Biomedical subjects

K Higo

Publications and source records attributed to K Higo.

At least 37 records · Page 2Linked to original sources

PubMed1996

Cloning and characterization of the rice CatA catalase gene, a homologue of the maize Cat3 gene.

We isolated and sequenced a genomic clone (CatA) encoding CAT-A catalase, a homologue of the maize catalase isozyme 3 (CAT-3) from rice (Oryza sativa L.). The 5'-upstream non-coding region had very low similarity with the maize Cat3 gene and possible cis elements and sequence motifs in the maize Cat3 gene were not evident, except for TATA and CAAT motifs. Several sequence motifs found in the promoters of plant seed-specific genes were identified in the 5'-upstream non-coding region of the CatA gene. Northern blotting showed that the CatA gene is expressed at high levels in seeds during early development and also in young seedlings. Methyl viologen (paraquat) resulted in the 3-fold induction of the CatA gene in the leaves of young seedlings, whereas abscisic acid, wounding, salicylic acid, and hydrogen peroxide had no or only slight effects. The 1.9 kb 5'-upstream fragment (-1559 to +342) of the CatA gene was fused with the Escherichia coli beta-glucuronidase (GUS) gene and introduced by electroporation into protoplasts prepared from rice suspension-cultured cells, then the transient expression of the GUS gene was examined. Deletion analysis of this chimeric gene suggested that a weak silencer is located in the region between -1564 to -699. Abscisic acid (ABA) at a final concentration of 10(-6) M doubled GUS activity in protoplasts electroporated with the chimeric DNAs having 1.9 to 1.2 kb 5'-upstream regions. A sequence highly similar to the Sph box, a motif found in genes modulated by ABA, was found at -266 to -254. Deletion of this region however, did not eliminate the responsiveness to ABA. Expression of the chimeric gene in the protoplasts was not enhanced by stress such as low and high temperature, hydrogen peroxide, methyl viologen, salicylic acid, elicitor, and UV light. The chimeric CatA-GUS plasmid DNAs amplified in the methylation-positive strain, E. coli DH5alpha, showed GUS gene activities, whereas all the chimeric DNAs amplified in the methylation-deficient E. coli JM110 were completely inactive in the presence or absence of ABA in the culture medium. DNA methylation, especially of either one or both of the deoxyadenosines at the two GATC motifs (one in the first exon and the other in the first intron of the rice CatA gene), appeared to be responsible for the CatA promoter activity identified in the transient assay.

Abscisic Acid↗

PubMed1996

Regional difference in sympathetic neurotransmitter- and Ca2+ channel-mediated responses in rat vas deferens.

1. Pharmacological properties were compared in the epididymal and prostatic portions of the rat vas deferens. 2. Contractile response to norepinephrine (NE) was larger in the epididymal portion, in spite of the smaller diameter in this region. In contrast, contraction evoked by ATP or alpha,beta-methylene ATP (alpha,beta-mATP) was larger in the prostatic portion. The sensitivities to NE but not to alpha,beta-mATP are different in these portions. 3. ATP or NE facilitated the contraction induced by the other agonist, suggesting that they cooperatively elicit smooth muscle contraction. This cooperation was observed in both portions. 4. Neither the contraction elicited by the addition of Ca2+ to smooth muscle depolarized with 63.7 mM extracellular K+ nor the relaxation by nifedipine of the depolarized smooth muscle precontracted with 1.8 mM extracellular Ca2+ was regionally different. However, Bay k 8644 elicited contraction only in the epididymal portion. A combination of 5 mMK+ with Bay k 8644 also caused oscillatory contraction in the prostatic portion. 5. A radioligand binding study was performed using the microsomal fraction prepared separately from these portions. Both the dissociation constant and the maximum binding for 3H-nimodipine were smaller in the epididymal fraction than in the prostatic fraction. 6. These results suggest that 1. the NE-elicited contraction is more pronounced in the epididymal portion, 2. the purinoceptor-mediated contractions along the vas deferens are less heterogeneous, and 3. although the sensitivity to Bay k 8644 is higher in the epididymal portion, Ca2+ channel-mediated responses are not regionally different when they are fully activated.

