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Biomedical subjects

H Kasai

Publications and source records attributed to H Kasai.

At least 73 records · Page 4Linked to original sources
PubMed2000

Molecular cloning of the bovine MYOC and induction of its expression in trabecular meshwork cells.

PURPOSE: Myocilin gene (MYOC) was identified as one of the disease-causing genes of primary open-angle glaucoma. This study was conducted to establish a system for the investigation of the biological role of MYOC in vitro by using bovine eyes, which are easy to obtain and have been widely used to examine the aqueous outflow system. The cDNA sequence of the bovine MYOC was determined and its expression in bovine eyes was examined with a quantitative polymerase chain reaction (PCR) assay. METHODS: Bovine MYOC cDNA was obtained from cultured bovine trabecular meshwork cells, and part of its sequence was determined using a primer pair designed based on the known sequence of the human MYOC gene. The 3' and 5' ends of this sequence were determined using the method of 3' and 5' rapid amplification of cDNA ends. The induction of the MYOC gene in cultured bovine trabecular meshwork cells after exposure to dexamethasone was quantitatively examined with real-time quantitative PCR using a probe designed according to the sequence of the determined bovine MYOC gene. RESULTS: Bovine MYOC protein was composed of 490 amino acids, which was 81.6% identical with that of human MYOC protein. Most of the amino acid residues of which mutation was reported to cause glaucoma were conserved in the bovine MYOC protein. After 2 weeks of treatment with 500 nM dexamethasone, expression of bovine MYOC mRNA was amplified 14-fold (14.1+/-5.1-fold, mean +/- SEM) measured by real-time quantitative PCR. CONCLUSIONS: The cDNA sequence of the bovine MYOC gene had a high degree of similarity to that of the human MYOC gene. Investigation of the function of bovine MYOC may contribute to identifying the role of MYOC protein in the aqueous outflow system.

Amino Acid Sequence↗
PubMed2000

[Problems of ALS patients in home health care].

We cared for nine amyotrophic lateral sclerosis (ALS) patients at home during the period from April 1995 to May 2000. Three patients died without mechanical ventilation. Four patients were supported by home mechanical ventilation but two others refused or could not clearly express their desire. It is important that ALS patients should received accurate knowledge of this disease from doctors, who should ask them repeatedly whether they want mechanical ventilation or not.

Aged↗
PubMed2000

[Shift to home treatment and the function of pharmacies].

A crisis point has been reached in the medical health system in Japan, where there has been a rapid aging of the population. The method of providing health system benefits is being reconsidered. As a result, medical treatment has been classified into inpatient, outpatient, and home care systems according to treatment functions. In concrete terms, the insurance system now classifies medical treatment and care separately, payment for medical fees has changed from payment at piece rates to a fixed amount system, and the number of days patients stay in hospital has been reduced. It is predicted that hospital treatment will increasingly rely on early discharge, and patients highly reliant on medical treatment will be shifted to home treatment. For this shift to home treatment, therefore, supplies of injectable agents such as used in total parenteral nutrition, and equipment by specialist pharmacies have become necessary. However, to dispense injectable agents, "standards of compounded sterile pharmaceuticals by licensed pharmacies" is applied, meaning there are very few pharmacies that can meet these standards. In the present survey, we found that 40 pharmacies have been recognized as meeting these standards.

Home Care Services, Hospital-Based↗
PubMed2000

[Role of the pharmacy for home cancer patients].

Otsukadai Pharmacy compounded morphine chloride for injection in 32 cancer patients. In 22 cases we compounded morphine into a TPN bag. However we recommend using an infusion pump to control the patient's pain at home. We think that we need a system with a pharmacy for cancer patients before they leave the hospital for home care.

Female↗
PubMed1999

Formation of 5-formyl-2'-deoxycytidine from 5-methyl-2'-deoxycytidine in duplex DNA by Fenton-type reactions and gamma-irradiation.

