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Biomedical subjects

Y Fujisawa

Publications and source records attributed to Y Fujisawa.

At least 109 records · Page 6Linked to original sources
PubMed1994

Novel subtype of human angiotensin II type 1 receptor: cDNA cloning and expression.

Angiotensin II (AII) plays a major role in regulation of cardiovascular function and fluid homeostasis through the action of an AII type 1 receptor (AT1R). The cDNA encoding a novel subtype of human AT1R (AT1bR) was cloned from a human placental cDNA library. The full-length cDNA clone (1563 bp) encoded a polypeptide that consists of 359 amino acid (aa) residues with 97.2% aa identity to the human AT1aR. All the aa replacements between two human AT1Rs reside within the C-terminal half region of AT1R molecule. The 2.4-knt AT1bR mRNA is expressed in the lung, placenta and liver, and differs from AT1aR mRNA in its tissue distribution. The AT1bR expressed in COS-7 cells is pharmacologically distinct from the human AT1aR.

Amino Acid Sequence↗
PubMed1994

Novel subtype of human angiotensin II type 1 receptor: analysis of signal transduction mechanism in transfected Chinese hamster ovary cells.

We examined the intracellular signal transduction of two subtypes of human angiotensin II type 1 receptor (AT1aR and AT1bR) by means of the stable expression of each receptor cDNA in Chinese hamster ovary cells. Both receptors showed a rapid stimulation of phosphatidylinositol hydrolysis, a transient increase of intracellular Ca2+ concentration and an inhibitory action on the forskolin-induced cyclic AMP formation. Interestingly, at high AII concentrations (> 1 microM), these AT1bR-mediated responses were inhibited, whereas the AT1aR-mediated responses were not. Thus, the two AT1Rs are considered to be coupled to the same signal transduction cascades, but to be regulated differently on the post-translational level (presumably desensitization).

Angiotensin II↗
PubMed1994

Fermentable and nonfermentable carbon sources sustain constitutive levels of expression of yeast triosephosphate dehydrogenase 3 gene from distinct promoter elements.

The triosephosphate dehydrogenase 3 gene (TDH3) is a glycolytic enzyme gene and is abundantly transcribed in Saccharomyces cerevisiae. The promoter region of the TDH3 gene is known to exhibit high transcriptional activity regardless of the fermentability of the carbon source and has been widely utilized to synthesize heterologous gene products in S. cerevisiae. To clarify the mechanism of constitutive transcription by the promoter, we constructed mutant promoters and analyzed the in vivo transcriptional activity of these promoters. The majority of the transcriptional potential is contained within a DNA fragment extending from nucleotides -524 to -255 (-524/-255; relative to the translation initiation codon), which consists of three cis-acting elements: a fermentable carbon source-dependent upstream activation sequence (UAS) 1 (-524/-426), a fermentable carbon source-dependent upstream repression sequence (URS) (-426/-393), and a nonfermetable carbon source-dependent UAS2 (-305/-255). This result indicates that the promoter involves two apparent promoter elements. One is fermentable carbon source-dependent, and another is nonfermentable carbon source-dependent. Southwestern analyses indicated that a novel 20-kDa protein is induced in yeast cells by shifting from a fermentable to nonfermentable carbon source. The protein interacts with two UAS1 13-base pair elements and one URS 13-base pair element, one of which had been previously designated GPE (general regulatory factor 1 (GRF1) binding site potentiator element) (Bitter, G. A., Chang, K. K. H., and Egan, K. M. (1991) Mol. Gen. Genet. 231, 22-32). We therefore termed the 20-kDa protein GPEB (GRF1-binding site potentiator element-binding protein). In addition, mutational analyses strongly suggested that UAS1, URS, and UAS2 interact with GRF1 and GPEB, GPEB, and the GCR1 (glycolysis regulation 1) gene product, respectively. We therefore concluded that constitutive transcription by the TDH3 promoter is sustained by two promoter elements and that the switch between them might be controlled by the nonfermentable carbon source-inducible GPEB.

Base Sequence↗
PubMed1994

Eclosion hormone-mediated signal transduction in the silkworm abdominal ganglia: involvement of a cascade from inositol(1,4,5)trisphosphate to cyclic GMP.

