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H Usui

Publications and source records attributed to H Usui.

At least 19 recordsLinked to original sources
PubMed1992

Human glial fibrillary acidic protein (GFAP): molecular cloning of the complete cDNA sequence and chromosomal localization (chromosome 17) of the GFAP gene.

We isolated three glial fibrillary acidic protein (GFAP) cDNA clones from a glioma cell line, U-251 MG. One clone isolated from a U-251 MG cDNA library was long, but lacked both ends. Using poly(A)+ RNA and primers synthesized according to the sequence of this clone, we used the polymerase chain reaction-assisted rapid amplification of cDNA ends (PCR-RACE) method, which is a strategy to isolate cDNA ends, and obtained cDNA clones for the 5' and 3' ends. From the sequences of these overlapping clones, the complete nucleotide sequence of human GFAP cDNA was established. The start (ATG) and the stop (TGA) signals were seen at nucleotide positions 15 and 1311, respectively, and divided the entire sequence of 3027 bp into 14 bp of 5' non-coding, 1296 bp of coding and 1717 bp of 3' non-coding regions. Using cDNA probes made from both the coding and the 3' non-coding regions, Northern blot hybridization was performed with two different stringencies on RNAs from human and rodent brains and human GFAP-positive and -negative cells. It was shown that the 3' non-coding region probe was more specific for human GFAP than the coding region probe which was specific only under higher stringency conditions. This was also suggested by homology analysis of the sequence with those of various intermediate filament proteins. Based on these findings, we performed spot blot hybridization of sorted human chromosomes and Southern blot hybridization of PCR-amplified DNAs of a panel of hamster-human somatic cell hybrids and localized the human GFAP gene to chromosome 17.

Amino Acid Sequence↗
PubMed1992

Nifedipine-resistant Ca(++)-induced contraction in tail artery of spontaneously hypertensive rats.

Nifedipine-resistant Ca(++)-induced contractions (NR-Ca(++)-contraction) were compared in the tail arteries from SHRs and WKYs (5 and 13 week old). NR-Ca(++)-contraction of tail artery was defined as follows: Ca(++)-induced contraction in the presence of norepinephrine (NE) (10(-5) M) or 5-hydroxytryptamine (5-HT) (10(-5) M) in Ca(++)-free medium containing EGTA (0.1 mM) and nifedipine (10(-6) M). NR-Ca(++)-contractions in arteries from 5 week old SHRs and WKYs were not different. In contrast, NR-Ca(++)-contractions in arteries from 13 week old SHRs were about 2-fold greater than in arteries from 13 week old WKYs. In arteries from 13 week old WKYs and SHRs, nitroglycerin (10(-5) M) significantly reduced the NR-Ca(++)-contraction in the presence of 5-HT but not in the presence of NE. The reduction was inhibited by the presence of methylene blue (3 x 10(-6) M). 8-Bromo-cGMP (10(-4) M) reduced significantly the NR-Ca(++)-contraction in the presence of 5-HT in arteries from 13 week old SHRs and WKYs. The present experiments clearly demonstrated that the NR-Ca(++)-contractions (both in the presence of NE and 5-HT) in 13 week old SHRs were significantly greater than those in arteries from 13 week old WKYs. These results suggest that in addition to an increase in voltage-operated Ca++ mobilization reported by others, an increase in NR-Ca++ mobilization may contribute to the development of hypertension in SHR.

Animals↗
PubMed1992

Structure and expression of human and rat D2 dopamine receptor genes.

