Biomedical subjects

T Kurata

Publications and source records attributed to T Kurata.

At least 145 records · Page 8Linked to original sources

PubMed1997

Enhancement of guanine-nucleotide exchange activity of C3G for Rap1 by the expression of Crk, CrkL, and Grb2.

Crk is an adaptor protein that consists almost entirely of SH2 and SH3 domains. We have previously demonstrated, by using in vivo and in vitro systems, that C3G, which was identified as a Crk SH3 domain-binding guanine nucleotide exchange factor, specifically activates Rap1. C3G also binds to other adaptor proteins, including CrkL and Grb2. In the present study, we analyzed the effect of Crk, CrkL, and Grb2 on the C3G-Rap1 pathway. Expression of Crk, CrkL, and Grb2 with C3G in Cos1 cells significantly increased the ratio of GTP/GDP bound to Rap1. Both the SH2 and SH3 domains of Crk were required for this activity. However, Crk did not stimulate the guanine nucleotide exchange activity of C3G for Rap1 in vitro, suggesting that Crk does not activate C3G by an allosteric mechanism. The requirement of the SH2 domain of Crk for the enhancement of guanine nucleotide exchange activity for Rap1 could be compensated for by the addition of a farnesylation signal to Crk, indicating that Crk enhanced the guanine nucleotide exchange activity of C3G by membrane recruitment of C3G. These results demonstrate that Crk, CrkL, and Grb2 positively modulate the C3G-Rap1 pathway primarily by recruiting C3G to the cell membrane.

Adaptor Proteins, Signal Transducing↗

PubMed1997

Generation of neutralizing antibody to the reverse transcriptase of human immunodeficiency virus type 1 by immunizing of mice with an infectious vaccinia virus recombinant.

Antibodies inhibiting the reverse transcriptase (RT) of human immunodeficiency virus type-1 (HIV-1) were found to be generated in the serum of mice repeatedly infected with a vaccinia virus recombinant, WRRT, expressing the enzyme. A monoclonal antibody (mAb), 7C4, which specifically and almost completely inhibits the RNA-dependent DNA polymerase activity of HIV-1 RT was produced from a mouse repeatedly immunized with WRRT. 7C4 seems to be specific for HIV-1 among retroviruses: 7C4 inhibited RT activity of three strains of HIV-1 (IIIB, Bru, and IMS-1) but not of two strains of HIV-2 (GH-1 and LAV-2) or two strains of SIV (MAC and MND). The immunoglobulin isotype of three out of four mAbs produced from spleen cells of the immunized mouse were IgG2a. This immunization method that avoids protein denaturation may preferentially induce a T helper type-1 immune response and increase the chances of producing the only occasionally obtainable mAb capable of recognizing a conformational epitope and completely inhibiting enzyme activity.

Animals↗

PubMed1997

Isolation and characterization of mouse nasal-associated lymphoid tissue.

A method for isolation of mouse nasal-associated lymphoid tissue (NALT), which is a principal mucosal lymphoid tissue of the respiratory tract in rodents, was developed. The paired lymphoid organs could be separated from the upper jaw by peeling away the palate where NALT was localized bilaterally on the posterior side. About 3 x 10(5) lymphocytes could be obtained from one NALT fragment. The NALT lymphocyte fraction from normal BALB/c mice contained T- and B-cells in about equal numbers, and contained about 4 times as many CD4+ T-cells as CD8+ T-cells when analyzed with a FACScan fluorescence analyzer. The composition of the NALT lymphocytes was similar to that of the lymphocytes from the portion of the nasal cavity remaining after isolation of the NALT. The NALT lymphocyte fraction from mice infected 7 days previously with influenza virus was also characterized. The numbers of NALT T- and B-cells from the infected mice were approximately 2 and 3 times higher than those of non-infected mice, respectively. In parallel with the cell increase, NALT lymphocytes produced IFN-gamma when cultured for 24 h and contained cells secreting influenza virus-specific IgA and IgG antibodies. The results suggest that this method can be successfully used for investigating cellular dynamics of mucosal immunology in the upper respiratory tract.

Animals↗

PubMed1997

Enhanced cytopathic effect of human cytomegalovirus on a retinal pigment epithelium cell line, K-1034, by serum-free medium.