The neuropeptide eclosion hormone triggers ecdysis behavior in lepidopteran insects. We have previously shown that the eclosion hormone stimulates the formation of two intracellular second messengers, cGMP and inositol(1,4,5)trisphosphate in the abdominal ganglia of Bombyx mori. In order to elucidate the intracellular signaling pathway involving these second messengers, we studied the eclosion hormone-mediated signal transduction using saponin-treated abdominal ganglia. We obtained the following results; i) eclosion hormone activated nitric oxide synthase, ii) the eclosion hormone-induced cGMP increase was inhibited by various enzyme inhibitors such as NG-nitro-arginine; a nitric oxide synthase inhibitor, EGTA; a calcium chelating reagent, W-5; a calmodulin inhibitor and compound 48/80; a phospholipase C inhibitor and iii) the inositol(1,4,5)-trisphosphate stimulated the formation of cGMP, in the Bombyx abdominal ganglia. Based on these findings we tentatively propose a hypothetical pathway: The signal initially triggered by eclosion hormone and eclosion hormone receptor complex induces activation of phospholipase C which produces inositol(1,4,5)trisphosphate. Inositol(1,4,5)trisphosphate increases intracellular Ca2+, followed by subsequent activation of nitric oxide synthase through the formation of Ca(2+)-calmodulin complex. The reaction product, nitric oxide acts on soluble guanylate cyclase to stimulate cGMP formation which induces the ecdysis behavior in Bombyx pharate adults.

Amino Acid Oxidoreductases↗
PubMed1994

Immunotherapy of acute and recurrent herpes simplex virus type 2 infection with an adjuvant-free form of recombinant glycoprotein D-interleukin-2 fusion protein.

Previous studies demonstrated that the adjuvant-free form of a fusion protein consisting of a truncated herpes simplex virus type 1 (HSV-1) glycoprotein D and human interleukin-2 (tgD-IL-2) elicited superior protective immunity in mice. In this study, the immunotherapeutic efficacy of tgD-IL-2 against vaginal HSV-2 infection was investigated using a guinea pig model. Footpad injections of tgD-IL-2 (12.5 micrograms/dose) after the onset of primary lesions strongly suppressed recurrence in the chronic phase of infection; consequently, the number of days with lesions was reduced 65%. Continuous medication with 100 mg/kg/day acyclovir for 5 days failed to suppress recurrent infection. In a UV radiation-induced recurrence model, prophylactic tgD-IL-2 significantly suppressed both duration and severity of disease. A single injection of tgD-IL-2 plus acyclovir produced an additive effect on the suppression of the disease in the acute phase. These results suggest that tgD-IL-2 is a promising immunotherapeutic agent against HSV-2 genital infections.

Acute Disease↗
PubMed1994

Role of vasopressin on cardiovascular changes during hemorrhage in conscious rats.

Hypotensive hemorrhage decreases heart rate (HR) and renal sympathetic nerve activity (RSNA). Hemorrhage is a potent stimulus for arginine vasopressin (AVP) release; therefore, AVP may contribute to such inhibitory action of HR and RSNA during hemorrhage. We evaluated the roles of vasopressin on the regulation of blood pressure (BP), HR, and RSNA during hemorrhage using nonpeptide and selective V1- and V2-receptor antagonists (OPC-21268 and OPC-31260) in conscious rats. After hemorrhage (20 ml/kg body wt) BP decreased by 62 +/- 10 mmHg along with bradycardia (-110 +/- 15 beats/min) and renal sympathoinhibition (-50 +/- 8). Pretreatment of V1-receptor antagonist (5 mg/kg iv) did not affect the initial fall of BP but attenuated subsequent BP recovery. Bradycardic and renal sympathoinhibitory responses following hemorrhage were abolished (-14 +/- 24 beats/min and -7 +/- 9) by V1-receptor antagonist. Pretreatment of V2-receptor antagonist (1 mg/kg iv) did not affect the response of BP; however, it did slightly strengthen bradycardia and prolong renal sympathoinhibition. Hemorrhage increased the plasma AVP concentration more than 50-fold. These results indicate that when the plasma concentration of AVP is extremely high during hemorrhage, vasopressin via V1 receptor contributes to BP recovery by the peripheral vasoconstriction and exerts an inhibitory action on RSNA, and vasopressin via V2 receptor exerts opposite stimulatory action on RSNA.

Animals↗
PubMed1993

Saccharomyces cerevisiae can release hepatitis B virus surface antigen (HBsAg) particles into the medium by its secretory apparatus.

