Biomedical subjects

N Wakamiya

Publications and source records attributed to N Wakamiya.

At least 37 records · Page 2Linked to original sources

PubMed1997

Detection of proviruses and viral RNA in the early stages of feline immunodeficiency virus infection in cats: a possible model of the early stage of HIV infection.

Feline immunodeficiency virus (FIV) infection in cats has been reported to be a useful animal model for human AIDS studies, especially in the early stages of infection. We examined the temporal changes in provirus detection in peripheral blood mononuclear cells (PBMC) and the distribution of FIV-DNA and RNA in feline tissues by the polymerase chain reaction at 10, 35, 70 days after intravenous inoculation of FIV. Viral DNA in the PBMC was detected three to four weeks after infection and its fluctuation was demonstrated for the first time. Ten days after infection, before seroconversion, proviruses were detected only in the mesenteric lymph nodes and intestines. At 35 and 70 days after infection, after seroconversion, proviruses were detected in most lymphoid organs and the salivary glands, but the expression of FIV-RNA was limited to the thymus at 70 days after infection. These results show that FIV-RNA is transcribed from proviral DNA exclusively in the thymus at this stage. We suggest that the quantitative changes in detectable proviruses in the PBMC depend on the relation between the decrease in infected cells caused by cytolytic T lymphocytes and/or apoptosis and their increase caused by the release of a new supply of lymphocytes from the thymus.

Animals↗

PubMed1997

Contribution to the regulation of virus replication in cells latently infected with human immunodeficiency virus 1.

Monocytes/macrophages have been known to play an important role in the initiation and propagation of human immunodeficiency virus 1 (HIV-1) infection. To analyze the function of these cells during the clinical asymptomatic period of infection, we examined the effect of murine peritoneal macrophages and human peripheral blood macrophages on two cell lines latently infected with HIV-1, a promonocytic cell line, U1, and a T-cell line, ACH-2. Monokines of the murine peritoneal macrophages induced significant viral expression in U1, but not in ACH-2 cells. Experiments employing transient transfection of U937 and CEM cells with HIV long terminal repeat (LTR)-chloramphenicol acetyl transferase (CAT) plasmids indicated that the effect of these monokines was due to specific activation of the HIV LTR. In contrast, supernatants of human macrophages induced viral expression in both ACH-2 and U1 cells. These results suggest that several monokines are active in regulating the transition from the clinical asymptomatic period of HIV infection to progression to acquired immunodeficiency syndrome (AIDS).

Acquired Immunodeficiency Syndrome↗

PubMed1996

Recombinant bovine conglutinin, lacking the N-terminal and collagenous domains, has less conglutination activity but is able to inhibit haemagglutination by influenza A virus.

Conglutinin is a bovine serum protein which was first described as a vertebrate lectin. This protein belongs to the family of C-type lectins. These lectins are composed of four characteristic domains: (1) an N-terminal cysteine-rich domain, (2) a collagen-like domain, (3) a neck domain and (4) a carbohydrate recognition domain (CRD). Recently lectins have been shown to function as immunoglobulin-independent defence molecules due to a complement-mediated mechanism or opsonization. Our previous study showed that bovine conglutinin can inhibit haemagglutination by influenza A viruses and act by directly neutralizing them due to its lectin properties. In order to elucidate the biological role of the collagen-like domain, a recombinant partial conglutinin lacking this collagen-like domain was produced in an Escherichia coli system and its biological activities were examined. A 497 bp sequence, consisting of a short collagen region (two repeats of G-X-Y amino acid sequences), the neck domain, and the CRD of conglutinin cDNA, was amplified by the reverse-transcriptase PCR technique. The cDNA was transferred to a bacterial expression vector system (pRSET-A) and stable transfectants with a high level of conglutinin production were obtained. SDS/PAGE and Western blotting analyses showed a recombinant fusion protein of 27 kDa. Results of a cross-linking study and gel-filtration assay indicated that the recombinant conglutinin can form a trimeric structure and that it has sugar binding activity and specificity similar to that of native conglutinin. The recombinant conglutinin was also found to inhibit haemagglutination caused by influenza A virus as well as to possess less conglutination activity. These results suggest that in order for conglutinin to inhibit haemagglutination caused by the influenza virus, as well as to have sugar binding activity or to form trimers, it does not require the N-terminal and collagenous domains; however, they are essential for full conglutination activity.

