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K Hamajima

Publications and source records attributed to K Hamajima.

At least 55 records · Page 3Linked to original sources
PubMed1994

Expression and characterization of the alpha 2 subunit of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-selective glutamate receptor channel in a baculovirus system.

Using a baculovirus expression vector system, the alpha 2 subunit of the mouse alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-selective glutamate receptor (GluR) channel was expressed in Spodoptera frugiperda insect cells. Immunoblotting using the antibody made to the synthetic peptide corresponding to the C-terminus of GluR alpha 2 and [35S]methionine/[35S]cysteine metabolic radiolabeling revealed the major 102-kDa and the minor 98-kDa protein bands. Metabolic radiolabeling with tunicamycin suggested that the two bands correspond to glycosylated and unglycosylated forms, respectively. The recombinant GluR alpha 2 proteins expressed in insect cells were also identified by immunofluorescence staining. The results of [3H]AMPA binding assay using whole cells suggested that, in infected Sf21 cells, binding sites of the GluR alpha 2 proteins were possibly located on the extracellular side. Scatchard analysis of AMPA binding showed the following parameters: Kd = 16 nM, Bmax = 1.9 x 10(5) binding sites per cell or 1 pmol/mg protein in the total particulate fraction. The ligand binding characteristics of the receptors expressed in insect cells were examined. From the effect of various agonists on [3H]AMPA binding of the receptors expressed in insect cells, the rank order potency of agonists was quisqualate > AMPA > L-glutamate > kainate. Thus, the baculovirus-insect cell expression system provides high-efficiency expression of the receptor sufficient to permit structural and functional analyses.

Animals↗
PubMed1994

Ligand-binding properties and N-glycosylation of alpha 1 subunit of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate(AMPA)-selective glutamate receptor channel expressed in a baculovirus system.

The alpha 1 subunit of the mouse alpha-amino-3-hydroxy-5-methyl-4-isoxazole- propionate(AMPA)-selective glutamate receptor channel has been expressed in insect Spodoptera frugiperda cells using a baculovirus system. The recombinant receptor proteins were identified by immunocytochemical detection, Western-blot analysis, and [35S]methionine/[35S]cysteine metabolic labeling experiments. The effect of tunicamycin on the metabolic labeling and immunoblots suggested that the two products, a major protein species of approximately 104 kDa and a minor species of approximately 100 kDa, correspond to glycosylated and non-N-glycosylated forms, respectively, which was also supported by the enzymic deglycosylation experiments. The lack of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate-binding activity of non-N-glycosylated glutamate receptor expressed in the presence of tunicamycin suggested that N-glycosylation is required, directly or indirectly, for functional expression in insect cells for ligand binding. Scatchard analysis of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate binding showed a single binding site with Kd 30 nM and a Bmax value of 2.6 x 10(5) binding sites/cell or 1.5 pmol/mg protein in the total particulate fraction. Among the compounds tested in the competition studies, beta-(3,5-dioxo-1,2,4-oxadiazolidin-2-yl)-L-alanine (quisqualate) was the most potent inhibitor of the 3H-labeled alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate binding (IC50 = 30 nM), followed in decreasing order by alpha-amino-3-hydroxy-5- methyl-4-isoxazole propionate, L-glutamate, 6,7-dinitroquinoxaline-2,3-dione, 6-cyano-7-nitroquinoxaline-2,3-dione, and 2-carboxy-4-(1-methylethenyl)-3-pyrrolidineacetate (kainate). Thus, in this study we present detailed analysis of alpha-amino-3-hydroxy-5-methyl-4- isoxazole-propionate-binding activity of the homomeric (single subunit) glutamate receptor channel of mouse alpha 1 subunit and discuss possible roles of N-glycosylation of the glutamate receptor channel alpha 1 subunit.

Amino Acid Sequence↗
PubMed1993

Strong immunogenicity of a multicomponent peptide vaccine developed with the branched lysine oligopeptide method for human immunodeficiency virus infection.

