Biomedical subjects

S Kojima

Publications and source records attributed to S Kojima.

At least 181 records · Page 10Linked to original sources

PubMed1999

Isolation and characterization of rice MADS box gene homologues and their RFLP mapping.

Thirty-five MADS box gene homologues were identified through a large-scale cDNA analysis in rice. Based on the nucleotide sequences of the 3'-untranslated region, these clones were classified into 11 independent species. Seven species were found to be new among the rice MADS box gene family, and the other 4 corresponded to the previously reported OsMADS1, OsMADS2, OsMADS4, and OsMADS5. The full nucleotide sequences of the 7 new species were determined. Each clone encoded a deduced protein of 164-267 amino acids. The K-domain of the MADS protein was conserved in all clones though with lower degree in clone S10304. Reverse transcription PCR analysis showed that clones E31254 and E31864 were expressed mainly in panicles. Dendrogram analysis suggested that E31254 and E31864 are close to Arabidopsis AGL9 and AP1, respectively. Restriction fragment length polymorphism (RFLP) linkage mapping revealed that the rice MADS box gene homologues reported here are not clustered but are located throughout the genome. The locus of E31864 on the RFLP map was closely linked to the long sterile lemma gene, g-1.

Amino Acid Sequence↗

PubMed1999

Optical detection of synaptically induced glutamate transport in hippocampal slices.

Although it has long been believed that glial cells play a major role in transmitter uptake at synapses in the CNS, the relative contribution of glial and neuronal cells to reuptake of synaptically released glutamate has been unclear. Recent identification of the diverse glutamate transporter subtypes provides an opportunity to examine this issue. To monitor glutamate transporter activity, we optically detected synaptically induced changes of membrane potential from hippocampal CA1 field in slice preparations using a voltage-sensitive dye, RH155. In the presence of ionotropic glutamate-receptor blockers, synaptic inputs gave rise to a slow depolarizing response (SDR) in the dendritic field. The amplitude of SDR correlated well with presynaptic activities, suggesting that it was related to transmitter release. The SDR was found to be caused by the activities of glutamate transporters because it was not affected by blockers for GABAA, nACh, 5-HT3, P2X, or metabotropic glutamate receptors but was greatly reduced by dihydrokainate (DHK), a specific blocker for GLT-1 transporter, and by D, L-threo-beta-hydroxyaspartate (THA), a blocker for EAAC, GLAST, and GLT-1 transporters. When SDR was detected with RH482 dye, which stains both glial and neuronal cells, 1 mM DHK and 1 mM THA were equally effective in suppressing SDR. The SDR was very small in GLT-1 knockout mice but was maintained in gerbil hippocampi in which postsynaptic neurons were absent because of ischemia. Because GLT-1 transporters are exclusively expressed in astrocytes, our results provide direct evidence that astrocytes play the dominant role in sequestering synaptically released glutamate.

ATP-Binding Cassette Transporters↗

PubMed1999

Molecular cloning of rat GADD45gamma, gene induction and its role during neuronal cell death.

To study the molecular mechanism of neuronal cell death, we carried out the screening of genes which were induced during the neuronal cell death of neuronal PC12. We cloned the cDNA of rat GADD45gamma, the third member of the GADD45 family. Induction of GADD45gamma mRNA was observed in the neuronal cell death caused by depletion of neurotrophic factor and Ca2+ ionophore treatment. Overexpression of GADD45gamma in superior cervical ganglion neurons caused cell death. These results suggest that GADD45gamma plays an important role in neuronal cell death.

Amino Acid Sequence↗

PubMed1999

Involvement of the C-terminal region of yeast proteinase B inhibitor 2 in its inhibitory action.