Amino Acid Sequence↗

PubMed1996

Characterization of microsomal cytochrome P450 enzymes involved in the oxidation of xenobiotic chemicals in human fetal liver and adult lungs.

Levels and catalytic activities of cytochrome P450 (P450) enzymes involved in the oxidation of drugs and carcinogens were determined in human adult lungs and fetal livers and compared with those in microsomes from adult livers. P450s immunoreactive with anti-human P4501A1 and anti-human P4503A antibodies were detected in fetal liver microsomes by immunoblotting analysis, and P450s related P4501A1, 2A6, 2C9, 2E1, and 3A4 were determined in adult lung microsomes; all of these P450 enzymes were detected in much higher amounts in adult liver microsomes except that P4501A2 was only the 1A subfamily of P450 found in adult livers. Drug oxidation activities with the substrates ethoxyresorufin, coumarin, 7-ethoxycoumarin, bufuralol, and testosterone were determined in these microsomes, and we found that none of the activities were higher in microsomes of adult lungs and fetal livers than in adult livers. Activation of procarcinogens to reactive metabolites that induce umu gene expression in Salmonella typhimurium TA1535/pSK1002 or NM2009 was also examined and it was found that activities with (+)- and (-)-enantiomers of 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene were higher in fetal liver microsomes than adult lung or liver microsomes. The adult liver and lung activities for these two procarcinogens were similar on the basis of microsomal protein contents despite the fact that p450 contents are higher in liver than lung microsomes. alpha-Naphthoflavone, a known inhibitor of P4501A-related activities, did not affect these procarcinogen activation in fetal liver microsomes. Fetal liver microsomes catalyzed activation of aflatoxin B1 and sterigmatocystin, two procarcinogens known to be activated by P4503A4/7 in humans, although activation of carcinogenic arylamines that are good substrates for P4501A2 was much lower in microsomes of fetal livers and adult lungs than in adult livers. These results suggest that in human fetal livers at least two P450 enzymes, a form of P450 that is immunoreactive P4501A1 and P4503A7, are actually expressed and these enzymes are suggested as being involved in the activation of the (+)- and (-)-enantiomers of 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene and the carcinogenic mycotoxins, respectively. The exact nature of the former enzyme in fetal livers is unknown. In adult human lungs, several P450 enzymes are expressed, although the precise roles of these enzymes in the oxidation of xenobiotics were not determined due to the low level of expression of these P450s.

Antibodies↗

PubMed1996

Neutralization of human immunodeficiency virus type 1 (HIV-1) with antibody from carriers' plasma against HIV-1 protein p17.

It was investigated whether human antibody against HIV-1 protein p17 (anti-p17) in HIV carriers' plasma has the ability to neutralize the infectivity of HIV. By the pretreatment of HIV-1 with anti-p17 from HIV carriers, progeny HIV-1 production from cells infected with virus pretreated with anti-p17 was suppressed and/or delayed. The neutralizing activity of anti-p17 was decreased in the presence of recombinant p17. The latter obviously masked the neutralizing activity of anti-p17. The relevant epitope(s) on p17 is located apparently on the surface of HIV virions and the binding of anti-p17 to p17 impairs the infectivity of HIV. This implies that anti-p17, if stably present in HIV carriers' plasma, may also play an important role in reducing the infectivity of HIV-1 in vivo.

Antibody Specificity↗

PubMed1995

Expression of the T antigen on a T-lymphoid cell line, supT1.

