-1) strains replicated more efficiently in IL-12-induced Th1-type cultures derived from normal CD4(+) T cells than did T-cell-line-tropic (T-tropic) strains. In contrast, T-tropic strains preferentially infected IL-4-induced Th2-type cultures derived from the same donor CD4(+) T cells. Additional studies using chimeric viruses demonstrated that the V3 region of HIV-1 gp120 was the principal determinant for efficiency of replication. Cell fusion analysis showed that cells expressing envelope protein from a T-tropic strain effectively fused with IL-4-induced Th2-type culture cells. Flow cytometric analysis showed that the level of CCR5 expression was higher on IL-12-induced Th1-type culture cells, whereas CXCR4 was highly expressed on IL-4-induced Th2-type culture cells, although a low level of CXCR4 expression was observed on IL-12-induced Th1-type culture cells. These results indicate that HIV-1 isolates exhibit differences in the ability to infect CD4(+)-T-cell subsets such as Th1 or Th2 cells and that this difference may partly correlate with the expression of particular chemokine receptors on these cells. The findings suggest that immunological conditions are one of the factors responsible for inducing selection of HIV-1 strains.

Cell Fusion↗
PubMed1999

Inhibitory mechanism of the CXCR4 antagonist T22 against human immunodeficiency virus type 1 infection.

We recently reported that a cationic peptide, T22 ([Tyr(5,12), Lys(7)]-polyphemusin II), specifically inhibits human immunodeficiency virus type 1 (HIV-1) infection mediated by CXCR4 (T. Murakami et al., J. Exp. Med. 186:1389-1393, 1997). Here we demonstrate that T22 effectively inhibits replication of T-tropic HIV-1, including primary isolates, but not of non-T-tropic strains. By using a panel of chimeric viruses between T- and M-tropic HIV-1 strains, viral determinants for T22 susceptibility were mapped to the V3 loop region of gp120. T22 bound to CXCR4 and interfered with stromal-cell-derived factor-1alpha-CXCR4 interactions in a competitive manner. Blocking of anti-CXCR4 monoclonal antibodies by T22 suggested that the peptide interacts with the N terminus and two of the extracellular loops of CXCR4. Furthermore, the inhibition of cell-cell fusion in cells expressing CXCR4/CXCR2 chimeric receptors suggested that determinants for sensitivity of CXCR4 to T22 include the three extracellular loops of the coreceptor.

Amino Acid Sequence↗
PubMed1998

Intracellular localization and release of eotaxin from normal eosinophils.

Eotaxin is a potent and selective CC chemokine for eosinophils and basophils. We established several monoclonal antibodies (Mabs) allowing the neutralization and measurement of human eotaxin. Using the Mabs as probes, we demonstrated that normal eosinophils contained intracellular granule-associated eotaxin. Quantification of cell-associated eotaxin in different leukocyte subsets revealed that it was principally expressed in eosinophils. Finally, we showed that normal eosinophils released eotaxin upon stimulation with either of two secretagogues, C5a or ionomycin. These findings raise the possibility that eosinophil-derived eotaxin contributes to the local accumulation of eosinophils at the site of inflammation.

Animals↗
PubMed1998

Detection and delineation of CXCR-4 (fusin) as an entry and fusion cofactor for T-tropic [correction of T cell-tropic] HIV-1 by three different monoclonal antibodies.

A chemokine receptor, CXCR-4, has been identified as an entry cofactor for T cell line-tropic (T-tropic) HIV-1. To detect expression of CXCR-4 at the single cell level and dissect postbinding events of HIV-1 infection, we generated three mAbs against human CXCR-4. These mAbs inhibited SDF-1-induced intracellular Ca2+ mobilization, and one of the mAbs immunoprecipitated a specific 47-kDa component from CXCR-4+ cells. Flow cytometric analysis showed that most human cell lines examined expressed CXCR-4. A fraction of normal PBMC expressed CXCR-4, but neutrophils were negative. Two-color analysis revealed that the majority of T cells, virtually all B cells, and all monocytes expressed CXCR-4, while it was only weakly present on NK cells. Thus, expression of CXCR-4 is not ubiquitous but cell type specific in hemopoietic cells. The three mAbs were shown to suppress cell fusion mediated by envelope proteins of a T-tropic NL432 virus but not by those of an M-tropic JRCSF virus Likewise, they suppressed infection of NL432 but not that of an M-tropic NL162 virus. In both cases it was noted that the suppressive activity varied considerably among the mAbs. These data confirmed that CXCR-4 is directly involved in env-mediated entry and fusion of T-tropic HIV-1 and suggest that the epitopes on CXCR-4 recognized by the three mAbs may have different roles in interaction with the envelope proteins of T-tropic HIV-1.

