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Biomedical subjects

M Yoshida

Publications and source records attributed to M Yoshida.

At least 559 records · Page 31Linked to original sources
PubMed1999

Neuronal differentiation of neuro 2a cells by inhibitors of cell cycle progression, trichostatin A and butyrolactone I.

Trichostatin A (TSA, 17 nM), a specific and reversible inhibitor of histone deacetylase induced neurite network formation at and after 4 days. The networks were preserved for at least 3 weeks in the presence of TSA. Butyrolactone I (BLI, 23.6 microM), an inhibitor of cdc2 and cdk2 kinases, also induced neurite extension. Both compounds enhanced the acetylcholinesterase activity of the cells. Cell cycle progression of the cells was blocked by TSA (17 nM) at G1 phase alone. Furthermore, the level of histone hyperacetylation and p21(WAF1) expression in TSA-treated cells increased transiently. These findings suggest that the induction of the neuronal differentiation in Neuro 2a cells by these agents requires the cell cycle arrest at G1 phase, which is caused by inhibition of cycline dependent kinase, a target molecule of BLI and p21(WAF1).

4-Butyrolactone↗
PubMed1999

Pharmacological effects of tolterodine on human isolated urinary bladder.

Tolterodine, (R)-N,N-diisopropyl-3-(2-hydroxy-5-methylphenyl)-3-phenylpropanamine+ ++, is an antimuscarinic drug developed for the treatment of overactive bladder with symptoms of frequency, urgency and urge incontinence. We investigated the effects of tolterodine and its major active metabolite, DD 01 (PNU-200577), (R)-N,N-diisopropyl-3-(2-hydroxy-5-hydroxymethylphenyl)-3-phenylpropa namine, on the contractions induced by carbachol, KCl, CaCl2 and electrical field stimulation in human isolated urinary bladder smooth muscles, using the muscle bath technique. Specimens of human urinary bladder were obtained from 20 patients who underwent total cystectomy due to malignant bladder tumor. The detrusor preparations were taken from the intact part of the dome region of the bladder. Carbachol (10(-9)-10(-2) M) caused concentration-dependent contraction of human detrusor smooth muscles. Tolterodine (10(-9)-10(-6) M), DD 01 (10(-9)-10(-6) M), oxybutynin (10(-8)-10(-6) M), propiverine (10(-8)-10(-6) M), atropine (10(-9)-10(-6) M), pirenzepine (10(-8)-10(-5) M), methoctramine (10(-8)-10(-5) M) and 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) (10(-9)-10(-6) M) caused typical shifts to the right of the concentration-response curves for carbachol, except for higher concentrations (10(-5) M) of oxybutynin and propiverine, which caused a decrease of about 30% of the maximum contractile responses to carbachol. All the slopes of the regression lines of Schild plots were close to unity, and the rank order of pA2 values was: atropine = DD 01 = tolterodine = 4-DAMP = oxybutynin > propiverine = pirenzepine > methoctramine. Tolterodine (10(-9)-10(-6) M) and DD 01 (10(-9)-10(-6) M) did not inhibit the KCl-induced (80 mM) and CaCl2-induced (5 mM) contractions, while oxybutynin (10(-8)-10(-5) M) and propiverine (10(-8)-10(-5) M) significantly inhibited the contractions. Electrical field stimulation (2-60 Hz) caused frequency-dependent contraction of human detrusor smooth muscles, which were significantly inhibited by various drugs. In the presence of 10(-6) M atropine, tolterodine and DD 01 did not inhibit the residual contractions induced by electrical field stimulation at any of the frequencies, while oxybutynin (10(-5) M) and propiverine (10(-5) M) significantly inhibited the atropine-resistant part of the contractions. The results suggest that the inhibitory effects of tolterodine and DD 01 are mediated only by their antimuscarinic action, which is equal to that of oxybutynin and significantly greater than that of propiverine, and that tolterodine and DD 01 have neither Ca2+ channel antagonist action nor inhibitory effect on the atropine-resistant part of the contractions in human detrusor smooth muscles. These findings support the usefulness of tolterodine as a therapeutic drug for overactive bladder with symptoms of frequency, urgency and urge incontinence.

Aged↗
PubMed1999

Cross-linking of two beta subunits in the closed conformation in F1-ATPase.