We have measured glycosyltransferase activities of SupT1 cells, a T-lymphoid cell line shown to react with autoantibodies in the sera of many HIV patients. Since considerable alpha-N-acetylgalactosaminyl-transferase and beta 1, 3 galactosyltransferase activities were found in SupT1 cells, at least the O-glycan core 1 structure can probably be synthesized. FACS analysis using an anti-T monoclonal antibody showed expression of the T antigen (Gal beta 1-3 GalNAc). Glycoproteins with the T antigen were isolated by immunoprecipitation with the anti-T antibody from a SupT1 cell lysate labelled metabolically with 3H-glucosamine and then analysed by SDS-PAGE. It was revealed that the precipitate contained a glycoprotein with a molecular weight corresponding to that of leukosialin. O-glycans were prepared from the immunoprecipitate by alkaline-borohydride treatment and then fractionated on Bio-Gel P-2, GalNAcOH and Gal-GalNAcOH being identified inter alia. These results suggest that an anti-T antibody may be included in the autoantibodies found in HIV-1 infected individuals.

Antigens, CD↗

PubMed1993

Cloning and sequencing of a cDNA coding for bovine conglutinin.

A 912 bp bovine cDNA fragment encoding bovine conglutinin was amplified by the RT-PCR technique. cDNA clones encoding the bovine conglutinin were isolated from a bovine liver cDNA library using a specific probe obtained from the PCR product. These cDNAs carry an insert of 1113 bp coding for a protein of 371 amino acid residues with a signal peptide of 20 residues. The deduced amino acid sequence of cDNA agrees with that determined by conventional amino acid sequence analysis. Two polyadenylation signal sequences were detected in the DNA sequence downstream of the 3' end of the gene. Southern blot analysis of total bovine genomic DNA indicated that there is only one copy of the gene encoding bovine conglutinin. Northern blot analysis of bovine tissues showed that conglutinin mRNA of about 1.5 kb is expressed in the liver and also slightly in the lung.

Amino Acid Sequence↗

PubMed1993

Anti-tumor activity of ceramides and glycosphingolipids in a murine tumor system.

The anti-tumor activity of 7 sphingolipids, 2 ceramides and 5 glycosphingolipids against the syngeneic murine ascitic tumors MH134 and MM102 in C3H mice was examined. Five of these compounds showed anti-tumor activity against the tumors, ceramide type-IV (Cer-IV) having the highest activity without cytotoxic or cytostatic activity. These results indicate that the fatty acid in ceramide and sugar chains binding to it affect the anti-tumor activity in vivo. The anti-tumor activity of Cer-IV depended on the time of treatment. Mice treated with Cer-IV one day after tumor implantation showed the highest rate of survival. The cured mice were resistant to rechallenge with the same tumor (MH134-->MH134, MM102-->MM102) but not with a heterologous tumor (MH134-->X5563, MM102-->X5563), indicating that the effect of Cer-IV may be due to in vivo induction of specific immunity. Studies with various antibodies demonstrated that the anti-tumor effect of Cer-IV was inhibited by all the antibodies tested (L3T4, Lyt-2, and Thy-1,2 T cells, macrophages, and TNF alpha) in the induction phase (before Cer-IV administration) and by the antibodies of L3T4 and TNF alpha in the effector phase (after Cer-IV administration). Therefore, the anti-tumor effect of Cer-IV in this system depended on the host immune response rather than on its direct cytotoxic and/or cytostatic action.

Animals↗

PubMed1992

Isolation and characterization of conglutinin as an influenza A virus inhibitor.