5-methyl-2'-deoxycytidine (5-Me-dC) is formed by the enzymatic methylation of dC, primarily in CpG sequences in DNA, and is involved in the regulation of gene expression. In the present study, 5-Me-dC and double-stranded DNA fragments containing 5-Me-dC were either gamma-irradiated or aerobically treated with Fenton-type reagents, Fe(II)-EDTA, Fe(II)-nitrilotriacetic acid, Fe(III)-EDTA-H(2)O(2)-catechol or ascorbic acid-H(2)O(2) under neutral conditions. The formation of 5-formyl-2'-deoxycytidine (5-CHO-dC) was observed upon treatment of both 5-Me-dC and DNA fragments containing 5-Me-dC. The yields of 5-CHO-dC from 5-Me-dC and those of 5-formyl-2'-deoxyuridine from dT were comparable. These results suggest that 5-Me-dC in DNA is as susceptible to oxidation as dT in cells, and raise the possibility that 5-CHO-dC may contribute to the high mutagenic rate observed in CpG sequences in genomic DNA.

Ascorbic Acid↗
PubMed1999

Olfactory neurons expressing closely linked and homologous odorant receptor genes tend to project their axons to neighboring glomeruli on the olfactory bulb.

We have characterized two separate odorant receptor (OR) gene clusters to examine how olfactory neurons expressing closely linked and homologous OR genes project their axons to the olfactory bulb. Murine OR genes, MOR28, MOR10, and MOR83, share 75-95% similarities in the amino acid sequences and are tightly linked on chromosome 14. In situ hybridization has demonstrated that the three genes are expressed in the same zone, at the most dorsolateral and ventromedial portions of the olfactory epithelium, and are rarely expressed simultaneously in individual neurons. Furthermore, we have found that olfactory neurons expressing MOR28, MOR10, or MOR83 project their axons to very close but distinct subsets of glomeruli on the medial and lateral sides of the olfactory bulb. Similar results have been obtained with another murine OR gene cluster for A16 and MOR18 on chromosome 2, sharing 91% similarity in the amino acid sequences. These results may indicate an intriguing possibility that olfactory neurons expressing homologous OR genes within a cluster tend to converge their axons to proximal but distinct subsets of glomeruli. These lines of study will shed light on the molecular basis of topographical projection of olfactory neurons to the olfactory bulb.

Amino Acid Sequence↗
PubMed1999

NADH shuttle system regulates K(ATP) channel-dependent pathway and steps distal to cytosolic Ca(2+) concentration elevation in glucose-induced insulin secretion.

The NADH shuttle system is composed of the glycerol phosphate and malate-aspartate shuttles. We generated mice that lack mitochondrial glycerol-3-phosphate dehydrogenase (mGPDH), a rate-limiting enzyme of the glycerol phosphate shuttle. Application of aminooxyacetate, an inhibitor of the malate-aspartate shuttle, to mGPDH-deficient islets demonstrated that the NADH shuttle system was essential for coupling glycolysis with activation of mitochondrial ATP generation to trigger glucose-induced insulin secretion. The present study revealed that blocking the NADH shuttle system severely suppressed closure of the ATP-sensitive potassium (K(ATP)) channel and depolarization of the plasma membrane in response to glucose in beta cells, although properties of the K(ATP) channel on the excised beta cell membrane were unaffected. In mGPDH-deficient islets treated with aminooxyacetate, Ca(2+) influx through the plasma membrane induced by a depolarizing concentration of KCl in the presence of the K(ATP) channel opener diazoxide restored insulin secretion. However, the level of the secretion was only approximately 40% of wild-type controls. Thus, glucose metabolism through the NADH shuttle system leading to efficient ATP generation is pivotal to activation of both the K(ATP) channel-dependent pathway and steps distal to an elevation of cytosolic Ca(2+) concentration in glucose-induced insulin secretion.

Animals↗
PubMed1999

Kinetic control of multiple forms of Ca(2+) spikes by inositol trisphosphate in pancreatic acinar cells.