In the crystal structure of mitochondrial F1-ATPase, two beta subunits with a bound Mg-nucleotide are in "closed" conformations, whereas the third beta subunit without bound nucleotide is in an "open" conformation. In this "CCO" (beta-closed beta-closed beta-open) conformational state, Ile-390s of the two closed beta subunits, even though they are separated by an intervening alpha subunit, have a direct contact. We replaced the equivalent Ile of the alpha3beta3gamma subcomplex of thermophilic F1-ATPase with Cys and observed the formation of the beta-beta cross-link through a disulfide bond. The analysis of conditions required for the cross-link formation indicates that: (i) F1-ATPase takes the CCO conformation when two catalytic sites are filled with Mg-nucleotide, (ii) intermediate(s) with the CCO conformation are generated during catalytic cycle, (iii) the Mg-ADP inhibited form is in the CCO conformation, and (iv) F1-ATPase dwells in conformational state(s) other than CCO when only one (or none) of catalytic sites is filled by Mg-nucleotide or when catalytic sites are filled by Mg2+-free nucleotide. The alpha3beta3gamma subcomplex containing the beta-beta cross-link retained the activity of uni-site catalysis but lost that of multiple catalytic turnover, suggesting that open-closed transition of beta subunits is required for the rotation of gamma subunit but not for hydrolysis of a single ATP.

Adenine Nucleotides↗
PubMed1999

Evolution of visual pigments in geckos.

Most geckos are nocturnal forms and possess rod retinas, but some diurnal genera have pure-cone retinas. We isolated cDNAs encoding the diurnal gecko opsins, dg1 and dg2, similar to nocturnal gecko P521 and P467, respectively. Despite the large morphological differences between the diurnal and nocturnal gecko photoreceptor types, they express phylogenetically closely related opsins. These results provide molecular evidence for the reverse transmutation, that is, rods of an ancestral nocturnal gecko have backed into cones of diurnal geckos. The amino acid substitution rates of dgl and dg2 are higher than those of P521 and P467, respectively. Changes of behavior regarding photic environment may have contributed to acceleration of amino acid substitutions in the diurnal gecko opsins.

Amino Acid Sequence↗
PubMed1999

Mitochondrial phospholipid hydroperoxide glutathione peroxidase plays a major role in preventing oxidative injury to cells.

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is synthesized as a long form (L-form; 23 kDa) and a short form (S-form; 20 kDa). The L-form contains a leader sequence that is required for transport to mitochondria, whereas the S-form lacks the leader sequence. A construct encoding the leader sequence of PHGPx tagged with green fluorescent protein was used to transfect RBL-2H3 cells, and the fusion protein was transported to mitochondria. The L-form of PHGPx was identified as the mitochondrial form of PHGPx and the S-form as the non-mitochondrial form of PHGPx since preferential enrichment of mitochondria for PHGPx was detected in M15 cells that overexpressed the L-form of PHGPx, whereas no similar enrichment was detected in L9 cells that overexpressed the S-form. Cell death caused by mitochondrial injury due to potassium cyanide (KCN) or rotenone (chemical hypoxia) was considerably suppressed in the M15 cells, whereas the L9 cells and control RBL-2H3 cells (S1 cells, transfected with the vector alone) succumbed to the cytotoxic effects of KCN. Flow cytometric analysis showed that mitochondrial PHGPx suppressed the generation of hydroperoxide, the loss of mitochondrial membrane potential, and the loss of plasma membrane integrity that are induced by KCN. Mitochondrial PHGPx might prevent changes in mitochondrial functions and cell death by reducing intracellular hydroperoxides. Mitochondrial PHGPx failed to protect M15 cells from mitochondrial injury by carbonyl cyanide m-chlorophenylhydrazone, which directly reduces membrane potential without the generation of hydroperoxides. M15 cells were more resistant than L9 cells to cell death caused by direct damage to mitochondria and to extracellular oxidative stress. L9 cells were more resistant to tert-butylhydroperoxide than S1 cells, whereas resistance to t-butylhydroperoxide was even more pronounced in M15 cells than in L9 cells. These results suggest that mitochondria might be a target for intracellular and extracellular oxidative stress and that mitochondrial PHGPx, as distinct form non-mitochondrial PHGPx, might play a primary role in protecting cells from oxidative stress.

Animals↗
PubMed1999

beta-subunit of nuclear pore-targeting complex (importin-beta) can be exported from the nucleus in a Ran-independent manner.

The nuclear export of importin-alpha is mediated by CAS, which is related to importin-beta, whereas the mechanism for the export of importin-beta remains unclear. In this study, we demonstrate that the nuclear export of importin-beta is mediated by the nuclear pore complex-binding domain of this molecule. Insensitivity to leptomycin B indicates that its export is not mediated by a leucine-rich nuclear export signal-specific receptor, CRM1. Furthermore, the nuclear export of importin-beta was not inhibited by co-injection with a GTPase-deficient Ran mutant (G19V). The cell line tsBN2 contains a temperature-sensitive point mutation in the RCC1 gene, which encodes a guanine nucleotide exchange factor of Ran. At the nonpermissive temperature, importin-beta was exported from the nucleus of these cells, even when RanGAP1, a GTPase-activating protein for Ran, was co-injected. These results not only provide support for the view that Ran-dependent GTP hydrolysis is not required for the nuclear export of importin-beta but also indicate that nuclear RanGTP is not essential for its export. As a result, we propose that importin-beta can be recycled from the nucleus alone in a Ran-independent manner.