D2 dopamine receptor may be related with the pathogenesis of Parkinson's disease and schizophrenia. Furthermore, the antipsychotic drugs have high affinity for D2 dopamine receptor. We carried out the cloning of the genomic DNA for human D2 dopamine receptor and clarified the structure of this gene. Our isolated gene spans about 15 kbp and consists of seven exons interrupted by six introns. However, putative first exon was not yet identified. Spot blot hybridization analysis of cell sorter fractionated human chromosomal DNA with D2 receptor genomic DNA revealed the localization of this gene in the chromosome 11 fraction. We analyzed human genomic DNA by Southern blot hybridization with D2 dopamine receptor genomic DNA as a probe, but so far we could not find RFLP. Northern blot analyses of brain RNA of several animals and rat brain RNA after various treatments were carried out. Developmental changes of D2 dopamine receptor mRNA were observed in the rat brains.

Aging↗
PubMed1992

Syntheses and 5-HT2 antagonist activity of bicyclic 1,2,4-triazol-3(2H)-one and 1,3,5-triazine-2,4(3H)-dione derivatives.

A series of bicyclic 1,2,4-triazol-3(2H)-one and 1,3,5-triazine-2,4(3H)-dione derivatives with a 4-[bis(4-fluoro-phenyl)methylene]piperidine or 4-(4-fluorobenzoyl)piperidine group has been prepared and tested for 5-HT2 and alpha 1 receptor antagonist activity. Among the compounds prepared, 2-[2-[4-[bis(4-fluorophenyl)methylene]-piperidin-1-yl]ethyl]- 5,6,7,8-tetrahydro-1,2,4-triazolo[4,3-a]pyridin-3(2H)-one (7b) had the most potent 5-HT2 antagonist activity, which was greater than ritanserin (2), while 7b did not show alpha 1 antagonist activity in vivo. The central 5-HT2 receptor antagonism was approximately 1/30 that of 2 when tested for the ability to block head twitches induced by 5-hydroxytryptophan. Compound 21b, 3-[2-[4-(4-fluorobenzoyl)piperidin-1-yl]ethyl]-6,7,8,9-tetrahydro- 2H- pyrido[1,2-a]-1,3,5-triazine-2,4(3H)-dione also displayed potent 5-HT2 antagonist activity. The compound had moderate alpha 1 receptor antagonism, and the potency inhibiting head twitches was about one-third that of ketanserin (1). These results indicate that 5,6,7,8-tetrahydro-1,2,4-triazolo[4,3-a]pyrimidin-3(2H)-one and 6,7,8,9-tetrahydro-2H-pyrido-[1,2-a]-1,3,5-triazine-2,4(3H)-dione ring systems are useful components of 5-HT2 antagonists.

Animals↗
PubMed1992

Purification and characterization of a possible protooncogene fyn product, p59fyn, from a rat brain particulate fraction.

Four tyrosine-protein kinases that reacted with antibodies specific to p62c-yes, p60c-src, p60c-src+, and p59fyn, respectively, were solubilized from a rat brain particulate fraction and separated by casein-Toyopearl column chromatography. Possible p59fyn, with a pI of 6.5, was purified 490-fold as a single 59-kDa protein band on SDS-PAGE. The purified enzyme contained almost no phosphotyrosine residues but was autophosphorylated with Mg2+. ATP exclusively at tyrosine residues, with a concomitant increase in the kinase activity toward tyrosine-glutamate (1:4) copolymers. The rate of the copolymer phosphorylation was proportional to the square of the enzyme concentration, suggesting activation through intermolecular catalysis. In the presence of Mn2+, however, the reaction showed a first-order dependence on the enzyme concentration.

Animals↗
PubMed1992

Endothelium-dependent contraction produced by acetylcholine and relaxation produced by histamine in monkey basilar arteries.

The present experiments were carried out to investigate the endothelium dependence of the responses to acetylcholine (ACh), arachidonic acid, and histamine in monkey basilar arteries. ACh and arachidonic acid caused endothelium-dependent contraction (EC) in both monkey and canine basilar arteries. The endothelium-derived contracting factor (EDCF) was probably thromboxane A2 (TxA2), as the EDC was attenuated by a cyclooxygenase inhibitor, TxA2 synthetase inhibitors, and TxA2 antagonists. On the other hand, histamine caused endothelium-dependent relaxation (EDR) in monkey and EDC in canine basilar arteries. The EDR in monkey basilar arteries was attenuated by a nitric oxide synthase inhibitor. The EDR and EDC were antagonized by tripelennamine but not by cimetidine, indicating that they are mediated by H1-receptors. From these results, we suggest that in the monkey basilar artery, either there are two types of endothelium (an EDCF type for ACh and arachidonic acid and an EDRF type for histamine) or there is a single type of endothelium with two types of signalling processes (one for EDC and one for EDR).