Adenosine Triphosphate↗

PubMed1996

Effect of mutations on the intracellular localization of Bombyx mori cytoplasmic polyhedrosis virus polyhedrin.

We have already cloned the polyhedrin genes of the wild-type strain H Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) and its mutant, strain A. In this work, polyhedrin genes of mutant BmCPV strains C1 and C2 were cloned and their nucleotide sequences were determined. The polyhedrin amino acid sequences of strains C1 and C2 were compared with that of strain H. Strains C1 and C2 contained two and three sites of mutation in their polyhedrin genes, respectively. Four amino acids (249RLLV) were added at the carboxy terminus of the polyhedrin of strain A, C1 and C2 and the corresponding polyhedrin genes were introduced into a baculovirus expression vector. Intracellular localization of expressed polyhedrin as well as the morphology and localization of polyhedra were investigated by Western blot and microscopy analysis. Recombinant baculovirus containing the polyhedrin gene of strain H produced hexahedral polyhedra in both the cytoplasm and the nucleus. However, the hexahedral polyhedra of strain A were localized only in the nucleus. Normal polyhedra were not observed in cells infected with recombinant baculoviruses expressing strain C1 or C2 polyhedrin genes, but amorphous structures were found in infected cells. Results of expression of a chimaeric luciferase-containing carboxyl-terminal sequence of strain A demonstrated that this sequence was responsible for the nuclear localization. We suggest that a mutation at the carboxy terminus of BmCPV polyhedrin led to nuclear localization of polyhedrin and that several other mutations were responsible for modification of the crystallization pattern of polyhedrin.

Amino Acid Sequence↗

PubMed1996

Possible origin of murine AIDS (MAIDS) virus: conversion of an endogenous retroviral p12gag sequence to a MAIDS-inducing sequence by frameshift mutations.

The murine AIDS (MAIDS) virus has a unique sequence in its p12gag region, which is responsible for MAIDS development. A transcript hybridizing with this sequence is expressed in normal C57BL/6 mice. The transcript, designated Edv, has been previously cloned and sequenced (Y. Kubo, Y. Nakagawa, K. Kakimi, H. Matsui, K. Higo, L. Wang, H. Kobayashi, T. Hirama, and A. Ishimoto, J. Gen. Virol. 75:881-888, 1994). Compared with the nucleotide sequence of the helper LP-BM5 ecotropic virus, the pathogenic replication-defective MAIDS virus has a 16-bp deletion and a 1-bp insertion in the 5' and 3' regions of the p12gag sequence, respectively, and the Edv transcript contains only a 3-bp deletion. Therefore, the amino acid sequence of the defective MAIDS virus p12gag region is not homologous to that of the helper virus and the Edv transcript because of the frameshift. To determine whether the amino acid sequence resulting from the frameshift is critical for MAIDS development, we constructed chimeric viruses that contained the p12gag regions of the helper virus and the Edv transcript, respectively, with and without the same frame as the defective MAIDS virus by the artificial frameshift mutations. The mutant viruses with the frameshift mutations induced MAIDS in inoculated mice, but the viruses without the mutations did not. These results suggested that the MAIDS virus was generated by frameshift mutations in the p12gag region of Edv or a related sequence.

Animals↗

PubMed1995

Hepatitis C virus core region: helper T cell epitopes recognized by BALB/c and C57BL/6 mice.

In this study, we characterized the B cell and T cell responses to the hydrophilic portion of hepatitis C virus (HCV) core protein in two strains of mice and identified the respective antigen determinants. BALB/c (H-2d) and C57BL/6 (B6:H-2b) mice were immunized by a subcutaneous injection of recombinant HCV core protein together with Freund's complete adjuvant. The level of antibody production, as determined by ELISA, was consistently higher in BALB/c than in B6 mice. However, antibodies in sera from each strain bound to the N-terminal region of the core protein within amino acids 1 to 28 (MSTNPKPQRKIKRNTNRRPQDVKFPGGG), according to an experiment using non-overlapping peptides that covered the hydrophilic portion of HCV core protein. The T cell responses were also higher in BALB/c than in B6 mice with respect to the proliferative responses of the draining lymph node cells in vitro. By limiting dilution cultures of the draining lymph node cells in vitro repetitively stimulated with recombinant core protein, T cell clones were established from both strains of mice and characterized. The surface markers of these clones were Thy-1.2+, CD3+, TCR alpha beta+, CD4+ and CD8+. The proliferative responses were inhibited in the presence of anti-CD4 or anti-MHC class II monoclonal antibodies. The T cell lines in BALB/c mice recognized an epitope in HCV core at amino acids 72 to 91 (EGRAWAQPGYPWPLYGNEGL). The T cell lines in B6 mice recognized an epitope at amino acids 55 to 74 (RPQPRGRRQPIPKARQPEGR). Thus, mice with different MHC haplotypes recognized different non-overlapping T cell antigenic determinants of HCV core proteins.