Animals↗
PubMed1999

Trans-activator Tax of human T-cell leukemia virus type 1 enhances mutation frequency of the cellular genome.

Tax protein of human T-cell leukemia virus type 1 (HTLV-1) is a potent trans-activator of specific sets of target genes, and on the other hand, it is a trans-repressor of other sets of genes. It is also an inhibitor of the tumor suppressor protein p16(INK4a) and thus has been thought to contribute to induction of adult T-cell leukemia (ATL). We examined the mutagenic effects of Tax on a cellular gene, hypoxanthine guanine phosphoribosyltransferase (hprt), and LacI gene in lambda shuttle vector exogenously integrated in Big Blue Rat-2 (BBRat-2) cells. Expression of Tax in BBRat-2 cells enhanced the frequency of HPRT(-) phenotype severalfold. Tax-expressing cell clones, BBTax-1 and -2 established from BBRat-2 cells, gave rise to an average mutation frequency of 5.9 x 10(-5) in LacI gene, but Tax-negative cell clones, BBRat-C1 and -C2, showed 2. 1 x 10(-5). The 2.8-fold increase in mutation frequency in the presence of Tax indicates that Tax expression enhanced mutation frequency in chromosomal DNA. However, neither the mutation spectrum of base transitions, transversions, and deletions/insertions nor the loci of the mutations were significantly affected by Tax expression. These findings indicate that Tax has the capability to induce random mutations and suggest that Tax would be able to modulate cellular phenotypes through mutation of the cellular genome.

Animals↗
PubMed1999

Differences in DNA recognition and conformational change activity between boxes A and B in HMG2 protein.

High mobility group (HMG) 2 is a sequence-nonspecific DNA-binding protein consisting of a repeat of DNA-binding domains called HMG1/2 boxes A and B and an acidic C-terminal. To understand the mode of HMG2 interaction with DNA, we expressed various HMG2 peptides containing HMG1/2 box(es) in Escherichia coli cells and purified them. Gel retardation and DNA supercoiling assay indicated that the region essential for the preferential binding of HMG2 with negatively supercoiled DNA and DNA unwinding activity is located in box B, but not sufficient alone. The flanking C-terminal basic region or box A linked by a linker region is necessary to express activities. The SPR measurements certified that the intrinsic DNA binding affinity of box B is weaker (Kd = 170 microM), and these adjoining regions largely strengthen the affinity (Kd </= 1.2 microM). In contrast, box A, even in the presence of the adjoining basic linker region, showed no such activities, indicating that boxes A and B are different in their DNA recognition mode. The computer modeling suggested that the side chain of Phe-102 in box B is inserted into the base stack to cause DNA conformational changes, while the side chain of Ala-16 in box A is too small to intercalate. These represent that boxes A and B have similar tertiary structures but their activities for DNA conformational changes obviously differ. Box B is the main region for DNA recognition and conformational changes, and box A must play an assistant to increase its DNA recognition.

Animals↗
PubMed1999

Post marketing surveillance of 13 products: safety and effectiveness.

We surveyed the safety and effectiveness of 13 drug products under the reexamination system in Japan. Until the time of reexamination, a total of 106,328 case reporting forms were collected in the drug use investigations, while 1340 adverse drug reactions (ADRs) were reported in the spontaneous case reporting on ADRs. In the drug use investigations, the data were primarily obtained on the incidence of unexpected ADRs and the incidence of ADRs in special patient populations. The spontaneously reported ADR provided the data on serious and unexpected ADRs. We established proper use of the 13 products by examining the data for significant risk factors for serious ADRs, including agranulocytosis, jaundice, hypotension, and QT prolongation. We revised the package inserts of the 13 products with the new safety information a total of 50 times and passed on the revisions to health care professionals. We also assessed the safety and effectiveness of the 13 products compared with that before marketing. We summarized the data and submitted them to the Japanese Ministry of Health and Welfare (JMHW). For all 13 products, the same indications and dosage and administration as those approved previously were approved at the reexamination.

Journal Article↗
PubMed1999

Cholera toxin increases intracellular pH in B lymphoma cells and decreases their antigen-presenting ability.