Normal horse and guinea pig sera contain alpha 2-macroglobulin which inhibits the infectivity and hemagglutinating activity of influenza A viruses of the H2 and H3 subtypes. On the other hand, normal bovine serum contains a component termed beta inhibitor that inhibits the infectivity and hemagglutinating activity of influenza A viruses of the H1 and H3 subtypes. To investigate the nature of the beta inhibitor of influenza A virus, we purified the conglutinin and examined its characteristics. First, we found a high correlation between the hemagglutination inhibition(HI) titer and conglutinin titer in several bovine sera (r = 0.906, p less than 0.005). The HI of bovine serum was mainly dependent on conglutinin because the HI activity was abrogated by N-acetylglucosamine but not by D-mannose. The conglutinin, purified from bovine serum, had neutralizing-activity as well as HI activity on influenza A viruses of the H1 and H3 subtypes. The HI activity of conglutinin was heat stable (56 degrees C, 30 min), Ca(++)-dependent, and resistant to both neuraminidase and periodate treatments. The HI activity of purified conglutinin was blocked by N-acetylglucosamine but not by D-mannose. The conglutinin was bound to hemagglutinin which had high mannose and complex sugar chains and its binding was inhibited by N-acetylglucosamine and dependent on divalent cations. These data indicate that the beta-like inhibitor activity of bovine serum is mainly dependent on conglutinin which inhibits hemagglutination and neutralizes the virus infectivity by its binding to a carbohydrate site at the HA.

Animals↗

PubMed1992

Two chicken monoclonal antibodies specific for heterophil Hanganutziu-Deicher antigens.

Two chicken monoclonal antibodies (MAbs), HU/Ch2-7 and HU/Ch6-1, against heterophil Hanganutziu-Deicher (HD) antigens with N-glycolylneuraminic acid (NeuGc) at a terminal carbohydrate were established by cell fusions using chicken B cell lines lacking thymidine kinase and spleen cells from chickens immunized with II3NeuGc alpha-LacCer (HD3). The reactivities of these MAbs against several gangliosides including NeuGc-containing glycosphingolipids were examined by a thin-layer chromatography/immunostaining method. MAb HU/Ch2-7 (IgG) reacted strongly with HD3 and IV3NeuGc alpha-nLc4Cer (HD5) and weakly with VI3NeuGc alpha-nLc6Cer (HD7) and 4-O-acetyl-HD3. HU/Ch6-1 (IgG) reacted with HD3 and HD5, but did not react with the other HD antigens. The reactivities of these MAbs against HD antigen were greatly reduced by pre-treatment of the antigen with neuraminidase. These MAbs did not react with N-acetylneuraminic acid-containing gangliosides (GM1 and GM3). These results indicate that these two chicken MAbs are directed toward the antigenic epitope containing the NeuGc.

Animals↗

PubMed1992

A prospective study on correlation between the decrease in anti-P17 antibody level and progression to AIDS in asymptomatic carriers of HIV.

As the majority of human immunodeficiency virus (HIV) carriers are in asymptomatic stage for a long period of time, it is important to investigate the factors or surrogate markers for conversion from asymptomatic to symptomatic stage. Our study is designed to evaluate the relationship among virus isolation rate, anti-p17 antibody status and progression to AIDS. We studied anti-p17 antibody status along with virus isolation in 56 asymptomatic carriers and 46 AIDS cases. Progression to AIDS was markedly associated with high rate of virus isolation and loss of anti-p17 antibody. In order to know the meaning of loss of anti-p17 antibody during the clinical course, 15 anti-p17 antibody positive and 16 anti-p17 antibody negative cases were followed up prospectively for the development of AIDS. None of the anti-p17 antibody positive cases developed AIDS while 6 out of 16 anti-p17 negative cases developed AIDS during observation period (P < 0.05). Progression to AIDS was associated with loss of anti-p17 antibody. Identification of cases losing anti-p17 antibody in peripheral blood during asymptomatic period may help high-risk group who are in need of chemoprophylaxis. Moreover, study of anti-p17 antibody may be helpful in designing vaccine in future if it works as a neutralizing antibody to HIV in vivo.

Acquired Immunodeficiency Syndrome↗

PubMed1991

Expression of Hanganutziu-Deicher antigen in activated human T lymphocytes.