Amino Acid Sequence↗

PubMed1995

Regulation of cyclic AMP phosphodiesterase activity by particulate protein tyrosine kinase and phosphotyrosine phosphatase activities sensitive to sodium orthovanadate.

Sodium orthovanadate (vanadate) stimulated cAMP phosphodiesterase (PDE) and protein tyrosine kinase (PTK) activities and inhibited the phosphotyrosine phosphatase (PTPase) activity in the particulate of isolated rat fat pads. Okadaic acid never showed any increase in the PDE activity up to 1 microM. Amiloride inhibited in part both stimulations of PDE and PTK activities by vanadate. The particulate PTK activity had an optimal divalent ion requirement of 15 mM Mg+2+2 mM Mn+2 in the assay medium and was not inhibited by 1 mM N-ethylmaleimide, suggesting it to be a different type from the insulin receptor and cytosolic PTK activities. The PDE, PTK, and PTPase active fractions were separated from the solubilized particulate fraction on a DEAE-Sephacel column. PDE activity was increased by the addition of the PTK active fraction. A further increase was observed by using the PTK active fraction pretreated with 1 mM vanadate. In contrast, the addition of PTPase active fraction decreased the PDE activity. This decrease disappeared by using the PTPase active fraction pretreated with 1 mM vanadate. These results suggest that the PDE activity is in part regulated through a process involving the particulate PTK and PTPase activities sensitive to vanadate.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1994

Secondary infection with rescued M-MSV is requisite for focus formation of S+L- mink cells by murine leukemia virus.

Whether the foci on S+L- mink cells transformed by the superinfection of MuLV were induced by the secondary infection of M-MSV pseudotype generated in the cells or by the secondary infection of MuLV has remained unresolved since the requirement of secondary infection was first reported (A. Ishimoto, J. Virol. 36, 18-21, 1980). Here, we show that infection with ecotropic MuLV of S+L- mink cells transfected with the receptor gene for ecotropic MuLV induced transformed foci resembling those induced by xenotropic and amphotropic viruses. This observation and our previous results (A. Ishimoto, J. Virol. 36, 18-21, 1980) that clonal lines of S+L- mink cells chronically infected with ecotropic MuLV are morphologically indistinguishable from normal S+L- mink cells suggest that the focus formation of S+L- mink cells by superinfection with MuLV is not due to secondary spread of helper virus which transactivates the expression of the v-mos oncogene by the MuLV, but is due to the secondary infection of the defective M-MSV genome. A new S+L- mink cell line with a receptor gene for ecotropic MuLV, designated ID cells, provided a new method for titrating the ecotropic MuLV that develop few XC foci and to simultaneously detect viruses in various host ranges.

Animals↗

PubMed1994

Molecular cloning and characterization of a murine AIDS virus-related endogenous transcript expressed in C57BL/6 mice.