We constructed a plasmid that directs the synthesis and secretion of hepatitis B virus (HBV) surface antigen (HBsAg) particles by Saccharomyces cerevisiae. This plasmid contains a proteinase-resistant HBsAg M (M-P31c) gene fused at its 5'-terminus with a chicken-lysozyme signal peptide (C-SIG) gene, which is placed under the yeast GLD (glyceraldehyde-3-phosphate dehydrogenase gene) promoter. The products encoded by the "C-SIG+M-P31c" (LM-P31c) gene were synthesized and assembled themselves into HBsAg particles in yeast cells, and the particles were released into the medium along with poly-HSA (polymerized human serum albumin) binding activity. The HBsAg particles purified from the medium were very similar in density (1.19 g cm-3), size (19.2 +/- 0.8 nm in diameter) and shape (sphere) to human-plasma-derived HBsAg particles. When several sec (temperature-sensitive secretion-defective) mutants were used as host cells, the release of HBsAg particles into the medium was blocked at 37 degrees C but not at 25 degrees C, indicating that the HBsAg particles are exported through the normal yeast secretion pathway. To our knowledge, this is the first report that yeast cells are capable of secreting particles into the medium.

Amino Acid Sequence↗
PubMed1993

Adjuvant-independent enhanced immune responses to recombinant herpes simplex virus type 1 glycoprotein D by fusion with biologically active interleukin-2.

A truncated herpes simplex virus (HSV) type 1 glycoprotein D (t-gD) gene was fused to the human interleukin-2 (IL-2) gene (t-gD-IL-2 gene) and introduced into mouse myeloma Sp2/0 cells. The gene product, t-gD-IL-2, secreted from the cells was immunoprecipitated with five monoclonal antibodies specific for native gD. Purified t-gD-IL-2 supported the growth of IL-2-dependent cells, with a specific activity almost comparable to that of recombinant human IL-2. Mice immunized with t-gD-IL-2 in an adjuvant-free form showed superior anti-HSV antibody responses, and were completely protected against HSV challenge, whereas immunization with t-gD adsorbed onto aluminum hydroxide (alum) partially failed to prevent the virus infection. The high immunogenicity of t-gD-IL-2 was due to the biological activity of the fused IL-2 rather than to a hapten-carrier effect of the IL-2 moiety, because mice primed with t-gD-IL-2 showed delayed-type hypersensitivity against stimulation with gD, but not against that with IL-2 antigen, and because a booster immunization with t-gD-IL-2 extensively augmented the response of anti-gD antibody, but not that of the anti-human IL-2 antibody. The serological half-life of IL-2 activity in mice injected with t-gD-IL-2 was prolonged to about four times that of rIL-2. However, when t-gD-IL-2 was co-administered with human albumin (HSA), the mouse anti-HSA antibody response was slightly enhanced.(ABSTRACT TRUNCATED AT 250 WORDS)

Adjuvants, Immunologic↗
PubMed1993

A Mytilus peptide related to the small cardioactive peptides (SCPs): structure determination and pharmacological characterization.

1. An SCP-related peptide, Ala-Pro-Asn-Phe-Leu-Ala-Tyr-Pro-Arg-Leu-NH2, was isolated from the ABRMs of Mytilus edulis. The peptide was designated Mytilus SCP. 2. At 10(-10) M or higher, Mytilus SCP showed a potentiating effect on phasic contraction of the ABRM in response to repetitive electrical stimulation. In contrast, the peptide did not show any potentiating effect on contractures in response to ACh and the FMRFamide-related Mytilus decapeptide, suggesting that the potentiating effect on phasic contraction was brought about by an action of the peptide on the nerve elements in the ABRM. 3. At 10(-8) M or higher, Mytilus SCP showed a catch-relaxing effect in addition to the potentiating effect. The relaxing effect was blocked by mersalyl, suggesting that it was also brought about by a presynaptic action. 4. Various analogues of Mytilus SCP were examined on the ABRM. Leu-Ala-Tyr-Pro-Arg-Leu-NH2 was suggested to be the minimum structure required for both potentiating and relaxing activities on the ABRM. Leu-D-Ala-Tyr-Pro-Arg-D-Leu-NH2 and D-Leu-Ala-Tyr-Pro-Arg-D-Leu-NH2 were found to show strong potentiating and relaxing activities, though they were less potent than the Mytilus SCP.

Amino Acid Sequence↗
PubMed1993

Effects of several peptides with Pro-Arg-Leu-NH2 C-terminal sequence on invertebrate muscles.