Although human cytomegalovirus (HCMV) predominantly infects epithelial cells in vivo, the majority of studies of HCMV gene expression and replication have been conducted using non-epithelial cell lines in part because of the absence of a good experimental system using epithelial cells. To address the nature of epithelial cell infection, we investigated the susceptibility of an epithelial cell line (K-1034) established from the retinal pigment epithelium to HCMV infection. This cell line exhibited high susceptibility to HCMV, as evidenced by detection of one of the immediate early antigens, IE2, in the nuclei of more than 80% of K-1034 cells at 24 h following inoculation at a multiplicity of infection of 3 plaque forming units per cell. However, the yield after one-step growth of HCMV in K-1034 cells was about twenty-fold less than that in human embryonic lung fibroblast cells. Cytopathic effect (CPE) on K-1034 cells was not prominent in medium supplemented with 10% fetal bovine serum and viral late antigens were detected in less than 5% of K-1034 cells. Interestingly, infected cells expressing late antigens and exhibiting CPE were markedly increased in serum-free medium, even though the yield of infectious HCMV and viral genome copy numbers were almost the same in the different serum concentrations, due to viral instability in the absence of serum. Thus, the progression of late antigens expression and the induction of CPE in infected epithelial cells is influenced by physiological conditions, and are negatively regulated by some serum factor.

Antigens, Viral↗

PubMed1997

Isolation of a measles virus variant: protection of newborn mice from measles encephalitis by 24 h prior intracerebral inoculation with the variant.

A small plaque mutant with reduced neurovirulence in newborn mice was obtained from Edmonston strain measles virus after propagation for 5 months in NIH3T3 cells. It retained the antigenicity of the parental virus and tended to induce higher neutralizing antibody titers in the adult BALB/c mice. The intracerebral (but not intraperitoneal) inoculation of the live mutant virus one day before prevented the newborn BALB/c mice from encephalitis caused by the intracerebral challenge with the parental strain at a dose of 10-20 LD50. The intracerebral inoculation with the mutant virus whose replication capacity was inactivated by UV-irradiation was ineffective. The protection was not attributed to interferons nor to viral interference. The mechanism remains unknown.

3T3 Cells↗

PubMed1997

Suppression of delayed-type hypersensitivity and IgE antibody responses to ovalbumin by intranasal administration of Escherichia coli heat-labile enterotoxin B subunit-conjugated ovalbumin.

Oral administration of a small amount of antigen conjugated cholera toxin B subunit is known to induce tolerance to the antigen. In the present experiments, whether nasal administration of allergen conjugated to Escherichia coli heat-labile toxin B subunit (LTB) induced tolerance was examined in BALB/c mice. A single administration of a small amount of LTB-coupled ovalbumin (OVA) suppressed the induction of delayed-type hypersensitivity and IgE antibody responses to OVA which was administered parenterally after nasal administration of LTB-coupled OVA. The antigen-specific suppression was abrogated by the addition of the holotoxin to LTB-coupled OVA. The suppression, induced by nasal administration with a small amount of allergen conjugated to a mucosa-binding molecule, may be applicable for preventing the development of allergy.

Administration, Intranasal↗

PubMed1997

Effects of frequent intranasal administration of adjuvant-combined influenza vaccine on the protection against virus infection.

In previous papers, we have shown that Escherichia coli heat-labile enterotoxin B subunit, supplemented with a trace amount of the holotoxin (LTB*) could be used as a potent adjuvant for a nasal influenza HA (haemagglutinin) vaccine in humans. The present study was designed to determine whether the effectiveness of a combined LTB*-HA vaccine could be limited by preexisting immunity to LTB and how many times the adjuvant-combined vaccine could be administered intranasally without reducing its protective efficacy in BALB/c, C3H and B10 mice. The magnitude of both nasal and serum Ab responses to HA vaccine was correlated with the degree of protection against virus infection. Higher doses of LTB*-combined vaccine were required for inducing high enough levels of anti-HA Ab responses to provide complete protection in low responder mice. Repeated pretreatments with LTB* alone (more than six times), which provided high levels of preexisting Abs to LTB, inhibited the induction of anti-HA Ab responses and reduced the protective efficacy of the adjuvant-combined vaccine. However, the LTB*-combined vaccine could be given repeatedly (about ten times) to mice without reducing the effectiveness of the adjuvant-combined vaccine. These results suggest that the LTB*-combined nasal influenza vaccine can be given to humans once every few years when an epidemic of influenza may occur.

Adjuvants, Immunologic↗

PubMed1997

Phase I study of sequentially administered topoisomerase I inhibitor (irinotecan) and topoisomerase II inhibitor (etoposide) for metastatic non-small-cell lung cancer.