The murine AIDS (MAIDS) virus has a unique sequence in the gag p12 region, which could be responsible for MAIDS development. RNA preparations from the spleens of normal uninfected C57BL/6 mice contain a transcript hybridizing with this sequence. Levels of the transcript in the kidney of C57BL/6 mice were higher than in the spleen, liver or thymus. Although BALB/c, NFS, DBA/2 and SL murine strains also contained genomic sequences hybridizing with the MAIDS virus-specific probe, no transcript hybridizing with the probe was detected in these strains of mice. The cDNAs carrying the transcript expressed in C57BL/6 mice were molecularly cloned. The complete nucleotide sequence of the clone indicates that the transcript is one of the endogenous murine leukaemia virus-related sequences containing large deletions from the R and U5 regions of the 5' long terminal repeat (LTR) to gag p15, from the C-terminal region of pol p40 (integrase) to the N-terminal region of env p15E, and many short deletions in the 3' LTR U3 region. The nucleotide sequence in the gag p12 region of the transcript was closely similar to that of the MAIDS virus, but the amino acid sequence was less similar because of frameshifting, even when translated. As the MAIDS virus was isolated from C57BL/6 mice with radiation-induced leukaemia, this transcript may be the progenitor of the MAIDS virus. To determine whether the gag p12 region of the transcript contains a functional sequence, a recombinant virus was generated by replacing the gag p12 region of a replication-competent BM5eco virus with that of the endogenous transcript. The recombinant virus was replication-competent, and the p12 region of the transcript retained the functional sequence present in the BM5eco virus.

Amino Acid Sequence↗

PubMed1994

Inhibition of murine AIDS (MAIDS), development by the transplantation of bone marrow cells carrying the Fv-4 resistance gene to MAIDS virus-infected mice.

To examine whether the resistance allele of the Fv-4 gene (the Fv-4r gene) is a dominant inhibitory-product-encoding gene which an be used to prevent the development of murine AIDS (MAIDS), bone marrow cells from BALB/c-Fv-4wr mice were transplanted into BALB/c mice and C57BL/6 mice infected with MAIDS virus. Almost all of the virus-infected BALB/c and C57BL/6 mice developed MAIDS within 4 months and died 2 or 3 months later. However, when the virus-infected mice were subjected to cobalt irradiation and then given an intravenous injection of 10(7) BALB/c-Fv-4wr mouse bone marrow cells, the recipient mice survived much longer than the untreated mice, which suggests that the Fv-4 gene is a dominant inhibitory gene that is potentially useful in gene therapy of MAIDS.

Animals↗

PubMed1994

The p15gag and p12gag regions are both necessary for the pathogenicity of the murine AIDS virus.

The defective murine AIDS (MAIDS) virus has unique sequences in its p15gag and p12gag regions. To clarify whether these sequences are responsible for the development of MAIDS, we constructed recombinant viruses by replacing various regions of the gag gene of the nonpathogenic replication-competent LP-BM5 ecotropic virus with those of the MAIDS virus. Recombinants containing both unique sequences of the MAIDS virus were replication defective and induced MAIDS. However, a recombinant containing either the p15gag or p12gag region of the MAIDS virus was also replication defective but nonpathogenic in mice. A recombinant virus containing only the p30gag region of the MAIDS virus was replication competent and nonpathogenic. These results indicate that the p15gag and p12gag regions of the MAIDS virus do not function like those of replication-competent viruses and that both of the unique sequences in the p15gag and p12gag regions are required to develop MAIDS.

Animals↗

PubMed1994

The novel thromboxane A2 receptor antagonist KW-3635 abolishes the cyclic flow reduction in the canine carotid artery.

We tested the hypothesis that the abolition of the cyclic flow reduction (CFR) in the canine carotid artery is related to inhibition of ex vivo platelet aggregation following administration of KW-3635, a thromboxane A2 receptor antagonist, or aspirin. The CFR was induced in the carotid artery of anesthetized dogs by mechanical injury and narrowing of the artery. After induction of CFR, KW-3635 or aspirin was administered every 30 min at doses of 0,1,0.3, 1 and 3 mg/kg (i.v.). The ex vivo platelet aggregation, induced by sodium arachidonate and collagen, was also examined before and 15 min after each administration. KW-3635 and aspirin, at doses of 1 mg/kg i.v. and above, inhibited CFR and ex vivo platelet aggregation. These results that CFR in the canine carotid artery is platelet-dependent.

Animals↗

PubMed1994

Protective effects of KW-3635, a thromboxane A2 antagonist, on arachidonic acid-induced transient cerebral ischemia in dogs.