We synthesized one V3 peptide each from HTLV-IIIB, Thai A and Thai B, conjugating them to the T cell epitope of the env region, and we also synthesized a p17 protein peptide of the gag region (HGP-30). These peptides were then coupled to 8-lysine copolymers using N-succinimidyl maleimido carboxylate (M(r) = ca 60,000). We designated this the branched lysine oligopeptide method. The large peptide complexes constructed from these four macromolecular peptides were used with aluminium hydroxide or complete Freund's adjuvant to immunize mice and rabbits four times. ELISA assay showed high titres of anti-peptide antibodies to each V3 loop peptide and the HGP-30 peptide. Strong inhibition of CD4+ dependent cell fusion was obtained with these antisera when IIIB, Thai A and Thai B strains of the human immunodeficiency virus (HIV) were used. Strong anti-fusion inhibition was also observed with two other HIV strains. In addition, an increase of the anti-HIV effect was observed when we used sera obtained by multicomponent vaccine immunization. The same kind of inhibition was also observed in p24 assay systems using these immunized antisera. Activation of IL-2 production in lymphocytes was observed in mice immunized with this vaccine. These results suggest that immunization with macromolecular peptide complexes can result in strong immunogenicity towards HIV-1.

AIDS Vaccines↗
PubMed1993

Strong synergistic effects of multicomponent vaccine for human immunodeficiency virus infection.

Several peptides were synthesized, based on sequences of the HTLV-IIIB, HTLV-IIRF, and HTLV-IIIMN strains. These corresponded to the V3 region, the killer T cell activating site, the constant region of env, gag region protein and the helper T cell activating site. Some of these peptides were coupled to keyhole limpet hemocyanin (KLH), and some of them were polymerized with m-maleimido benzoyl-N hydroxysuccinimide ester (MBS). These peptides were used to immunize rabbits and mice. Antisera from immunized animals showed good levels of inhibition of CD4-dependent cell fusion by in vitro HIV infection. The antisera also inhibited the production of p24 protein in the HIV-infected culture system. Interestingly, a strong synergistic HIV growth inhibition by antiserum was observed when we immunized animals with multicomponent vaccine. In addition, substantial levels of HIV antigen-specific IL-2 producing and interferon-gamma (IFN-gamma) producing cells were also observed in lymph node cells from vaccinated mice. These results suggest that immunization with multicomponent vaccine can induce high levels of humoral antibodies as well as activate HIV-specific cellular immunity.

AIDS Vaccines↗
PubMed1991

Detection of elastase production in Escherichia coli with the elastase structural gene from several non-elastase-producing strains of Pseudomonas aeruginosa.

The elastase structural gene from Pseudomonas aeruginosa IFO 3455 has been cloned and sequenced. Using this gene as a probe, we cloned the DNA fragments (pEL3080R, pEL10, and pEL103R) of the elastase gene from non-elastase-producing strains (P. aeruginosa IFO 3080, N-10, and PA103 respectively). These three Pseudomonas strains showed no detectable levels of elastase antigenicity by Western blotting (immunoblotting) or by elastase activity. When elastase structural genes about 8 kb in length were cloned into pUC18, an Escherichia coli expression vector, we were able to detect both elastase antigenicity and elastolytic activity in two bacterial clones (E. coli pEL10 and E. coli pEL103R). However, neither elastolytic activity nor elastase antigenicity was detected in the E. coli pEL3080R clone, although elastase mRNA was observed. The partial restriction map determined with several restriction enzymes of these three structural genes corresponded to that of P. aeruginosa IFO 3455. We sequenced the three DNA segments of the elastase gene from non-elastase-producing strains and compared the sequences with those from the elastase-producing P. aeruginosa strains IFO 3455 and PAO1. In P. aeruginosa N-10 and PA103, the sequences were almost identical to those from elastase-producing strains, except for several nucleotide differences. These minor differences may reflect a microheterogeneity of the elastase gene. These results suggest that two of the non-elastase-producing strains have the normal elastase structural gene and that elastase production is repressed by regulation of this gene expression in P. aeruginosa. Possible reasons for the lack of expression in these two strains are offered in this paper. In P. aeruginosa IFO 3080, the sequence had a 1-base deletion in the coding region, which should have caused a frameshift variation in the amino acid sequence. At present, we have no explanation for the abnormal posttransciptional behavior of this strain.

Amino Acid Sequence↗
PubMed1990

Molecular cloning and partial DNA sequencing of the collagenase gene of Vibrio alginolyticus.

DNA fragments Vibrio alginolyticus chemovar iophagus, at least 7 kb in length, were ligated to Escherichia coli expression vectors. Three clones of Escherichia coli HB101 (pLCO-1, pLCO-2, pLCO-3) were obtained by the colony immunoblotting method using anti-collagenase antibody. In Escherichia coli, all these genes produced collagenase antigens which were detected with Western blotting. The amino acid sequence of chemically purified collagenase fragments was also analyzed. An approximately 2.5 kb DNA fragment of the pLCO-1 clone was sequenced, and we found that portions of the deduced amino acid sequence of the chemically analyzed fragments. Therefore, it is highly probable that the gene studied in the present experiment is truly a collagenase structural gene.