Hanganutziu-Deicher (HD) antigen is a heterophile antigen that is widely distributed in many animals other than humans and chickens and is highly immunogenic in humans and chickens. In the present study, we demonstrated expression of HD-antigenic glycoproteins in activated T lymphocytes by SDS-PAGE and immunoblotting. Treatment with IL-2 plus PMA induced 29kD glycoprotein antigen detected under reducing condition. It contained sialic acid epitope of HD antigen because of the expression being neuraminidase-sensitive. Treatment with PMA plus A 23187 or PHA treatment and then PHA plus IL-2 treatment also induced two proteins of Mr 50kD and 70kD. These expressions were not detected in all individuals examined. These results indicate that HD antigen is an activated T cell antigen and expressed as an isoantigen as it is expressed in cancerous tissues from some patients.

Antigens, Heterophile↗

PubMed1991

Tumor necrosis factor expression by human ovarian carcinoma in vivo.

Tumor necrosis factor (TNF) is a cytokine produced by monocytes and other cells with selective cytolytic activity against some but not all tumor cells. Cellular resistance to the cytolytic effects of TNF has been reported to be associated with autocrine production of TNF by the target cells. The purpose of this study was to determine whether or not human tumors produce tumor necrosis factor in vivo. Ovarian carcinoma tissue from 25 patients with ovarian carcinoma was examined for the presence of TNF. Four of 5 ascites fluid specimens and tissue sections of 16 of 20 patients were positive for TNF by immunoperoxidase staining. The source of the immunoreactive protein was further examined by in situ hybridization studies. TNF mRNA was detectable in each of the ascites specimens and 7 of 16 tissue sections that were positive by immunoperoxidase staining. These findings suggest that TNF is produced by some human tumors in vivo and that the association between TNF production and resistance to TNF antitumor action may be clinically relevant.

Ascites↗

PubMed1991

T cell receptor-independent cell-mediated cytotoxicity by nude mouse lymphokine-activated killer cells.

Lymphokine-activated killer (LAK) cells, which can lyse a variety of tumor cells, can be induced from both normal and athymic nude mouse spleen cells by culture with high doses of recombinant interleukin 2 (rIL-2). LAK cells generated from nude mouse spleen cells (Nude-LAK cells) express just Thy 1.2 antigen, but not CD4 and CD8 antigens. Nude-LAK cells express neither T3 molecule, T cell receptor (TCR) alpha beta nor TCR gamma delta on their cell surface. The lack of TCR expression on Nude-LAK cells was confirmed by the results of northern blot analysis. LAK cells generated from normal mouse spleen cells (Nor-LAK) express TCR alpha, beta transcripts, while Nude-LAK cells express only sterile TCR beta transcript, but not TCR alpha transcript. TCR gamma delta transcripts were scarcely detected in both Nor-LAK cells and Nude-LAK cells. Thus, it is strongly suggested that Nude-LAK cells can recognize and lyse tumor cells by TCR-independent mechanisms. Monoclonal antibody against lymphocyte function-associated antigen (LFA-1) molecule can block the cytotoxicity of Nude-LAK cells, indicating an important role of such accessory molecules in Nude-LAK cell-mediated cytotoxicity.

Animals↗

PubMed1990

Antitumor efficacy of vaccinia virus-modified tumor cell vaccine.

The antitumor efficacies of vaccinia virus-modified tumor cell vaccines were examined in murine syngeneic MH134 and X5563 tumor cells. UV-inactivated vaccinia virus was inoculated i.p. into C3H/HeN mice that had received whole body X-irradiation at 150 rads. After 3 weeks, the vaccines were administered i.p. 3 times at weekly intervals. One week after the last injection, mice were challenged i.p. with various doses of syngeneic MH134 or X5563 viable tumor cells. Four methods were used for preparing tumor cell vaccines: X-ray irradiation; fixation with paraformaldehyde for 1 h or 3 months; and purification of the membrane fraction. All four vaccines were effective, but the former two vaccines were the most effective. A mixture of the membrane fraction of untreated tumor cells and UV-inactivated vaccinia virus also had an antitumor effect. These results indicate that vaccine with the complete cell structure is the most effective. The membrane fraction of UV-inactivated vaccinia virus-absorbed tumor cells was also effective. UV-inactivated vaccinia virus can react with not only intact tumor cells but also the purified membrane fraction of tumor cells and augment antitumor activity.