We investigated the effect of KW-3635, a selective thromboxane (TX) A2-receptor antagonist, on the arachidonic acid (AA)-induced transient cerebral ischemia in anesthetized dogs. Intracarotid-arterial injection of AA (0.25-1 mg/kg) produced a transient and reversible decrease in electroencephalographic (EEG) activity. The reduction of EEG power was inhibited by the intravenous injection of KW-3635 or aspirin, a cyclooxygenase inhibitor. Local cortical perfusion (LCP) measured by a laser-doppler flow meter was also reduced concomitantly with the reduction of EEG power. Although KW-3635 at 1 and 3 mg/kg (i.v.) did not affect the maximum reduction of LCP, the duration of the reduction period of LCP was significantly shortened by KW-3635. On the other hand, aspirin at 1 and 3 mg/kg (i.v.) inhibited both the maximum and the delay of LCP reduction. The intravenous administration of KW-3635 or aspirin caused dose-dependent inhibition of ex vivo platelet aggregation stimulated by AA (150 microM) at the doses that improve the EEG activity. These data suggest that TXA2 is one of the important factors in the AA-induced transient reduction of EEG activity in anesthetized dogs. The TXA2-receptor antagonist may be useful for protection against the ischemic brain damage following transient ischemic attack.

Animals↗

PubMed1994

Antithrombotic effects of KW-3635, a thromboxane A2-receptor antagonist, in guinea pigs.

Antithrombotic effects of KW-3635, a newly synthesized thromboxane (TX) A2-receptor antagonist, were studied in guinea pigs. In the extracorporeal circulation thrombosis model, the shunt was filled with thrombi, and reduction of platelet count and increase in plasma TXA2 concentration were observed. KW-3635 (30 and 100 mg/kg, p.o.) inhibited the thrombus formation in the shunt and prevented the decrease in platelet count in the circulating blood without affecting the red blood cell count. BM13,505 (30, 100 mg/kg, p.o.), another TXA2-receptor antagonist, and ticlopidine (300 mg/kg, p.o.), an antiplatelet drug, also inhibited the thrombus formation, while aspirin (10, 300 mg/kg, p.o.) did not. Peripheral arterial occlusive disease was induced by injection of sodium laurate into the femoral artery in guinea pigs. Daily oral administration of KW-3635 (3-30 mg/kg) significantly prevented the progression of vascular lesions. BM13,505 (3-30 mg/kg, p.o.) and ticlopidine (100 mg/kg, p.o.) also ameliorated the vascular lesions, whereas aspirin (10, 100 mg/kg, p.o.) did not. KW-3635 at concentrations up to 10(-4) M did not affect coagulation parameters in vitro. These results suggest that TXA2 is involved in the pathogenesis of arterial thrombotic and ischemic disorders. KW-3635 may be useful for the treatment of thrombotic disease and peripheral arterial occlusive diseases.

Animals↗

PubMed1994

Effects of a thromboxane A2-receptor antagonist, a thromboxane synthetase inhibitor and aspirin on prostaglandin I2 production in endothelium-intact and -injured aorta of guinea pigs.

We examined the effects of KW-3635, a thromboxane (TX) A2-receptor antagonist, and OKY-046, a TX synthetase inhibitor, on the prostaglandin (PG) I2 production in endothelium-intact and -injured guinea pig aorta and compared them with those of aspirin. In the endothelium-intact aorta, both the low (3 mg/kg) and the high (100 mg/kg) dose of aspirin similarly reduced the PGI2 production, as measured ex vivo 1 hr after the injury. In contrast, neither KW-3635 (10 mg/kg) nor OKY-046 (30 mg/kg) inhibited the PGI2 production. The endothelial injury, induced by balloon catheterization, caused a reduction of PGI2 production in the aorta and decline of plasma PGI2/TXA2 ratio. In the endothelium-injured animals, the high dose of aspirin further reduced the PGI2 production in the aorta, whereas KW-3635 and OKY-046 did not affect it. KW-3635 and OKY-046 also ameliorated the reduced ratio of PGI2/TXA2 in the plasma. The present results demonstrate that aspirin, but not KW-3635 or OKY-046, reduces the PGI2 production in the aorta either in the endothelium-intact or -injured state. It is thus suggested that the TXA2-receptor antagonist and the TX synthetase inhibitor have some advantages over aspirin when used for the prevention of acute thrombosis after percutaneous transluminal angioplasty.