Yeast proteinase B inhibitor 2 (YIB2), which is composed of 74 amino acid residues, is an unusual serine protease inhibitor, since it lacks disulfide bonds. To identify its reactive site for proteases, we constructed an expression system for a synthetic YIB2 gene and then attempted to change the inhibitory properties of YIB2 by amino acid replacements. The purified wild-type YIB2 inhibited the activity of subtilisin BPN', a protein homologous to yeast proteinase B, although its binding ability was not strong, and a time-dependent decrease in its inhibitory activity was observed, demonstrating that wild-type YIB2 behaves as a temporary inhibitor when subtilisin BPN' is the target protease. Since YIB2 exhibits sequence homology to the propeptide of subtilisin, which inhibits a cognate protease using its C-terminal region, we replaced the six C-termi nal residues of YIB2 with those of the propeptide of subtilisin BPN' to make the mutant YIB2m1. This mutant exhibited markedly increased inhibitory activity toward subtilisin BPN' without a time-dependent decrease in its inhibitory activity. Replacement of only the C-terminal Asn of YIB2 by Tyr, or deletion of the C-terminal Tyr of YIB2m1, inhibited subtilisin, but the ability of these mutants to bind subtilisin and their resistance to proteolytic attack were weaker than those of YIB2m1, indicating that the C-terminal residue contributes to the interaction with the protease to a greater extent than the preceding five residues and that the resistance of YIB2 to proteolyic attack is closely related to its ability to bind a protease. These results demonstrate that YIB2 is a unique protease inhibitor that involves its C-terminal region in the interaction with the protease.

Amino Acid Sequence↗

PubMed1999

Na+-driven flagellar motor resistant to phenamil, an amiloride analog, caused by mutations in putative channel components.

The rotation of the Na+-driven flagellar motor is specifically and strongly inhibited by phenamil, an amiloride analog. Here, we provide the first evidence that phenamil interacts directly with the Na+-channel components (PomA and PomB) of the motor. The alterations in Mpar (motility resistant to phenamil) strains were mapped to the pomA and/or pomB genes. We cloned and sequenced pomA and pomB from two Mpar strains, NMB205 and NMB201, and found a substitution in pomA (Asp148 to Tyr; NMB205) and in pomB (Pro16 to Ser; NMB201). Both residues are predicted to be near the cytoplasmic ends of the putative transmembrane segments. Mutational analyses at PomA-Asp148 and PomB-Pro16 suggest that a certain structural change around these residues affects the sensitivity of the motor to phenamil. Co-expression of the PomA D148Y and PomB P16S proteins resulted in an Mpar phenotype which seemed to be less sensitive to phenamil than either of the single mutants, although motility was more severely impaired in the absence of inhibitors. These results support the idea that PomA and PomB interact with each other and suggest that multiple residues, including Asp148 of PomA and Pro16 of PomB, constitute a high-affinity phenamil-binding site at the inner face of the PomA/PomB channel complex.

Amiloride↗

PubMed1999

Prognostic value of internal tandem duplication of the FLT3 gene in childhood acute myelogenous leukemia.

BACKGROUND: Recently, an internal tandem duplication of the FLT3 gene (FLT3/ITD) was found in 20% of adult cases of acute myelogenous leukemia (AML), and this length abnormality was suggested to be associated with leukemic progression. PROCEDURE: We examined the mRNA expression of the FLT3 gene by using reverse transcription-polymerase chain reaction (RT-PCR) in 64 children with AML, and further abnormal transcripts were cloned and sequenced. RESULTS: An unexpected longer product was found in seven patients (11%) by RT-PCR of the FLT3 gene. Sequence analysis of these abnormal products revealed the presence of tandemly duplicated fragments in all seven patients. Three factors were identified to be associated with high incidence of FLT3/ITD; older patients (> or = 10 years) (P = 0.049), high WBC count (> or = 50,000/microl) at presentation (P = 0.002), and M3 in FAB subtypes (P = 0.002). Induction failure was observed in 3 (43%) of 7 patients with FLT3/ITD. Only FLT3/ITD was identified as a significant risk factor for induction failure by univariate analysis (P = 0.013), although it was not significant by multivariate analysis (P = 0.11). The Kaplan-Meier estimate of event-free survival rate at 5 years was 14% for patients with FLT3/ITD, which was significantly lower in comparison with 69% for patients without FLT3/ITD (P = 0.003). This finding was also identified by multivariate analysis (P = 0.01). CONCLUSIONS: In this study, FLT3/ITD was observed in 11% of childhood AML and identified to be associated with poor prognosis. A large prospective study with uniform treatment is necessary to confirm our results.

Adolescent↗

PubMed1999

Benefit of intravascular ultrasound in Wiktor stent implantation.