The mechanisms of agonist-induced Ca(2+) spikes have been investigated using a caged inositol 1,4,5-trisphosphate (IP(3)) and a low-affinity Ca(2+) indicator, BTC, in pancreatic acinar cells. Rapid photolysis of caged IP(3) was able to reproduce acetylcholine (ACh)-induced three forms of Ca(2+) spikes: local Ca(2+) spikes and submicromolar (<1 microM) and micromolar (1-15 microM) global Ca(2+) spikes (Ca(2+) waves). These observations indicate that subcellular gradients of IP(3) sensitivity underlie all forms of ACh-induced Ca(2+) spikes, and that the amplitude and extent of Ca(2+) spikes are determined by the concentration of IP(3). IP(3)-induced local Ca(2+) spikes exhibited similar time courses to those generated by ACh, supporting a role for Ca(2+)-induced Ca(2+) release in local Ca(2+) spikes. In contrast, IP(3)- induced global Ca(2+) spikes were consistently faster than those evoked with ACh at all concentrations of IP(3) and ACh, suggesting that production of IP(3) via phospholipase C was slow and limited the spread of the Ca(2+) spikes. Indeed, gradual photolysis of caged IP(3) reproduced ACh-induced slow Ca(2+) spikes. Thus, local and global Ca(2+) spikes involve distinct mechanisms, and the kinetics of global Ca(2+) spikes depends on that of IP(3) production particularly in those cells such as acinar cells where heterogeneity in IP(3) sensitivity plays critical role.

Acetylcholine↗
PubMed1999

Possible involvement of a novel STAM-associated molecule "AMSH" in intracellular signal transduction mediated by cytokines.

STAM containing an SH3 (Src homology 3) domain and an immunoreceptor tyrosine-based activation motif was previously revealed to be implicated in signaling pathways immediately downstream of Jak2 and Jak3 tyrosine kinases associated with cytokine receptors. We molecularly cloned a novel molecule interacting with the SH3 domain of STAM, which was named AMSH (associated molecule with the SH3 domain of STAM). AMSH contains a putative bipartite nuclear localization signal and a homologous region of a c-Jun activation domain-binding protein 1 (JAB1) subdomain in addition to a binding site for the SH3 domain of STAM. AMSH mutant deleted of the C-terminal half conferred dominant negative effects on signaling for DNA synthesis and c-myc induction mediated by interleukin 2 and granulocyte macrophage-colony-stimulating factor. These results suggest that AMSH plays a critical role in the cytokine-mediated intracellular signal transduction downstream of the Jak2/Jak3.STAM complex.

Adaptor Proteins, Signal Transducing↗
PubMed1999

The oxidized forms of dATP are substrates for the human MutT homologue, the hMTH1 protein.

The possibility that Escherichia coli MutT and human MTH1 (hMTH1) hydrolyze oxidized DNA precursors other than 8-hydroxy-dGTP (8-OH-dGTP) was investigated. We report here that hMTH1 hydrolyzed 2-hydroxy-dATP (2-OH-dATP) and 8-hydroxy-dATP (8-OH-dATP), oxidized forms of dATP, but not (R)-8,5'-cyclo-dATP, 5-hydroxy-dCTP, and 5-formyl-dUTP. The kinetic parameters indicated that 2-OH-dATP was hydrolyzed more efficiently and with higher affinity than 8-OH-dGTP. 8-OH-dATP was hydrolyzed as efficiently as 8-OH-dGTP. The preferential hydrolysis of 2-OH-dATP over 8-OH-dGTP was observed at all of the pH values tested (pH 7.2 to pH 8.8). In particular, a 5-fold difference in the hydrolysis efficiencies for 2-OH-dATP over 8-OH-dGTP was found at pH 7.2. However, E. coli MutT had no hydrolysis activity for either 2-OH-dATP or 8-OH-dATP. Thus, E. coli MutT is an imperfect counterpart for hMTH1. Furthermore, we found that 2-hydroxy-dADP and 8-hydroxy-dGDP competitively inhibited both the 2-OH-dATP hydrolase and 8-OH-dGTP hydrolase activities of hMTH1. The inhibitory effects of 2-hydroxy-dADP were 3-fold stronger than those of 8-hydroxy-dGDP. These results suggest that the three damaged nucleotides share the same recognition site of hMTH1 and that it is a more important sanitization enzyme than expected thus far.

Bacterial Proteins↗
PubMed1999

Deficient nucleotide excision repair increases base-pair substitutions but decreases TGGC frameshifts induced by methylglyoxal in Escherichia coli.