Angioplasty, Balloon↗

PubMed1993

Blockade by calmodulin inhibitors of Ca2+ channels in smooth muscle from rat vas deferens.

1. Effects of three compounds which are used as calmodulin inhibitors (trifluoperazine, W-7 and calmidazolium) on Ca2+ channels were investigated in smooth muscle from rat vas deferens. 2. All three calmodulin inhibitors relaxed the smooth muscle precontracted by a high concentration of KCl (63.7 mM). The order of potency for the relaxation was trifluoperazine > W-7 > calmidazolium. 3. In binding studies using a microsomal fraction of vas deferens, all these calmodulin inhibitors displaced specific [3H]-nimodipine binding. Trifluoperazine and W-7 inhibited the binding at concentrations that relaxed the smooth muscle whereas calmidazolium inhibited at concentrations much lower than those necessary for muscle relaxation. 4. Ba2+ current flowing through voltage-gated Ca2+ channels was measured under whole-cell voltage-clamp conditions in isolated smooth muscle cells. The Ba2+ current was suppressed by the three calmodulin inhibitors in the concentration-range where inhibition of [3H]-nimodipine binding was observed. Neither voltage-dependence nor the inactivation time course of Ba2+ current were affected by these compounds. 5. The results suggest that the calmodulin inhibitors directly block Ca2+ channels in the smooth muscle cells. The channel inhibition by trifluoperazine and W-7, but perhaps not that by calmidazolium, may be responsible for the muscle relaxation observed with these compounds.

Animals↗

PubMed1993

Inhibitory effect of KW-3635, a new thromboxane A2-receptor antagonist, on arterial thrombosis in guinea pigs.

The antithrombotic effects of the thromboxane (TX) A2-receptor antagonist and aspirin were determined using a photochemically-induced arterial thrombosis model in the femoral arteries of guinea pigs. KW-3635 (sodium (E)-11-[2-(5,6-dimethyl-1-benzimidazolyl)ethylidene]-6,11- dihydrodibenz[b,e]-oxepine-2-carboxylate monohydrate) and BM-13505, both of which are TXA2-receptor antagonists, and aspirin inhibited the thrombus formation at the doses that inhibited the ex vivo platelet aggregation induced by sodium arachidonate (100 microM) or collagen (3 micrograms/ml). These results support the notion that TXA2 is an important mediator in the present model of arterial thrombogenesis.

Animals↗

PubMed1993

Expression of a chemically synthesized gene for human epidermal growth factor under the control of cauliflower mosaic virus 35S promoter in transgenic tobacco.

Nicotiana tabacum was transformed with a chemically synthesized gene encoding the human epidermal growth factor (hEGF) under control of the CaMV-35S promoter. The hEGF gene sequence was present at one to several copies in the primary transformant plants (R0), and a transcript with the expected length was produced. Slot blot analysis of total RNAs of the progeny (R1) seedlings, originating from self-pollination of the R0 plants, showed that the level of mRNA expression was generally, but not always, heritable. The highest hEGF peptide content per unit of total soluble protein in young (upper) R1 leaves so far examined by an immunological method was about 0.001%. These results suggest that either the hEGF peptide was less stable than the average leaf protein, or the hEGF mRNAs were not efficiently translated.

Base Sequence↗

PubMed1993

Changes in translatable mRNA populations induced in rice seedlings by exposure to freeze-thaw stress.

In order to examine whether changes in gene expression are induced by injury in seedlings of rice (Oryza sativa) after a lethal freeze-thaw stress. mRNAs were extracted and their in vitro translation products were compared with those of the control plants by polyacrylamide gel electrophoresis. An mRNA encoding an entirely new protein with M(r) of ca. 37 kDa was detected as early as 30 min of thawing at 28 degrees C. Two-dimensional gel electrophoresis revealed the induction of mRNAs for at least 10 additional proteins, including those in a range of 15 to 20 kDa. Most of these proteins appear to be distinct from heat-shock proteins. The steady state level of mRNAs encoding putative major light-harvesting chlorophyll a/b binding protein-I and -II were, on the other hand, very low as compared with those of the control plants. The changes observed resemble those previously seen in cold-sensitive plants exposed to sub-lethal chilling temperatures (4-10 degrees C).

Cell Death↗