Animals↗
PubMed1998

Involvement of human CRM1 (exportin 1) in the export and multimerization of the Rex protein of human T-cell leukemia virus type 1.

We investigated the role of human CRM1 (hCRM1) (exportin 1) in the function of Rex protein encoded by human T-cell leukemia virus type 1. hCRM1 promoted the export of Rex protein from the nucleus to the cytoplasm. A Rex protein with a mutation in the activation domain, RexM90, lost both the ability to bind to hCRM1 and the ability to multimerize. The overexpression of hCRM1 complemented the functional defects of RexM64, which contains a mutation in the multimerization domain of Rex. A dominant-negative mutant of Rex which sequesters cofactors of Rex abrogated multimerization as well as the activity of the wild-type Rex protein. These two functions were simultaneously restored by the overexpression of hCRM1. Taken together, these results suggest that hCRM1 plays important roles in the multimerization and export of Rex protein.

Biological Transport↗
PubMed1997

CXCR4/fusin is not a species-specific barrier in murine cells for HIV-1 entry.

Since some murine cells expressing human CD4 fail to internalize HIV-1, another block was thought to be located at the level of viral entry in addition to CD4. Recently, CXCR4 was shown to function as a coreceptor for T cell line-tropic HIV-1 entry. Here we demonstrated that cells expressing murine CXCR4 and human CD4 fused with cells expressing the env proteins derived from T cell line-tropic HIV-1 and were infected with T cell line-tropic HIV-1 strains. In contrast, the same cells were not infected with chimeric clones constructed by substitution of monocyte- or macrophage-tropic strain-derived env region or V3 region into T cell line-tropic HIV-1, indicating V3 loop of envelope protein is required for murine CXCR4mediated HIV-1 entry. We conclude that murine CXCR4 is not a species specific barrier to the entry of T cell line-tropic HIV-1.

3T3 Cells↗
PubMed1997

Functional expression of human mannan-binding proteins (MBPs) in human hepatoma cell lines infected by recombinant vaccinia virus: post-translational modification, molecular assembly, and differentiation of serum and liver MBP.

Human mannan-binding proteins (MBPs) occur in two forms, serum MBP (S-MBP) and liver MBP (L-MBP), both of which are synthesized in the liver from a single form of human MBP mRNA. To investigate further the mechanisms of post-translational modification, molecular assembly and differentiation of S-MBP and L-MBP in vitro, we expressed a full-length human MBP cDNA in three human hepatoma cell lines, using the vaccinia virus expression system. The expression of human MBP cDNA reproduced the native MBP differentiation of S-MBP and L-MBP in human hepatoma cells. The recombinant S-MBP was secreted into the medium, and the recombinant L-MBP retained in the cells. The former had the ability to activate the complement through the classical or lectin pathway but the latter did not. Furthermore, one notable difference between the two MBPs was the degree of oligomerization through interchain disulfide bonds between subunits. In addition, we showed that both S-MBP and L-MBP undergo hydroxylation of lysine and proline residues in collagen-like sequences, and that the hydroxylysine is glycosylated to form glucosylgalactosylhydroxylysine (GluGalHyl) and galactosylhydroxylysine (GalHyl). Hydroxylation was required for S-MBP to be assembled into large complexes, the apparent molecular sizes of which were estimated to be 200-1,300 kDa by SDS-PAGE under non-reducing conditions and gel filtration under non-denaturing conditions. The hydroxylation and subsequent glycosylation and oligomerization were inhibited by alpha,alpha'-dipyridyl, an inhibitor of collagen lysyl and prolyl hydroxylases. These results suggested that newly synthesized lectins undergo post-translational modifications unique to the two forms of MBP, S-MBP, and L-MBP, in human hepatocytes and hepatoma cells, and that the collagen-like domains of the MBPs play an important role in promoting molecular assembly.

Aged↗
PubMed1997

Long-term persistence of protective immunity in cynomolgus monkeys immunized with a recombinant vaccinia virus expressing the human T cell leukaemia virus type I envelope gene.