We conducted a phase I study of irinotecan (CPT-11) and etoposide (VP-16) given sequentially to untreated patients with metastatic non-small-cell lung cancer. Arm A: CPT-11 was given over 90 min on days 1-3 and VP-16 was given over 60 min on days 4-6. Arm B: VP-16 was given on days 1-3 and CPT-11 on days 4-6. G-CSF was given to all patients daily on days 7-17. Twenty-seven patients were entered randomly at the two arms. The major dose-limiting toxicities in arms A and B were granulocytopenia and diarrhoea. Transient elevations of transaminases and bilirubin were observed in both arms. The degree of the toxicities did not differ between the two arms. The maximum tolerated doses (MTDs) were 60 mg m-2 CPT-11 and 60 mg m-2 VP-16 in both arms. Of the 13 patients who received more than two cycles, two out of five achieved partial response (PR) at the first level of arm A and one out of four achieved PR at the second level of arm B. We conclude that these schedules of sequential CPT-11 and VP-16 administration were inappropriate because of severe toxicities.

Adult↗

PubMed1997

Unexpected correlation in the sensitivity of 19 herpes simplex virus strains to types I and II interferons.

We compared the sensitivity of 19 herpes simplex virus (HSV) strains to type I (IFN-alpha and IFN-beta) and type II (IFN-gamma) human interferons in cultures of human retinal epithelial (K-1034) and lung (HEL) cells. Their sensitivities proved to be well correlated, even though type I and type II IFN have been reported to have different antiviral actions. The correlation was not because IFN-gamma stimulated the formation of IFN-beta, for an antibody that neutralized IFN-beta did not reduce its inhibitory effects. Our results show that each HSV strain has a characteristic and similar sensitivity to type I and type II IFN and suggest some common pathway in the mechanism of their antiviral actions.

Cells, Cultured↗

PubMed1997

Interleukin-4 production in Epstein-Barr virus-transformed B cell lines from peripheral mononuclear cells of patients with atopic dermatitis.

Interleukin-4 (IL-4) is known as an immunomodulatory cytokine secreted by relatively few cell types, for example, activated T lymphocytes, basophils, and mast cells, but not by B cells. It plays an important role in promoting the production of the IgE antibody. We established Epstein-Barr virus (EBV)-positive B cell lines from peripheral blood mononuclear cells of patients with atopic dermatitis (AD) and tested the production of several cytokines in the cell lines. We found that IL-4 was produced in a cell line, OB, by an IL-4-specific enzyme-linked immunosorbent assay. IL-4 mRNA was detected in OB and two other AD-derived cell lines by IL-4-specific reverse transcription-polymerase chain reaction. IgE was also produced by the OB cells. The production of IL-4 and IgE was enhanced in the cells treated with 12-O-tetradecanoyl phorbol-13-acetate. This is the first evidence that IL-4 is produced by an EBV-transformed B cell line.

B-Lymphocytes↗

PubMed1997

Presence of a novel hamster oral papillomavirus in dysplastic lesions of hamster lingual mucosa induced by application of dimethylbenzanthracene and excisional wounding: molecular cloning and complete nucleotide sequence.

A combination of 9,10-dimethyl-1,2-benzanthracene (DMBA) application and excisional wounding on the lingual tips of Syrian Golden hamsters (Mesocricetus auratus) induces dysplastic and malignant mucosal lesions. Papillomavirus genus-specific antigen and viral particles, measuring 55 nm in diameter, were demonstrated in the nuclei of squamous cells of dysplastic lesions showing koilocytotic change. In this study, we cloned a circular genome at a single Kpnl site from one of these dysplastic lesions. The genomic sequence of this clone, consisting of 7647 bp, was shown to be that of a novel papillomavirus with a conserved genomic organization. We named the new virus hamster oral papillomavirus (HOPV). All dysplastic lesions induced by this combination of DMBA application and excisional wounding contained viral DNA. Although Southern blot hybridization analysis could not detect the HOPV genome, PCR analysis demonstrated the latent HOPV genome in the tongue and skin of an untreated hamster. These results suggest that latently present HOPV genome is reactivated by the DMBA/wounding procedures. Lingual HOPV infection may be an important model for gaining insight into the interactions between papillomavirus infection, chemical carcinogens and physical irritations in carcinogenesis or malignant transformation.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1997

[Efficacy of nasal influenza vaccine combined with Escherichia coli heat-labile enterotoxin B subunit containing a trace amount of the holotoxin in healthy volunteers].