The goal of this study was to evaluate retrospectively the efficacy and safety of intravascular ultrasound (IVUS) during Wiktor stent implantation. Until 1996, the success of stent implantation was assessed by angiographic criteria only, but in 1997, the procedure was expanded to include pre- and postprocedural IVUS imaging. Sixty-six patients were included, 28 treated in 1996 (group A) and 38 treated in 1997 (group B). Stent size was larger in group B than in group A (3.6+/-0.4 vs. 3.1+/-0.2, P < 0.001). Acute gain was greater in group B than in group A (2.58+/-0.61 vs. 2.11+/-0.56, P < 0.001). Restenosis rate was 31% in group A and 14% in group B. No major acute complications due to IVUS examination occurred. IVUS is helpful in choosing optimal stent size.

Aged↗

PubMed1999

Expression of a zinc-binding domain of boar spermatidal transition protein 2 in Escherichia coli.

Transition protein 2 (TP2; 137 amino acid residues) from boar late spermatid nuclei has three potential zinc finger motifs in the N-terminal 34 region. Gel shift assays revealed that boar TP2 recognized a CpG island sequence in a zinc-dependent manner. However, there was some nonspecific recognition of the oligonucleotide. Then, we constructed the expression system of zinc-binding domain of TP2 (TP2Z) (residues 1-103) in Escherichia coli. Double-stranded DNA fragments encoding TP2Z were synthesized as 18 fragments with 103 residues, annealed, and cloned into the expression plasmid pET11d. TP2Z was expressed upon induction with 1 mM isopropylthiogalactoside and extracted with acid including 0.71 M 2-mercaptoethanol. TP2Z was purified by ion-exchange chromatography on Fractogel EMD SO(-)(3) and HPLC on Nucleosil 300 7C18 and on Diol-120. Atomic absorption and CD spectroscopy showed that TP2Z bound three atoms of zinc per molecule of the protein and underwent a zinc-dependent conformational change in a manner similar to that for intact TP2. Gel shift assays indicated that TP2Z recognized a CpG island sequence more specifically than intact TP2 and that the specificity is dependent on zinc.

Amino Acid Sequence↗

PubMed1999

The role of Bcl6 in mature cardiac myocytes.

OBJECTIVE: The Bcl6 gene encodes a sequence-specific transcriptional repressor and is ubiquitously expressed in adult murine tissues including heart muscle. The objective of this study was to examine the role of Bcl6 in cardiac myocytes. METHOD: We developed Bcl6-deficient (Bcl6-/-) mice and histologically examined hearts from these mice. RESULTS: Massive myocarditis with eosinophilic infiltration occurred in Bcl6-/- mice after 4-6 weeks of age. Since expression of the Bcl6 gene was induced in normal cardiac myocytes after 2 weeks of age and thereafter detected through adulthood, loss of Bcl6 in mature cardiac myocytes may be related to the induction of eosinophilic myocarditis. To examine the effects of eosinophils from Bcl6-/- mice on normal hearts, bone marrow cells from Bcl6-/- mice were adoptively transferred into sublethally irradiated RAG1-deficient mice. Although massive eosinophilic infiltration was detected in conjunctivas and spleens from the chimeric mice, myocarditis was never observed. Electron microscopic analysis of cardiac myocytes from Bcl6-/- mice revealed a spectrum of degenerative changes prior to eosinophilic infiltration. CONCLUSION: Bcl6 maynot be essential for the maturation of cardiac myocytes but may play a role in protecting mature cardiac myocytes from eosinophilic inflammation.

Adoptive Transfer↗

PubMed1999

Experimental optic cup enlargement caused by endothelin-1-induced chronic optic nerve head ischemia.