To investigate the mutation spectrum of a well-known mutagen, methylglyoxal, and the influence of nucleotide excision repair (NER) on methylglyoxal-induced mutations, we treated wild-type and NER-deficient (uvrA or uvrC) Escherichia coli strains with methylglyoxal, and analyzed mutations in the chromosomal lacI gene. In the three strains, the cell death and the mutation frequency increased according to the dose of methylglyoxal added to the culture medium. The frequencies of methylglyoxal-induced base-pair substitutions were higher in the NER-deficient strains than in the wild-type strain, in the presence and absence of mucAB gene. Paradoxically, the frequency of methylglyoxal-induced TGGC frameshifts was higher in the wild-type strain than in the NER-deficient strains. When the methylglyoxal-induced mutation spectra in the presence and absence of mucAB gene are compared, the ratios of base-pair substitutions to frameshifts were increased by the effects of mucAB gene. In the three strains, more than 75% of the base-pair substitutions occurred at G:C sites, independent of the mucAB gene. When the mucAB gene was present, G:C-->T:A transversions were predominant, followed by G:C-->A:T transitions. When the mucAB gene was absent, the predominant mutations differed in the three strains: in the wild-type and uvrC strains, G:C-->A:T transitions were predominant, followed by G:C-->T:A transversions, while in the uvrA strains, G:C-->T:A transversions were predominant, followed by G:C-->A:T transitions. These results suggest that NER may be involved in both the repair and the fixation of methylglyoxal-induced mutations.

Base Pair Mismatch↗
PubMed1999

Grf40, A novel Grb2 family member, is involved in T cell signaling through interaction with SLP-76 and LAT.

We molecularly cloned a new Grb2 family member, named Grf40, containing the common SH3-SH2-SH3 motif. Expression of Grf40 is predominant in hematopoietic cells, particularly T cells. Grf40 binds to the SH2 domain-containing leukocyte protein of 76 kD (SLP-76) via its SH3 domain more tightly than Grb2. Incidentally, Grf40 binds to linker for activation of T cells (LAT) possibly via its SH2 domain. Overexpression of wild-type Grf40 in Jurkat cells induced a significant increase of SLP-76-dependent interleukin (IL)-2 promoter and nuclear factor of activated T cell (NF-AT) activation upon T cell receptor (TCR) stimulation, whereas the COOH-terminal SH3-deleted Grf40 mutant lacked any recognizable increase in IL-2 promoter activity. Furthermore, the SH2-deleted Grf40 mutant led to a marked inhibition of these regulatory activities, the effect of which is apparently stronger than that of the SH2-deleted Grb2 mutant. Our data suggest that Grf40 is an adaptor molecule involved in TCR-mediated signaling through a more efficient interaction than Grb2 with SLP-76 and LAT.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Correlation of antiangiogenic and antitumor efficacy of N-biphenyl sulfonyl-phenylalanine hydroxiamic acid (BPHA), an orally-active, selective matrix metalloproteinase inhibitor.

The antiangiogenic activity and antitumor efficacy of a newly developed matrix metalloproteinase (MMP) inhibitor were examined. N-biphenyl sulfonyl-phenylalanine hydroxiamic acid (BPHA) potently inhibits MMP-2, -9, and -14, but not MMP-1, -3, or -7. In contrast, (-)BPHA, an enantiomer of BPHA, was inactive against all MMPs tested. Daily oral administration of 200 mg/kg BPHA, but not (-)BPHA in mice resulted in potent inhibition of tumor-induced angiogenesis, primary tumor growth, and liver metastasis. The growth inhibition activity of BPHA was 48% and 45% in a B16-BL6 melanoma and F2 hemangio-endothelioma model, respectively. BPHA also showed 42% inhibition of the liver metastasis of C-1H human colon carcinoma cells. These results indicate that selective MMP inhibition is correlated with antiangiogenic and antitumor efficacy and that the selective MMP inhibitor BPHA has therapeutic potential.

Animals↗
PubMed1999

Multiple kinetic components and the Ca2+ requirements of exocytosis.