Amino Acid Sequence↗
PubMed1988

Trans-activation of class II (I-A alpha) gene by I-A beta gene transfection using bovine papilloma virus as a shuttle vector system.

We have engineered a bovine papilloma virus to carry the class II MHC (A beta d) gene. The recombinant plasmid was introduced into various mouse B cell hybridomas by co-transfecting with the neomycin-resistance gene. Several transfectants which received only the beta-chain gene of I-Ad, expressed I-Ad proteins on the cell surface. Their presence was determined by direct immunofluorescence and by Northern blot hybridization as well as RNA dot blot hybridization. These I-Ad molecules could induce IL-2 production in I-Ad restricted T cell hybridomas. When I-Ad molecules are expressed on the cell surface of the transfectants, they become fully active in inducing the I-Ad restricted T cell response. The original B cell hybridomas used for transfection possessed the alpha and beta chain genes of the I-Ad molecule but there are no transcripts of these genes in the cell. We suppose that many copies of the transfected beta-chain gene or its product may also induce production of cellular alpha-chain gene products and thereby induce expression of the I-Ad molecule on the cell surface.

Animals↗
PubMed1988

Detection of specificity of a new antigen in Candida tropicalis and its evaluation by taxonomic DNA analyses.

Monospecific factor serum for identifying Candida tropicalis was obtained either from rabbit antiserum to heated cells of C. tropicalis M 1519 (S 96) or from antiserum to C. tropicalis IFO 1400, by adsorption with heated cells of Candida albicans serotype A, or C. albicans (A) and Candida krusei, respectively. We designated this adsorbed serum factor t serum. The monospecific factor serum reacted with 31 out of 32 strains of C. tropicalis, only when tested on heat-treated cell antigens, whereas it did not react with any of 72 strains of the six other medically important species of Candida. The morphological and physiological characteristics of the one strain of C. tropicalis that did not react with the factor t serum, designated the t- -strain, were shown to be similar to those of the type strain of C. tropicalis by most of the methods employed for identifying Candida. Therefore, cell wall mannan from the t- -strain was compared with that from several typical strains of C. tropicalis for its specificity by the precipitation reaction and also for its 1H-nuclear magnetic resonance spectrum. The results showed that these mannans are similar to each other serologically and physicochemically, suggesting that the new antigen t is not mannan. Taxonomic characterization of the t-- and several typical strains of C. tropicalis was carried out by determining the mol% G+C of their DNA and also their DNA homology. Although the mol% G+C values of four typical strains of C. tropicalis were fairly similar (35.2 to 36.2 mol% by the Tm method and 35.5 to 36.4 mol% by the HPLC method), the t- -strain had a G+C content of 44.1 (Tm) and 43.3 (HPLC) mol%. Furthermore, the DNAs of the t- -strain and the type strain of C. tropicalis showed only 18.2% relatedness. These results suggest that the antigen corresponding to serum factor t exists only in the cell wall of C. tropicalis strains, not in those of the other medically important Candida, and that the t- -strain should not be classified as C. tropicalis. In conclusion, the taxonomic value and usefulness of factor t serum is primarily for differentiating C. tropicalis from C. albicans serotype A serologically.

Antigens, Fungal↗
PubMed1986

Effects of two cannabinoids on hepatic microsomal cytochrome P-450.

The effects of cannabidiol (CBD) and delta 9-tetrahydrocannabinol (delta 9-THC) on the synthesis and degradation of hepatic microsomal cytochrome P-450 were studied in mice. Cannabinoids used (10, 50 and 100 mg/kg, i.p.) did not affect delta-aminolevulinic acid synthetase activity in the liver. delta 9-THC-treatment (10, 50 and 100 mg/kg, i.p.) markedly stimulated heme oxygenase activity in hepatic 18000 X g supernatant fractions in a dose-dependent manner, whereas CBD-treatment was without effect. In vitro experiments, CBD and delta 9-THC (40 to 160 microM) markedly inhibited nicotinamide adenine dinucleotide phosphate (NADPH)-induced lipid peroxidation in hepatic microsomes. When CBD was incubated with the hepatic microsomes in the presence of an NADPH-generating system, cytochrome P-450 content decreased significantly. However, delta 9-THC showed no effects in similar experiments. The rate of decrease in the cytochrome P-450 content using CBD (160 microM) was 0.212 nmol/mg protein/20 min in microsomes from control mice. This value increased significantly in microsomes from phenobarbital-treated mice (0.792 nmol/mg protein/20 min) but not in those from 3-methylcholanthrene-treated mice (0.190 nmol/mg protein/20 min). The metabolic rate (per nmol cytochrome P-450) of CBD was also increased significantly by phenobarbital-treatment but not by 3-methylcholanthrene-treatment. These results suggest that CBD metabolites rather than CBD itself, play some role in the decreasing effect on cytochrome P-450 content in the hepatic microsomes in vitro, and that the microsomal formation of reactive metabolite of CBD is increased by phenobarbital-treatment.