PURPOSE: Vascular insufficiency of the optic nerve head may contribute to glaucomatous optic neuropathy, especially in normal-tension glaucoma. We investigated the effect of chronic optic nerve head ischemia, created by repeated intravitreal injection of endothelin-1 (ET-1), on the morphology and function of the optic nerve. METHODS: In pigmented rabbits, we injected ET-1 (10(-6) M, 10 microL) into the posterior vitreous of one eye twice a week for 4 weeks (N = 7). The vehicle for ET-1 was injected into the contralateral eye as a control (N = 7). The subsequent observation period was set at 8 weeks. The microcirculation of the optic nerve head was noninvasively monitored with a laser speckle circulation analyzer. To evaluate the changes of visual function, visual-evoked potentials were recorded. Morphologic changes of the optic nerve head were analyzed with stereography, and the ratio of cup area (CA) to disk area (DA) was measured by calculating the number of pixels in each area with a microcomputer. RESULTS: Capillary blood flow in the optic nerve head was continuously below 80% of the baseline throughout the study. The visual-evoked potential latency was significantly delayed in ET-1-treated eyes. The CA/DA ratio was significantly increased relative to baseline in the ET-1 treated eyes. Histologic examination showed axonal loss and demyelination affecting the prelaminar portion of the optic nerve. The intraocular pressure was not significantly different from the control value. CONCLUSION: Optic nerve head ischemia could contribute to the enlargement and excavation of the disk cup independent of the intraocular pressure level.

Animals↗

PubMed1999

Histological characterization of lip and tentacle nerves in Lymnaea stagnalis.

The lip and tentacle nerves of the pond snail, Lymnaea stagnalis, were characterized using histological techniques. Anatomical drawings showed the detailed distributions of the superior lip, median lip, and tentacle nerves in the lip and tentacle; in particular it was found that the mouth is mainly innervated by the superior lip nerve. The tentacle nerve was clarified to form a zigzag structure along the extension direction in a shrinking tentacle. By backfilling of the superior lip nerve and/or the median lip nerve with fluorescent dyes, the neurons in the CNS made some clusters, whereas those stained from the tentacle nerve made other clusters. These stained neurons were not part of the central pattern generator or its regulatory neurons for feeding. The present results, therefore, suggest that the superior lip nerve may be employed as a principal factor in the chemosensory transduction from the mouth, and that no direct inputs occur through the lip and tentacle nerves to the central pattern generator or its regulatory neurons for feeding.

Animals↗

PubMed1999

Physiological characterization of lip and tentacle nerves in Lymnaea stagnalis.

The lip and tentacle nerves of the pond snail, Lymnaea stagnalis, were characterized using electrophysiological techniques. When the activity of those nerves was induced in lip-tentacle preparations, aversive taste signals were transmitted through all the lip and tentacle nerves, but appetitive signals could be recorded only through the superior lip nerve. In the CNS immersed in high Mg2+ -high Ca2+ saline, electrical stimuli applied to any of the nerves failed to induce action potentials in one of the regulatory neurons (cerebral giant cell: CGC) involved in feeding responses, implying that the signals are polysynaptically transmitted to the CGC. Intracellular recordings revealed that the CGCs in semi-intact half-body preparations received both appetitive and aversive taste signals not only through the superior lip nerve but also through the median lip nerve. In addition, an osphradium was ruled out as a candidate for appetitive reception. The present results, together with our preceding data arrived at by the histochemical analyses, indicate that the appetitive taste transduction responsible for generating feeding responses is performed through the superior lip nerve with some contribution of the median lip nerve. The data showing that the CGC can receive various taste signals suggests that it may play a crucial role in feeding behavior as demonstrated in the study of conditioned taste-aversion.

Animals↗

PubMed1999

Increased 9,13-di-cis-retinoic acid in rat hepatic fibrosis: implication for a potential link between retinoid loss and TGF-beta mediated fibrogenesis in vivo.

BACKGROUND/AIMS: During hepatic fibrosis, hepatic stellate cells (HSCs) transform into myofibroblastic cells and lose their intracellular droplets of retinyl esters, the storage form of vitamin A. Recently, we have demonstrated that 9,13-di-cis-retinoic acid (RA), a geometric isomer identified as a stable and major metabolite of vitamin A in circulation, stimulates the synthesis of plasminogen activator (PA) and induces PA/plasmin-dependent latent transforming growth factor (TGF)-beta activation in HSC cultures, probably via induction and activation of RA receptor (RAR) alpha. The aim of the present study was to address a potential link between the loss of retinyl esters to increased formation of RA(s), which might play a role in facilitating TGF-beta-mediated liver fibrogenesis in vivo. METHODS: We examined the effect of 9,13-di-cis-RA on transactivating activity of RARalpha in HeLa cells as well as its effect on PA- and TGF-beta-dependent collagen synthesis in rat and human HSC cultures. We measured the changes in 9,13-di-cis-RA levels both during activation of rat HSCs in vitro and during porcine serum-induced rat hepatic fibrosis in vivo and correlated this with RAR alpha/beta, PA, TGF-beta and type I procollagen mRNA expression in the fibrotic liver. RESULTS: 9,13-di-cis-RA transactivated RARalpha, and provoked PA/plasmin and TGF-beta-dependent procollagen synthesis in HSCs. 9,13-di-cis-RA levels were increased both in activated HSCs in vitro and in fibrotic liver accompanying the enhanced expression of RAR alpha/beta, PA, TGF-beta and procollagen in vivo. CONCLUSIONS: These findings suggest a potential link between 9,13-di-cis RA formation and hepatic fibrosis via formation of TGF-beta in vivo, and thus provide further insight into the biologic role of retinoids during hepatic fibrogenesis.