Animals↗

PubMed1990

Heterogeneity of Hanganutziu-Deicher antigen glycoproteins in different species animal sera.

A heterophilic Hanganutziu-Deicher (HD) antigen is present in many animal sera except human and chicken sera. To visualize the antigenic molecules, nine species animal and human sera were analyzed by SDS-PAGE, followed by Western blotting and immunostaining with avain anti-N-glycolylneuraminyl-lactosyl ceramide antibody which recognizes the terminal N-glycolylneuraminic acid moiety of glycoconjugates as an epitope of the HD antigen. Several HD antigen-active glycoprotein bands were detected in the sera of fetal calf, calf, horse, goat, monkey, rabbit, guinea pig, rat and mouse, except for human serum. The HD antigenic proteins showed heterogeneities in their molecular weights and were not identical with any major band visualized with silver-staining, indicating that they are minor components of serum proteins in each animal. Neuraminidase treatment destroyed the antigenicity of all proteins, confirming that N-glycolylneuraminic acid (NeuGc) at the non-reducing terminal of carbohydrate chains is the antigenic epitope in serum glycoprotein molecules as already confirmed in glycosphingolipid (GSL) antigens. The finding of HD-antigenic glycoproteins in animal sera suggests that they also stimulate HD antibody production in patients who received animal antiserum for therapeutic aim.

Animals↗

PubMed1989

Detection of glycoproteins as tumor-associated Hanganutziu-Deicher antigen in human gastric cancer cell line, NUGC4.

NUGC4 cells derived from a human gastric cancer gave 6% Hanganutziu-Deicher (HD) antigen-positive cells by flow cytometric analysis using an affinity-purified chicken antibody to N-glycolyneuraminyl-lactosyl-ceramide (HD3 ganglioside). The cells showed no HD antigenic ganglioside by thin-layer chromatography enzyme-immunostaining; however, they were revealed to contain HD antigenic proteins with molecular masses of 150, 100, 90, 70, 65, 60, 47, and 40 kDa, by both immunoblotting after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoprecipitation of [35S]-methionine-labeled proteins, followed by SDS-PAGE and autoradiography. Neuraminidase treatment destroyed the antigenicity of all proteins, indicating that these molecules are glycoproteins and have N-glycolyneuraminic acid at the non-reducing terminal of carbohydrate chains as an HD antigenic epitope.

Antigens, Heterophile↗

PubMed1989

Tumor cells treated with vaccinia virus can activate the alternative pathway of mouse complement.

Vaccinia virus has been shown to render mouse tumor cells highly immunogenic. Since we have demonstrated that induction of complement activating capacity on guinea pig tumor cells by Sendai virus infection causes the tumor cells to become immunogenic, we assumed that vaccinia virus infection of mouse tumor cells might render them reactive with homologous mouse complement. Therefore, murine tumor cells, MH134 and X5563, infected with vaccinia virus (VV) were incubated with mouse plasma and C3 deposition was determined by staining with fluorescein isothiocyanate-labeled anti-C3. We found that VV-infected tumor cells possess the ability to activate the alternative complement pathway (ACP) of murine complement. For induction of complement activating ability, at least a 3 h incubation of the infected MH134 cells was required indicating that the generation of ACP-activating capacity on MH134 infected with VV is time-dependent. Furthermore, ultraviolet-irradiated vaccinia virus was able to induce ACP-activating capacity on tumor cells as well.

Animals↗