1. Effects of the following PRLamide peptides on some invertebrate muscles were examined: APNFLAYPRLamide (a peptide of the bivalve mollusc Mytilus), AAPLPRLamide (a peptide of the echiuroid Urechis), FTPRLamide (a fragment of a peptide of the insect Pseudaletia), YFSPRLamide (a fragment of a peptide of the insect Heliothis) and pQTSFTPRLamide (a peptide of the insect Leucophaea). 2. In the ABRM of Mytilus, the Mytilus and Urechis peptides potentiated phasic contraction by repetitive electrical stimulation. The former was more potent than the latter. The three insect-origin peptides did not show any potentiating effect but showed a weak antagonistic action on the potentiating effect of the Mytilus peptide. Except the Mytilus peptide, the PRLamide peptides showed little or no relaxing effect on catch tension, though they accelerated catch-relaxing response to repetitive electrical stimulation. 3. In the inner circular body-wall of muscle of Urechis, the PRLamide peptides showed a potentiating effect on twitch contraction. The Urechis peptide was most potent. In the esophagus of the annelid Perinereis, the PRLamide peptides showed a contractile effect. The Urechis peptide was also most potent. 4. In the crop of the cricket Gryllus, the insect-origin peptides markedly potentiated spontaneous rhythmic contractions. The Mytilus peptide showed a weak potentiating effect, but the Urechis peptide showed little or no effect.

Amino Acid Sequence↗
PubMed1993

Further identification of bioactive peptides in the anterior byssus retractor muscle of Mytilus: two contractile and three inhibitory peptides.

1. Two contractile and three inhibitory peptides were newly isolated from the anterior byssus retractor muscles (ABRMs) of the bivalve mollusc Mytilus edulis by using the muscle as the bioassay system. 2. The structures of the two contractile peptides were GPFGTHIKamide (GPFG-8) and GPFGLNKHGamide (GPFG-9). The contractile response of the ABRM to the first-time application of GPFG-8 or GPFG-9 was of considerable size. The threshold concentrations of the peptides were around 10(-9) M. However, the contractile response to the second-time application was far smaller than that to the first-time application in both cases. Namely, the muscle showed tachyphylaxis to the peptides. 3. Two of the three inhibitory peptides were members of the Mytilus-inhibitory-peptide (MIP) family. Their structures were RAPLFIamide (MIP6) and RSPMFVamide (MIP7). The peptides, as well as the other MIPs previously identified, showed a potent inhibitory effect on phasic contraction of the ABRM in response to repetitive electrical stimulation. The remaining one was an MIP-related peptide (MIP-RP) having the sequence of MRYFVamide. The MIP-RP was less potent than the two MIPs in inhibiting the contraction of the ABRM.

Amino Acid Sequence↗
PubMed1993

An episode of silent thyroiditis in a patient with chronic thyroiditis and papillary adenocarcinoma following alpha interferon treatment for hepatitis C.

In the present paper we described the first case report of silent thyroiditis following alpha-interferon (IFN-alpha) treatment for chronic type C hepatitis in Japan. A 51-year-old woman with chronic type C hepatitis was treated with 6 million units of IFN-alpha three times a week for 24 weeks. Thyroid function was within normal limits and thyroid autoantibodies were negative before IFN therapy. Sixteen weeks after initiation of the treatment, she complained of increasing fatigue, palpitation and losing 7 kg in weight. Thyroid function tests at that time revealed an increase in serum T3, T4, free T3 and free T4 and a markedly suppressed TSH concentration. Both antithyroglobulin antibody (TgAb) and antimicrosomal antibody (McAb) were positive in a dilution of 1: 400. The computed tomographic (CT) scan of the thyroid showed a decrease in the CT number (Hounsfield unit; H.U.) to 58 H.U. (normal, 95-167 H.U.). The 24-h thyroid uptake of 123I was 0.75%. Aspiration biopsy specimens from a nodule in the right lobe and the remaining struma disclosed papillary adenocarcinoma and Hashimoto thyroiditis, respectively. Thyroid function spontaneously returned to normal two months after the onset of thyrotoxicosis through the subclinical hypothyroid stage. After recovery of thyroid function, patient had an operation of papillary cancer without any complications. These clinical features and laboratory findings led to the diagnosis of silent thyroiditis developing in the course of the long-term IFN therapy, which, to our knowledge, has not been reported before in Japan.