Animals↗

PubMed1999

Antimalarial activity of extracts of Malaysian medicinal plants.

In vitro and in vivo studies revealed that Malaysian medicinal plants, Piper sarmentosum, Andrographis paniculata and Tinospora crispa produced considerable antimalarial effects. Chloroform extract in vitro did show better effect than the methanol extract. The chloroform extract showed complete parasite growth inhibition as low as 0.05 mg/ml drug dose within 24 h incubation period (Andrographis paniculata) as compared to methanol extract of drug dose of 2.5 mg/ml but under incubation time of 48 h of the same plant spesies. In vivo activity of Andrographis paniculata also demonstrated higher antimalarial effect than other two plant species.

Animals↗

PubMed1999

Functional expression of the ascofuranone-sensitive Trypanosoma brucei brucei alternative oxidase in the cytoplasmic membrane of Escherichia coli.

Trypanosome alternative oxidase (TAO) is the terminal oxidase of the respiratory chain of long slender bloodstream forms (LS forms) of African trypanosoma, which causes sleeping sickness in human and nagana in cattle. TAO is a cytochrome-independent, cyanide-insensitive quinol oxidase and these properties are quite different from those of the bacterial quinol oxidase which belongs to the heme-copper terminal oxidase superfamily. Only little information concerning the molecular structure and enzymatic features of TAO have been available, whereas the bacterial enzyme has been well characterized. In this study, a cDNA encoding TAO from Trypanosoma brucei brucei was cloned into the expression vector pET15b (pTAO) and recombinant TAO was expressed in Escherichia coli. The growth of the transformant carrying pTAO was cyanide-resistant. A peptide with a molecular mass of 37 kDa was found in the cytoplasmic membrane of E. coli, and was recognized by antibodies against plant-type alternative oxidases from Sauromatum guttatum and Hansenula anomala. Both the ubiquinol oxidase and succinate oxidase activities found in the membrane of the transformant were insensitive to cyanide, while those of the control strain, which contained vector alone, were inhibited. This cyanide-insensitive growth of the E. coli carrying pTAO was inhibited by the addition of ascofuranone, a potent and specific inhibitor of TAO ubiquinol oxidase. The ubiquinol oxidase activity of the membrane from the transformant was sensitive to ascofuranone. These results clearly show the functional expression of TAO in E. coli and indicate that ubiquinol-8 in the E. coli membrane is able to serve as an electron donor to the recombinant enzyme and confer cyanide-resistant and ascofuranone-sensitive growth to E. coli. This system will facilitate the biochemical characterization of the novel terminal oxidase, TAO, and the understanding on the mechanism of the trypanocidal effect of ascofuranone.

Animals↗

PubMed1999

Elevation of antioxidant potency in the brain of mice by low-dose gamma-ray irradiation and its effect on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced brain damage.

The elevation of endogenous thiol-related antioxidants and free radical scavenging enzymes in the brain of C57BL/6 female mice after low-dose gamma-ray irradiation and its inhibitory effect on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced brain damage were investigated. The brain level of the reduced form of glutathione (GSH) increased soon after irradiation with 50 cGy of gamma-rays, reached a maximum at 3 h post-treatment, and remained elevated until 12 h. Thioredoxin (TRX) was also transiently increased after irradiation. The activities of free radical scavenging enzymes, including Cu/Zn-superoxide dismutase, catalase and glutathione peroxidase, were significantly induced after irradiation as well. Cerebral malondialdehyde was remarkably elevated by MPTP treatment, and this elevation was suppressed by pre-irradiation (50 cGy). The contents of GSH and TRX were significantly decreased by MPTP treatment in comparison with those of the control group. These reductions both seemed to be attenuated by pre-irradiation with gamma-rays. These results suggest that low-dose gamma-ray irradiation induces endogenous antioxidative potency in the brain of mice and might be effective for the prevention and/or therapy of various reactive oxygen species-related neurodegenerative disorders, such as Parkinson's disease and Alzheimer's disease.