To develop effective vaccines against infection with human T cell leukaemia virus type I (HTLV-I), we constructed a recombinant vaccinia virus (WR-SFB5env) synthesizing the HTLV-I envelope (Env) gp46 protein under the control of a strong promoter, termed the ATI hybrid promoter. WR-SFB5env expressed a large quantity of gp46. In cynomolgus monkeys (Macaca fascicularis) immunized with WR-SFB5env, anti-HTLV-I Env antibody, including neutralizing antibody, was induced and remained at a high level until 136 weeks (2-6 years) post-infection (p.i.). These immunized monkeys had HTLV-I Env-specific cytotoxic T lymphocyte activity. At 136 weeks p.i., the immunized monkeys were challenged with an HTLV-I-producing cynomolgus T lymphocyte cell line. Neither HTLV-I antigen nor HTLV-I proviruses were detected in peripheral blood mononuclear cells, lymph nodes or spleens of the WR-SFB5env-immunized monkeys, in contrast to non-immunized control monkeys. These results indicate that a single immunization with WR-SFB5env induced prolonged humoral and cellular immune responses to HTLV-I and protected the monkeys against virus challenge.

Animals↗
PubMed1996

Identification of HTLV-1-specific CTL directed against synthetic and naturally processed peptides in HLA-B*3501 transgenic mice.

Previous studies of CTL responses to influenza peptides in HLA single transgenic mice resulted in the identification of at most one immunodominant epitope. Since HLA-B*3501 is known to present multiple HIV-1-specific T cell epitopes we tested the cellular immune response of HLA-B*3501 transgenic mice to synthetic HTLV-1 peptides mixed with the lipohexapeptide N-palmitoyl-S-[2,3-bis(palmitoyloxy)propyl]cysteinyl-seryl-lysyl-l ysyl- lysyl-lysine, which is a biocompatible, Th-epitopeindependent adjuvant. Eleven of 37 tested HLA-B*3501 binding peptides mounted a CTL response after three in vitro stimulations. The HLA-B*3501 affinity of peptides correlated with their ability to induce CTL in HLA-B*3501 transgenic mice. Seven peptides derived from env-gp46 (VPSPSSTPLL, VPSSSSTPL, YPSLALAPH, and YPSLALAPA), pol (QAFPQCTIL), gagp19 (YPGRVNEIL), and tax (GAFLTNVPY) proteins induced peptide-specific CTL Bulk CTL generated by four peptides derived from env-gp46 (SPPSTPLLY, VPSPSSTPLLY, and VPSPSSTPLL) and pol (QAFPQCTILQY) killed peptide-pulsed and recombinant vaccinia-infected target cells. The latter peptides therefore present T-cell epitopes and are vaccine candidates for our transgenic mouse model.

Animals↗
PubMed1996

Asymptomatic colorectal cancer detected by screening.

PURPOSE: Colorectal cancer screening has become prevalent. To discuss the efficacy of screening, we studied the characteristic of asymptomatic colorectal cancer detected by screening. METHODS: This is a retrospective review of patients with colorectal cancer treated at our institution. During the past 20 years, 96 of 1,046 cases of colorectal cancer were asymptomatic and detected by screening. Sixty-one of these cases were detected in the recent five years. The initial screening procedures were fecal occult blood test in 51 cases, sigmoidoscopy or colonoscopy in 18, barium enema in 9, and other tests in 18. RESULTS: Thirteen lesions (14 percent) were smaller than 1.0 cm and 32 (33 percent) were 1-2 cm in size. There were 34 Tis, 21 T1, and 8 T2 tumors. Of the 55 Tis or T1 lesions, 14 showed nonpolypoid growth (5 flat-elevated, 7 flat-elevated with depression, 1 flat, 1 depressed), and 12 of these were detected on endoscopy. Thirty-four cases were TNM Stage 0, 25 were Stage I, 16 were Stage II, 12 were Stage III, and 9 were Stage IV. Sixty-one percent of those detected by screening were in either Stage 0 or Stage I compared with 16 percent in the symptomatic group. Cumulative five-year disease-free survival rates were 100 percent for both Stage 0 and Stage I, 94 percent for Stage II, and 52 percent for Stage III. Overall cumulative five-year survival rate was 87 percent for those detected by screening, compared with 57 percent in symptomatic patients. CONCLUSIONS: Asymptomatic cancers detected by screening were at a less advanced stage. In particular, many nonpolypoid early cancers were detected by endoscopic screening.

Aged↗