Adenocarcinoma, Papillary↗
PubMed1993

The inhibitory effect of a neuropeptide, ManducaFLRFamide, on the midgut activity of the sphingid moth, Agrius convolvuli.

The effect of ManducaFLRFamide, a neuropeptide originally isolated from the central nervous system of a sphingid moth, Manduca sexta, was examined on the midgut motility of another sphingid moth, Agrius convolvuli. The peptide was shown to suppress the spontaneous contractions of the isolated gut in adult Agrius at low concentrations (> or = 10(-10) M). Immunoreactivity against anti-FMRFamide antiserum was also detected in the brain-suboesophageal ganglion and the retrocerebral neurohaemal organs of the moth. These observations suggest that ManducaFLRFamide, or its closely-related analog, is a neuropeptide which regulates the activity of the digestive tract in Agrius.

Amino Acid Sequence↗
PubMed1993

Intranasal immunization against herpes simplex virus infection by using a recombinant glycoprotein D fused with immunomodulating proteins, the B subunit of Escherichia coli heat-labile enterotoxin and interleukin-2.

To establish a novel strategy of mucosal immunization against herpes simplex virus type 1 (HSV-1) infection, we studied the immune responses elicited by intranasal immunization with several forms of a recombinant glycoprotein D (gD) of HSV-1. A truncated gD (t-gD) co-administered with heat-labile enterotoxin B subunit (LTB) from Escherichia coli induced both a mucosal immune response involving secretion of anti-gD IgA and serum IgG production. The levels of these responses are comparable to those in mice which have recovered from intranasal HSV-1 infections. The fusion protein (t-gD-LTB), consisting of t-gD and LTB, induced the responses more efficiently than did co-administration of t-gD and LTB, although GM1 ganglioside binding activity was significantly reduced in t-gD-LTB. We found that another fusion protein, consisting of t-gD and human interleukin-2 (t-gD-IL-2), also elicited antibody responses comparable to those induced by t-gD-LTB. Immunity acquired by intranasal immunization with t-gD-IL-2 protected mice from intraperitoneal HSV-1 infections, whereas t-gD-LTB or t-gD alone failed to provide protection against infection. Even in a mouse strain that responded highly to subcutaneously administered gD, intranasally administered t-gD did not elicit antibody responses. The lack of response to gD was clearly abrogated by co-administration with IL-2, and administration of t-gD-IL-2 induced an excellent level of antibody responses in this strain. These results suggest that the IL-2 fusion strategy yields a new type of mucosal immunization, the mechanism of which differs from that speculated for the mucosal adjuvant activity of LTB.

Administration, Intranasal↗
PubMed1992

Myristoylation-dependent membrane targeting and release of the HTLV-I Gag precursor, Pr53gag, in yeast.

The unprocessed HTLV-I Gag precursor, Pr53gag, was synthesized in yeast, Saccharomyces cerevisiae. The synthesized Pr53gag was myristoylated, associated with the cellular membrane, and released into the culture medium. The released Pr53gag was pelleted by centrifugation at 100,000 x g for 2 h. Conversion of Gly2 to Ala allowed synthesis of a non-myristoylated soluble Pr53gag which was not released into the culture medium. These results suggest that the release of the HTLV-I Gag precursors Pr53gag, occurs in yeast in a myristoylation-dependent manner.

Amino Acid Sequence↗
PubMed1992

Requirement of N- and C-terminal regions for enzymatic activity of human T-cell leukemia virus type I protease.

The requirement of N- and C-terminal regions for the enzymatic activity of human T-cell leukemia virus type I (HTLV-I) protease was investigated using a series of deletion mutants. The activity was analyzed by autoprocessing of the protease itself or by processing of the gag p53 precursor. The deletional analyses indicated that Asp38-Gly152 with an additional Met-Pro sequence at the N-terminus was probably sufficient for the enzymatic activity, although the mature HTLV-I protease consists of Pro33-Leu157. A molecular model of HTLV-I protease, which was constructed by comparison with the structure of Rous sarcoma virus protease, predicted that Pro33-Leu37 and Gly143-Leu147 would form a beta-sheet. Our experimental results and the model structure suggest that (a) five amino acids in the N-terminal region (Pro33-Leu37), which are thought to be involved in the beta-sheet, are not crucial for the enzymatic activity; (b) Pro153-Leu157 is not necessary but Pro148-Gly152 is important for the enzymatic activity, in addition to Gly143-Leu147 involved in the beta-sheet.

Amino Acid Sequence↗