We conducted a field trial to evaluate the efficacy of nasal influenza vaccine combined with Escherichia coli heat-labile enterotoxin B subunit (LTB) containing a trace amount of the holotoxin (LT) in preventing or attenuating influenza among volunteers during the winter season of 1994-1995. A trivalent inactivated influenza vaccine, composed of A/Yamagata/32/89 (H1N1), A/Kitakyusyu/159/93 (H2N2) and B/Mie/1/93 influenza virus strains, was administered intranasally together with recombinant LTB containing 1% recombinant LT (LTB*). Vaccination was done twice 4 weeks apart. Salivary secretory IgA and serum HI antibodies were measured before and 8 weeks after the primary vaccination. Thirty-two volunteers were enrolled in this study; 18 volunteers (mean age 37.7 +/- 11.3) were given LTB*-combined vaccine and 14 volunteers (mean age 44.1 +/- 11.3) given placebo. Outbreaks of H3N2 subtype and B type virus were observed during this study period. Six (42.9%) of the 14 volunteers in the placebo group and 3 (16.7%) of the 18 receiving the LTB*-combined vaccine contracted influenza. There was no statistically significant difference between the two groups, because the number of subjects was small. Higher percentage of positive IgA and HI antibody responses among vaccines given vaccine with LTB* were observed as compared with those in the placebo group. Positive IgA antibody response to all vaccine strains were observed in 46.7% (7/15) of the vaccine group. On the other hand, none of the placebo group showed positive IgA antibody response to all vaccine strains. These results suggest that nasal influenza vaccine with LTB* appears to be effective in preventing influenza.

Administration, Intranasal↗

PubMed1997

Angiomyolipoma: imaging findings in lesions with minimal fat.

PURPOSE: To investigate a method of diagnosing angiomyolipoma that contains minimal fat. MATERIALS AND METHODS: In six cases of angiomyolipoma with minimal fat, the attenuation on contrast material-enhanced and unenhanced computed tomographic (CT) images, the echogenicity on sonograms, the signal intensity on T2-weighted magnetic resonance (MR) images, and the gross configuration of the lesion were retrospectively analyzed. In 100 cases of renal cell carcinoma, the same parameters were analyzed, and results were compared with those of angiomyolipoma. RESULTS: When compared with the surrounding renal parenchyma, all six angiomyolipomas showed homogeneously high attenuation on unenhanced CT images, homogeneous enhancement on contrast-enhanced CT images, and homogeneous isoechogenicity on sonograms. Of the five angiomyolipomas examined with MR imaging, four were hypointense and one was isointense on T2-weighted images. All six angiomyolipomas protruded from the renal margin. None of the 100 renal cell carcinomas showed homogeneously high attenuation on unenhanced CT images, homogeneous enhancement on contrast-enhanced CT images, or homogeneous isoechogenicity on sonograms. CONCLUSION: In the kidney, homogeneously high attenuation on unenhanced CT images, homogeneous enhancement on contrast-enhanced CT images, and homogeneous isoechogenicity on sonograms are suggestive of angiomyolipoma that contains abundant muscle and minimal fat.

Adipose Tissue↗

PubMed1997

Role of Crk oncogene product in physiologic signaling.

v-Crk is a member of the family of adaptor-type signaling molecules that consist mostly of SH2 and SH3 domains. The cellular homologs of v-Crk includes CrkI, CrkII, and CrkL; these have been isolated from species ranging from lower vertebrates to man. Crk-family proteins are involved in a variety of signaling cascades such as those of growth factor receptor, integrin, T-cell receptor, B-cell antigen receptor, and cytokines. It has been postulated that the primary function of Crk is to recruit cytoplasmic proteins in the vicinity of tyrosine kinases through SH2-phosphotyrosine interaction. Thus, the output from Crk depends on the SH3-binding proteins, which include the C3G guanine nucleotide exchange protein for Rap1, Abl tyrosine kinase, DOCK180, and the Sos guanine nucleotide exchange protein for Ras. The variety of the Crk-binding proteins indicate the pleiotropic function of Crk.

Animals↗

PubMed1997

Activities of D- and L-xyloascorbic acid and D- and L-araboascorbic acid as a cofactor for dopamine beta-hydroxylase reaction.

L-Xyloascorbic acid (L-xylo-AsA) and its three stereoisomers, D-xyloascorbic acid (D-xylo-AsA), L-araboascorbic acid (L-arabo-AsA) and D-araboascorbic acid (D-arabc-AsA), have been considered to show some differences in vitamin C activity. In this paper the effect of L-xylo-AsA, D-xylo-AsA, L-arabo-AsA and D-arabo-AsA on the activity of dopamine beta-hydroxylase was studied to clarify whether or not the structural specificities of these stereoisomers have different effects on enzyme activity. The maximum velocity (Vmax) of the hydroxylation and Km for ascorbic acid were calculated using double-reciprocal plotting. Vmax for L-xylo-AsA was estimated to be 201 nmol/min/mg protein and those of D-xylo-AsA, L-arabo-AsA and D-arabo-AsA were 157 nmol/min/mg protein, 112 nmol/min/mg protein and 194 nmol/min/mg protein, respectively. Km for L-xylo-AsA was 1.5 mM and those for D-xylo-AsA, L-arabo-AsA and D-arabo-AsA were 2.3 mM, 2.7 mM and 1.4 mM, respectively. The effect of D-arabo-AsA on the activity of dopamine beta-hydroxylase was almost the same as that of L-xylo-AsA, while D-xylo-AsA and L-arabo-AsA showed smaller effects. Our results suggest that the configuration at carbon 4 might be more important than that of the hydroxyl group at carbon 5 for the development of the activity as a cofactor for dopamine beta-hydroxylase reaction.