The use of caged-Ca2+ compounds to stimulate Ca(2+)-dependent exocytosis has substantially increased our understanding of this complex process. By this approach, the existence of multiple kinetic components of exocytosis has been established. These components may correspond to a series of sequential steps that lead to a single fusion-ready state (sequential mechanism) or, alternatively, to heterogeneity in secretory vesicles or in fusion-ready states (parallel mechanism). It is suggested that both of these mechanisms can underlie exocytosis of a single type of vesicle (mixed sequential-parallel mechanism). Studies with caged-Ca2+ compounds have also indicated that the Ca2+ requirement for exocytosis is substantially greater than that suggested by conventional methodologies. This discrepancy is mainly attributable to the underestimation, by imaging studies with high-affinity Ca2+ indicators (due to dye saturation), of the local increases in cytosolic Ca2+ concentration that trigger the exocytosis of individual vesicles. The effects of local saturation of such indicators are explored by means of a simple theory.

Animals↗
PubMed1999

Role of NADH shuttle system in glucose-induced activation of mitochondrial metabolism and insulin secretion.

Glucose metabolism in glycolysis and in mitochondria is pivotal to glucose-induced insulin secretion from pancreatic beta cells. One or more factors derived from glycolysis other than pyruvate appear to be required for the generation of mitochondrial signals that lead to insulin secretion. The electrons of the glycolysis-derived reduced form of nicotinamide adenine dinucleotide (NADH) are transferred to mitochondria through the NADH shuttle system. By abolishing the NADH shuttle function, glucose-induced increases in NADH autofluorescence, mitochondrial membrane potential, and adenosine triphosphate content were reduced and glucose-induced insulin secretion was abrogated. The NADH shuttle evidently couples glycolysis with activation of mitochondrial energy metabolism to trigger insulin secretion.

Adenosine Triphosphate↗
PubMed1999

Multiple and diverse forms of regulated exocytosis in wild-type and defective PC12 cells.

Regulated exocytosis triggered by the photolysis of a caged Ca2+ compound, DM-nitrophen, was investigated by patch-clamp capacitance measurements in two clones of PC12, the first wild-type and the second (PC12-27) defective of both types of classical secretory vesicles together with the neuronal-type receptors for the attachment proteins of the N-ethylmaleimide-sensitive fusion protein, the so called SNAREs. Moreover, the electrophysiological data were correlated with the ultrastructure of resting quick-frozen-freeze-dried cells of the two clones. Wild-type PC12 exhibited two-component capacitance responses, time constants of 30-100 ms and >10 s, that previous studies had suggested to reflect primarily the fusion of the small and large secretory vesicles, each contributing cell surface increases of approximately 10%. Both of these components were largely and specifically inhibited whether cells previously were microinjected with tetanus toxin light chain. In the defective clone, large responses also were recorded ( approximately 19% surface expansion; time constant, approximately 1 s) that, in contrast to those of the wild-type, were entirely resistant to the toxin. Although secretory organelles, i.e., large vesicles and also profiles of small vesicles, were abundant at the cell periphery and often docked to the plasmalemma of resting wild-type PC12, in the defective clone, no superficial accumulation of vesicles was observed. Our coordinate structural and functional results have revealed diversities between the two classical forms of regulated secretion in wild-type PC12 and have provided evidence of a toxin-insensitive form of Ca2+-induced exocytosis, prominent in the defective clone, that may play an important role(s) in cellular physiology.

Acetates↗
PubMed1999

Post-priming actions of ATP on Ca2+-dependent exocytosis in pancreatic beta cells.

The role of cytosolic ATP in exocytosis was investigated by using amperometric measurement of insulin exocytosis in pancreatic beta cells, which were stimulated with photolysis of caged Ca2+ compounds. Insulin exocytosis occurred with two rates. We found that ATP hastened and augmented the exocytosis via selective enhancement of the exocytosis with the faster rate. A nonhydrolysable analog of ATP, adenosine 5'-O-(3-thiotriphosphate), which blocks ATPase, was even more effective than ATP, indicating that the phosphorylation event occurred downstream of ATP-dependent vesicle transportation and priming. The action of ATP was eliminated by a competitive antagonist of cAMP, and by an inhibitor of adenylate cyclase. These data characterize an ATP sensing mechanism for the Ca2+-dependent exocytosis involving adenylate-cyclase, cAMP-dependent protein kinase, and, possibly, the fusion machinery itself. Thus, the fast exocytotic machinery requires both phosphorylation and Ca2+ for the final triggering and likely constitutes a distal ATP sensor for insulin exocytosis that acts in concert with ATP-sensitive K+ channels.

Adenosine Triphosphate↗