Acetylcholine↗
PubMed1992

Transcription of the rat cholecystokinin gene is initiated at multiple sites: verification by an in vitro transcription system.

The cDNA that accommodates the most distal 5'-end region of cholecystokinin mRNA was isolated from an internally primed cDNA library. Using primer extension and S1 nuclease protection analyses, we demonstrated multiple RNA molecules generated from the rat cholecystokinin gene, a single-copy sequence. The longest RNA is transcribed at position--225 upstream relative to the translation start site. The major transcription, more than 95% of the total cholecystokinin mRNA in rat brain, occurred at--59 and its promoter activity was determined by in vitro RNA synthesis in a HeLa cell extract. Deletion to--105 demonstrated an approximately 60% decrease in transcriptional level compared with the full promoter activity. At least the upstream region between--254 and--105 is necessary for transcription initiated at--59 of the cholecystokinin gene by the cell-free system.

Animals↗
PubMed1992

Relaxant response of isolated basilar arteries to calcitonin gene-related peptide in stroke-prone spontaneously hypertensive rats.

The relaxant effects of calcitonin gene-related peptide (CGRP) and other drugs were compared in basilar artery rings obtained from stroke-prone spontaneously hypertensive rats (SHRSP) and Wistar-Kyoto rats (WKY). In addition, the relaxant effect of CGRP on basilar arteries from spontaneously hypertensive rats (SHR) was examined. Relaxation induced by CGRP was independent of the presence of endothelium, and it was markedly increased in SHRSP when compared to WKY. In contrast, acetylcholine-induced relaxation was endothelium-dependent and did not differ between the two groups. Enhanced CGRP-induced relaxation was also found in SHR when compared to WKY. However, the relaxant response was greater in SHRSP than in SHR. No significant differences were found in the relaxation induced by isoproterenol, forskolin, dibutyryl cyclic AMP, and 3-isobutyl-1-methylxanthine in endothelium-rubbed arteries of WKY and SHRSP. These results suggest that CGRP produces endothelium-independent relaxation in the rat basilar artery, and that the enhanced CGRP-induced relaxation found in SHRSP may not be associated with alterations of vasodilation mediated by cyclic AMP.

1-Methyl-3-isobutylxanthine↗
PubMed1992

[Clinical pathology of hypopharyngeal cancer--comparison between TN and pTN].

Seventy patients with the hypopharyngeal cancer who underwent pharyngolaryngoesophagectomy with bilateral neck dissection between 1978 and 1990 were examined retrospectively to compare TN and pTN in these patients and clarify the clinical pathology of the hypopharyngeal cancer. Among the 70 cases of hypopharyngeal cancer, there were 46 cases of piriformis sinus cancer (PSC) and 24 cases of postcricoid cancer (PCC). The pathological diagnosis of all these cases was squamous cell carcinoma (SCC). The following results were obtained: 1. Invasion of the thyroid gland was seen in 8 cases of PCC (33.3%) and 6 cases of PSC (13.0%). The thyroid gland can be preserved in PSC, whereas its removal is indicated in PCC. 2. Twenty-nine cases of N0 necks proved to be pN0-2b in 27 cases (93.1%) which may be controlled by homolateral neck dissection, and pN2c in 2 cases (6.9%), which requires bilateral neck dissection. On the other hand, 29 cases of N1-2b necks which represent one-sided neck metastasis were pN0-2b in 15 cases (51.7%) and pN2c in 14 cases (48.3%). These results demonstrate that N0 necks can, in the majority of cases, be controlled by homolateral neck dissection alone but that N1-2b necks require bilateral neck dissection. 3. Occult neck metastases were observed in PCC more often than in PSC, because paratracheal metastases of PCC were difficult to expose before surgery. 4. Pathological neck metastases of both PSC and PCC were most commonly situated in the superior and middle internal jugular nodes. Paratracheal metastases of PCC was found pathologically in 10 cases (41.7%). Paratracheal nodes must be dissected meticulously during the resection of PCC.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1992