Analysis of Variance↗

PubMed1999

Advantages of concurrent use of anabolic and antiresorptive agents over single use of these agents in increasing trabecular bone volume, connectivity, and biomechanical competence of rat vertebrae.

The purpose of this study was to evaluate the usefulness of a combination regimen of anabolic and antiresorptive agents in increasing skeletal quantity and quality in comparison to a single-drug regimen with these agents. We examined histomorphometrically and biomechanically the effects of separate and combined administration of intermittent parathyroid hormone (PTH) and estrogen or bisphosphonate on both axial and appendicular skeletons of male Wistar rats, which were 4 months old and weighed approximately 300 g at the beginning of the treatment. The animals were untreated or injected with vehicle, recombinant human PTH(1-84) (PTH; 100 microg/kg daily), 17beta-estradiol (E2, 500 microg/kg every other day), incadronate disodium (YM175, 80 microg/kg every other day), combined PTH and E2 (PTH + E2), or a combination of PTH and YM175 (PTH + YM175). After 1 month of treatment, the three groups in which PTH was administered (PTH, PTH + E2, and PTH + YM175) had significantly higher trabecular bone volume and connectivity in the proximal tibial metaphyses (PTMs) compared with the untreated and vehicle-treated groups, whereas only combination groups (PTH + E2 and PTH + YM175) showed significant increases in these indices in the lumbar vertebrae. This site-related discrepancy was attributed to the fact that PTH significantly elevated bone resorption not in the PTMs but in the vertebrae. This increased bone resorption in the vertebrae was suppressed by the addition of an antiresorptive agent. As a result, trabecular bone mass, connectivity, and mechanical strength of the vertebrae were significantly increased from control levels only in the concurrent treatment groups (PTH + E2 and PTH + YM175). The superior skeletal effects of PTH cotherapy over PTH monotherapy were not seen with regard to bone mass, but with increased connectivity and mechanical strength. The concurrent use of PTH and an antiresorptive agent has been shown to be superior to the single use of PTH for enhancing these properties of rat vertebrae, which encourages future research, especially in larger animals.

Animals↗

PubMed1999

The effect of excipients on the molecular mobility of lyophilized formulations, as measured by glass transition temperature and NMR relaxation-based critical mobility temperature.

PURPOSE: The dependence of the molecular mobility of lyophilized formulations on pharmaceutical polymer excipients was studied. Molecular mobility as determined by NMR relaxation-based critical temperature of molecular mobility (Tmc) and glass transition temperature (Tg) is discussed in relation to the plasticizing effect of water in formulations. METHODS: The Tmc and Tg of lyophilized gamma-globulin formulations containing 6 different polymer excipients such as dextran, polyvinylpyrrolidone (PVP) and methylcellulose (MC) was determined by NMR and DSC. The molecular mobility of water in the formulations was determined by proton NMR and dielectric relaxation spectrometry (DRS). RESULTS: Tmc varied with polymer excipients. Tmc increased as the ratio of bound water to mobile water increased and as the molecular mobility of mobile water decreased. The formulation containing MC exhibited a lower Tmc than the formulation containing dextran because of the smaller ratio of bound water and the higher molecular mobility of mobile water. The Tmc of the formulation containing PVP was higher than that expected from the higher T2 values of water because of the lower molecular mobility of mobile water regardless of the higher ratio of mobile water. The Tmc of these lyophilized formulations was higher than their T(g) by 23 degrees C to 34 degrees C, indicating that the formulations became a NMR-detected microscopically liquidized state below their T(g). CONCLUSIONS: The quantity and the molecular mobility of mobile water in lyophilized formulations can be considered to affect the T(mc) of lyophilized formulations, which in turn governs their stability.

Calorimetry, Differential Scanning↗