Animals↗

PubMed1997

Distribution of herpes simplex virus types 1 and 2 genomes in human spinal ganglia studied by PCR and in situ hybridization.

Clinical data indicate that the recurring herpes simplex virus (HSV) from oro-labial lesions is HSV subtype 1 and that the virus from genital lesions is HSV-2. This suggests that HSV-1 and HSV-2 reside in latent forms in the trigeminal ganglia and sacral ganglia, respectively. However, the distribution of latent HSV-1 and HSV-2 infections in human spinal ganglia has not been fully examined. This report concerns the application of polymerase chain reaction (PCR) and in situ hybridization (ISH) to such a study. By using PCR and employing the respective primers, HSV-1 and HSV-2 DNAs were detected in 207 of 524 samples from 262 spinal ganglia (from the cervical to the sacral ganglia) examined on both sides. The percentages of HSV-1 and HSV-2 detected in a given set of ganglia were similar, indicating an absence of site preference. By ISH, few but positive hybridization signals were detected evenly in sacral ganglia sections. The data suggest that regional specificity of recurrent HSV infections is not due to regional distribution of latent virus, but that local host factors may be important for recurrences.

Adult↗

PubMed1997

The role of topoisomerase I in HIV-1 replication.

The mechanisms involved in the restriction of the cellular tropism of HIV-1 to cells of primate origin remain to be clearly defined. However, a number of studies have shown that this is determined not only at the level of the cellular receptor(s) or virus entry, but at a number of additional and later stages in virus replication. We have recently reported that the reverse transcription of HIV-1 RNA is markedly enhanced by the association of the gag encoded nucleocapsid p15 protein and cellular topoisomerase 1. In the present study we have now investigated if the recruitment of cellular topoisomerase I during virus replication is important in determining the cellular tropism of HIV-1. Employing a stable murine cell line, L929, expressing both human CD4 and topoisomerase I, it could be demonstrated that effective proviral DNA synthesis occurred following infection. In contrast in cells expressing only human CD4 proviral DNA synthesis was not detected. In addition we have co-expressed fusin, a protein known to act as an accessory factor as the virus entry stage in infection of T cell tropic HIV-1, to support viral entry completely. However no progeny virus could be detected after HIV-1 infection. These results suggest that reverse transcription in vivo is critically dependent on the presence of cellular topoisomerase I, and support the view that involvement of this enzyme is in HIV-1 replication. Moreover the findings suggest that other factors which remained to be identified, are involved in restricting HIV-1 replication in non-primate cells.

Animals↗

PubMed1997

The role of topoisomerase I in HIV-1 replication.

The mechanisms involved in the restriction of the cellular tropism of HIV-1 to cells of primate origin remain to be clearly defined. However, a number of studies have shown that this is determined not only at the level of the cellular receptor(s) or virus entry, but at a number of additional and later stages in virus replication. We have recently reported that the reverse transcription of HIV-1 RNA is markedly enhanced by the association of the gag encoded nucleocapsid p15 protein and cellular topoisomerase I. In the present study we have investigated if the recruitment of cellular topoisomerase I during virus replication is important in determining the cellular tropism of HIV-1. Employing a stable murine cell line, L929, expressing both human CD4 and topoisomerase I, it could be demonstrated that effective proviral DNA synthesis occurred following infection. In contrast in cells expressing only human CD4, proviral DNA synthesis was not detected. In addition we have co-expressed fusin, a protein known to act as an accessory factor as the virus entry stage in infection of T cell tropic HIV-1, to support viral entry completely. However no progeny virus could be detected after HIV-1 infection. These results suggest that reverse transcription in vivo is critically dependent on the presence of cellular topoisomerase I, and support the view that involvement of this enzyme is important in HIV-1 replication. Moreover the findings suggest that other factors which remained to be identified, are involved in restricting HIV-1 replication in non-primate cells.

Animals↗