5-Aminolevulinate Synthetase↗
PubMed1986

Central effects of N-allyl,N'-methyl-barbital and N-allylmethyprylon in mice.

Pharmacological activities (hypnotic, anticonvulsant and convulsant activities, acute toxicity and pentobarbital (PB) synergism) of N-allyl,N'-methyl derivatives of barbiturates and related N-allyl substituted compounds were studied in mice. N-Allylhexobarbital (AHB), N-allylglutethimide (AGI) and N-allylmethyprylon (AMP) showed hypnotic activity. N-Allyl,N'-methyl derivatives of barbital (B), phenobarbital (PheB) and PB (AMB, AMPheB and AMPB), AHB and AMP exhibited anticonvulsant activity against pentylenetetrazol (PTZ)-induced seizures. AMP was the most potent agent among the allyl compounds tested in the protective effect against PTZ-induced seizures. N-Allyl, N'-methylallobarbital (AMAlloB) showed a weak convulsant activity at a dose of 640 mg/kg, i.p. All allyl compounds tested showed potentiation of the PB-induced sleep. AMB prolonged the PB-induced sleep 30-fold as compared with the control. These results indicate that AMB, AMP and the other N-allyl compounds possess some depressant effects on the central nervous system.

Allyl Compounds↗
PubMed1985

Cross-tolerance to the hypothermic effect of delta 8-tetrahydrocannabinol 11-hydroxy-delta 8-tetrahydrocannabinol and chlorpromazine in the mouse.

When a 5 mg/kg i.v. dose of delta 8-tetrahydrocannabinol (delta 8-THC) and 11-hydroxy-delta 8-tetrahydrocannabinol (11-OH-delta 8-THC) was administered daily to mice, tolerance to their hypothermic effects developed quickly. Cross-tolerance to the hypothermic effect also developed between delta 8-THC and 11-OH-delta 8-THC. Tolerance development was dose-dependent and dose-effect experiments indicated that the magnitude of the tolerance developed to 11-OH-delta 8-THC was greater than that to delta 8-THC. Both cannabinoids were cross-tolerant to chlorpromazine in the hypothermic effect, but not to morphine, pentobarbital and reserpine under the conditions used. The present results suggest that 11-OH-delta 8-THC has some role in the development of tolerance to the hypothermic effect of delta 8-THC in mice.

Animals↗
PubMed1978

Screening of anti-B cell antibodies in the sera of renal transplant recipients by using Daudi cell line.

In order to examine the presence of antibodies against B cells, 83 sera obtained from 55 renal transplant recipients were screened by microcytotoxicity test using Daudi cell line. Out of 83 samples, 12 were pretransplant sera, 62 were post-transplant sera and 9 were from the patients whose grafts has been removed due to rejection. For the purpose of improving the reliability of the tests, 5 different rabbit complements were selected and cytotoxicity tests with them were carried out simultaneously. Pretransplant sera without blood transfusion did not show any cytotoxicity against Daudi cells. Although 42% of post-transplant sera showed cytotoxicity against Daudi cells, cytotoxic activity of the sera showed no relation with the occurrence of rejection episodes. Three serum samples out of 5 patients whose grafts had been removed due to rejection showed strong cytotoxicity against Daudi cells and the sera of 2 other patients did not. There was no relation between the cytotoxicity against Daudi cells and antibodies against Epstein-Barr virus capsid antigen in the post-transplant sera.

Animals↗
PubMed1978

A histopathological study of lymphoid tissue reaction to metastatic nasopharyngeal carcinoma in nude mice.