[Maxillary prosthesis for better QOL--early setting and faster construction of maxillary prosthesis].

The maxillofacial prosthesis (MFP) is well accepted as one of the modalities to ameliolate the postsurgical crippling in the patients with maxillary malignancy. In this report, we analyzed 55 primary cases of MFP out of 100, from July, 1981 to July, 1987 in terms of the time of start after operation, and the duration and procedure of the MFP-making. MFP-making set about within 4 weeks in 35% of patients, 8 weeks in 25%, 12 weeks in 22%, and more than 12 weeks in 18%. The completion of MFP-making averaged 10 days. As a result of simplifying of MFP-making, we shortened a period requiring MFP-making within 3 days in recent 10 cases. There were no adverse effects of early wear of MFP after operation. We believe that early wear of MFP after operation improve the quality of life of patients with maxillary malignancy and considered that wear of MFP is not the completion of treatment, but is one of the procedures of treatment for the patients undergone maxillectomy.

Adolescent↗
PubMed1991

Purification and characterization of a rat liver membrane tyrosine-protein kinase, the possible protooncogene c-yes product, p60c-yes.

A tyrosine-protein kinase was purified more than 270-fold from the rat liver plasma membrane fraction by successive column chromatographies on Sephacryl S-300, wheat germ agglutinin-agarose, casein-Sepharose, and hydroxylapatite, followed by isoelectrofocusing electrophoresis. The enzyme with pI of 6.2 was a 60-kDa single polypeptide which represented 42% of total protein. The enzyme reacted quantitatively with a monoclonal antibody to the amino-terminal sequence (Cys-3 to Ser-66) specific to the human c-yes protein, but not with antibodies to the specific amino-terminal sequences of the c-src, fyn, and lck proteins. The purified enzyme contained almost no phosphotyrosine residue but was autophosphorylated with Mg.ATP exclusively at tyrosine residues with concomitant increase in the kinase activity. The rates of autophosphorylation of the enzyme and phosphorylation of tyrosine-glutamate (1:4) copolymers, catalyzed by the enzyme were proportional to the square of enzyme concentration, suggesting that p60c-yes undergoes autophosphorylation through intermolecular catalysis, resulting in stimulation of the enzyme activity. Although the enzyme reaction showed an essential requirement for Mg2+ or Mn2+ with optimal concentrations of 20 and 3 mM, respectively, autophosphorylation significantly activated the enzyme only in the presence of Mg2+. Autophosphorylation of the enzyme reduced the Km for tyrosine-glutamate copolymers and tubulin, but not for ATP, and increased the Vmax of copolymer and tubulin phosphorylation.

Animals↗
PubMed1991

In situ hybridization histochemistry of Spot 35 protein, a calcium-binding protein, in the rat brain.