In attempts to heterotransplant human NPc into nude mice, using seven cultured cell lines, we have succeeded in growing a carcinoma simplex, composed of Epstein-Barr virus-determined nuclear antigen-positive and Epstein-Barr virus genome-positive cancer cells, at the injected site with two of the cell lines. These originated from a spindle-cell carcinoma (Chinese NPC-204) and from a combined-cell carcinoma (Chinese NPC-501), respectively. During the first few passages, wandering macrophages were prevalent and increased in number in response to the presence of the tumours. In conjunction with a gradual decrease in the number of wandering macrophages in the medullary sinuses, diffuse hyperplasia of lymphocytes occurred in regional lymph nodes. As a result of the release of lymphocytes and macrophages into the peripheral lymph nodes, the spleen underwent extensive change, as manifested by the collapse of the splenic cords and the formation of septa studded with granulomas. Under these conditions of immunosuppression, lymphatic metastases were observed during the periods between the 11th and 14th generations and the 24th and 30th generations with NPC-204 and between the 9th and 14th generations with NPC-501. The neighbouring lymph nodes, like the spleen, were often studded with epithelioid-cell granulomas, formed by the aggregation of macrophages around nuclear debris in the subcortical areas. We assume that the clumps of debris are the remnants of metastatic cancer cells which were probably killed by macrophages or by sensitized lymphocytes. If the lymph nodes contain a barrier of granulomas, they are not invaded by tumour cells from the cortical sinuses, except in the rare case of retrograde metastasis from the hilus. It would appear that macrophages can replace T lymphocytes, which are found in very small numbers in the nude mice used in this study, in killing tumour cells and, furthermore, in protecting the lymph nodes from the spread of metastases. Metastasis cannot occur in these nude mice when their lymphoreticular system, especially that of the spleen, is working in a stable balance.

Animals↗
PubMed1978

Augmented mitotic response of human peripheral lymphocytes in the presence of lymphocytes from nude mice: detection of a small number of functional human lymphocytes in the explanted host.

In the present paper, authors showed a method to detect a small number of functional human peripheral lymphocytes (HPL) in the presence of lymphoid cells of nude mouse (n-ML). HPL was mixed with n-ML to give a total number of 5 X 10(4) cells and cultured in vitro in the presence of PHA for 5 days. HPL responded will to PHA while n-ML did not, as assessed by 3H-TdR incorporation in the presence of the mitogen. Synergistic response between these two cell population was observed. Five--ten folds enhancement was shown by mixing 5 X 10(3) HPL and 4.5 X 10(4) n-ML (10% of HPL) and 500 HPL became detectable by comixing with n-ML, while 5,000 HPL was the lowest limit to be detected in a single cell culture with this mitogen. The results seem to indicate that this method could detect functional HPL as small number as one percent of the cell mixture. On the other hand, xenogeneic mixed lymphocyte reaction was observed when the percentage of HPL exceeded 40%. Mitomycin-C treatment of either HPL or n-ML revealed that a major part of cells incorporating 3H-TdR was HPL which could be activated by the stimulation with xenogeneic murine nucleated cells and that a smaller part of murien B cells were also stimulated by the HPL.

Animals↗
PubMed1976

Nasopharyngeal carcinoma and Epstein-Barr virus. III. The detection of anti-nuclear antibodies.

The presence of anti-nuclear antibodies (ANA) has been studied in 433 sera from 305 patients with anaplastic nasopharyngeal carcinoma (NPC) in Taiwan, by means of indirect fluorescent antibody method. Another 134 sera from normal adults or from patients with other diseases served as controls. Patients with NPC showed positive rate of 23.1%. Forty-two patients with other malignant and 25 patients with benign tumors of the head and neck showed positive rates of 9.5% and 8.0%, respectively. Forty-eight patients with inflammatory diseases and 19 normal adults were negative. The presence of ANA in 103 NPC patients before treatment was analyzed in detail. The positive rate was not significantly influenced by the age and sex of the patients, by the duration of the disease, by the extent of primary tumor and the presence of neck metastasis. But the positive rate rose as the anti-EB-VCA titer increased. This positive correlation of ANA and anti-EB-VCA antibodies was also significant in all the NPC sera. During and at the end of irradiation therapy, the ANA positive rates showed no remarkable variation. After treatment of the disease, ANA positive rate decreased gradually and this reduction became apparent after three years of remission of the disease. But the presence of ANA in NPC patients was not related to the prognosis of the patients. The possible mechanism in production of ANA in NPC patients was discussed with special reference to EB virus.

Adult↗