Using in situ hybridization, we analyzed the localization of mRNA for Spot 35 protein (Spot 35), a calcium-binding protein of the EF-hand type, in the rat cerebellum at various developmental stages. A cDNA fragment corresponding to part of the 3'-noncoding region was 35S-labeled and used as a hybridization probe. Autoradiographic signals for Spot 35 mRNA were detected in all the Purkinje cells, but not in any other neurons or glial cells in the adult rat cerebellum. There was no significant difference in signal intensity among individual cells. The signals were observed exclusively in Purkinje cell bodies, but not in their processes, in striking contrast to previous immunohistochemical studies in which Spot 35 protein was demonstrated in both cell bodies and processes. In the time-course study, signals for Spot 35 mRNA were detected in Purkinje cell bodies weakly at embryonic day 19, thereafter more intensely at more developed stages and most intensely at postnatal days 30 and 60 (adulthood). The signal intensities of individual cells were similar at each of these developmental stages except for the very early stages at which signals were weak and slightly variable among cells. These findings, especially that of the characteristic coordinated expression of Spot 35 mRNA at given stages, should prove useful in studies of degenerative diseases in the cerebellum in experimental animals and man. A weak expression of Spot 35 mRNA in some of non-Purkinje cells was also noted.

Animals↗
PubMed1991

Endothelium-independent and -dependent contractions induced by endothelin-1 in canine basilar arteries.

Endothelium-dependence of contractile responses to endothelin-1 was examined in isolated canine basilar arteries. Within 2 hrs after mounting tissue preparations, endothelin-1 (10(-9) M) caused a monophasic tonic contraction that developed very slowly and was sustained in intact and endothelium-removed arteries. More than 5 hrs after tissue mounting, endothelin-1 (10(-9) M) caused a biphasic contraction consisting of phasic and tonic components in intact arteries, and caused a monophasic tonic contraction in endothelium-removed arteries. This phasic component was significantly decreased by aspirin (5 x 10(-5) M,), OKY-046 (10(-5) M) (a TXA2 synthetase inhibitor) and ONO-3708 (10(-8) M) (a TXA2 antagonist). The present experiments demonstrate that endothelin-1 causes an endothelium-independent tonic contraction and an endothelium-dependent phasic contraction in canine basilar arteries, and suggest that TXA2 plays a role as an endothelium-derived contracting factor.

Animals↗
PubMed1991

Vasoinhibitory effect of NP-252, a new dihydropyridine derivative, in canine cerebral artery.

The vasoinhibitory effect of NP-252, a 1,4-dihydropyridine derivative Ca++ antagonist, was examined in canine cerebral artery, and this effect was compared with that of nifedipine. NP-252 (10(-7)M) and nifedipine (10(-6) M) nearly abolished the contraction induced by addition of Ca++ to Ca(++)-free medium containing KC1. NP-252 (10(-6)M) and nifedipine (10(-6)M) attenuated the contraction produced by thromboxane A2 agonist (STA2) in normal medium, and the resultant contractions were 22% (n = 6) and 35% (n = 6) of the control contraction, respectively. The vasoinhibitory effects of NP-252 were significantly stronger than those of nifedipine in canine cerebral artery. NP-252 (10(-7) and 10(-6) M) dose-dependently attenuated nifedipine-resistant Ca(++)-contraction in the presence of STA2 in both canine cerebral and coronary arteries. The inhibitory effect of combined treatment with NP-252 (10(-6) M) and nitroglycerin (10(-6) M) on nifedipine-resistant Ca(++)-contraction in the cerebral artery was additive. These results indicate that NP-252 possesses a stronger vasoinhibitory effect than that of nifedipine in canine cerebral artery.

Animals↗
PubMed1991

Structure and expression of rat S-100 beta subunit gene.

The gene structure for S-100 beta subunit has been elucidated. The gene spans about 8 kbp and consists of 3 exons and 2 introns. The transcription initiation site was determined by an S1 nuclease mapping. The promoter region contains TATA-box-like and CAAT-box-like sequences. To examine the activity of the promoter sequence, a transfection of pS100 beta-lacZ fused gene to the cultured cells was carried out. C6 glioma cells showed a positive expression of beta-galactosidase. Gene-deletion experiments suggested the functional importance of the DNA fragment (22 bp) containing TATA-box-like and CAAT-box-like sequences. A factor protein that binds to the 100 bp DNA fragment containing the promoter sequence was specifically detected in the rat brain nuclear extract.

Animals↗