Cholera toxin (CT) can function as a potent adjuvant in the mucosal immune response. However, we have found that treatment of A20-HL murine B lymphoma cells with CT severely inhibits the presentation of ovalbumin (OVA) to cells of the T cell clone 42-6A specific for OVA(323-339)/I-Ad, whereas it does not affect the presentation of OVA(323-339) peptide. CT treatment did not affect the expression of B7-1, B7-2, ICAM-1, LFA-1 or MHC class II on, or the internalization of OVA into A20-HL cells. In CT-treated A20-HL cells, degradation of OVA was decreased, and intracellular pH was raised to a level approximately equivalent to that in CH3NH2-treated cells. Treatment with CH3NH2 is known to raise the pH in endocytic structures and thus inhibits antigen processing. Treatment of A20-HL cells with dibutyryl-cAMP similarly increased intracellular pH. The increase in intracellular pH following CT treatment was inhibited by a cAMP inhibitor, 2',3'-dideoxyadenosine. These results strongly suggest that CT treatment of A20-HL cells inhibits their antigen-presenting cell function by triggering the cAMP cascade, increasing intracellular pH, and reducing the degradation of OVA.

Animals↗
PubMed1999

Evolution of visual pigments and related molecules.

The molecular phylogenetic tree of vertebrate visual pigments, constructed on the basis of amino acid sequence identity, suggests that the visual pigments can be classified into five groups (L, ML, MS, S and Rh) and that their genes have evolved along these five gene lines. Goldfish has a UV-sensitive visual pigment (S group) localized in miniature single cone cells. Medaka has one type of rod cell containing rhodopsin (Rh group) and four types of cone cells, each of which contains a specific visual pigment with an absorption maximum that differs from those of the others. Frogs have a violet-sensitive visual pigment (S group) in small single cone cells and a blue-sensitive visual pigment (MS group) in green rod cells. Although nocturnal and diurnal geckos have rod- and cone-based retinas, respectively, they have phylogenetically closely related visual pigments. The pigments in each line may have restricted absorption maxima. We have cloned cDNAs encoding molecules involved in the phototransduction system of visual cells, such as phosphodiesterase, opsin kinase and arrestin. We then constructed phylogenetic trees of these molecules with the deduced amino acid sequences. The resulting phylogenetic trees show that these molecules are classified into two groups; one is expressed in cones and another in rods, suggesting that rods and cones contain homologous molecules with different amino acid sequences. These differences may result in the different light responses of rods and cones.

Animals↗
PubMed1999

A histone deacetylase inhibitor, trichostatin A, suppresses myofibroblastic differentiation of rat hepatic stellate cells in primary culture.

Hepatic stellate cells are the major cellular sources of extracellular matrix in chronic liver diseases leading to fibrosis. We explored the antifibrogenic effect of two histone deacetylase inhibitors, sodium butyrate and trichostatin A (TSA), on this cell type in vitro. Primary hepatic stellate cells as well as culture activated cells were exposed to butyrate (0.01-1 mmol/L) or TSA (1-100 nmol/L); their effect on collagen types I and III and smooth muscle alpha-actin was examined by quantitative immunoprecipitation and by Northern analysis. Their antiproliferative effect was examined by 3H-thymidine incorporation and cell counting. Hyperacetylation of histones was demonstrated by acid urea/Triton-X-100 (AUT) polyacrylamide gel electrophoresis. Possible cytotoxic effects were judged on stellate cells by evaluating de novo total protein synthesis, and on hepatocytes by measuring lactate dehydrogenase (LDH) leakage, albumin secretion, and epoxide hydrolase and ethoxycoumarin O-deethylase activity. TSA at 100 nmol/L and butyrate at 1 mmol/L retarded the morphological changes characteristic for activation of primary stellate cells. TSA at 100 nmol/L inhibited synthesis of collagen types I and III and smooth muscle alpha-actin by 62%, 70%, and 88%. Butyrate at 1 mmol/L showed a modest inhibitory effect on collagen type III and smooth muscle alpha-actin, but had no effect on collagen type I. Northern analysis suggested that these inhibitory effects on collagen type III and smooth muscle alpha-actin were transcriptional, while the effect on collagen type I was largely posttranscriptional. At 100 nmol/L, TSA strongly suppressed proliferation of primary hepatic stellate cells. Inhibition of activation of stellate cells was preceded by hyperacetylation of histone H4. When tested on cells at day 14 in culture, butyrate had no inhibitory effects on the synthesis of collagens or smooth muscle alpha-actin. One hundred or 10 nmol/L TSA modestly inhibited the synthesis of collagens type I (-24%,-22%) and III (-34%,-22%), and smooth muscle alpha-actin (-27%,-12%). We conclude that TSA inhibits transdifferentiation of stellate cells into myofibroblasts by interfering with the level of acetylation of histone H4.

Actins↗
PubMed1999

Postictal blockade of ischemic hippocampal neuronal death in primates using selective cathepsin inhibitors.

This paper is to study the participation of cathepsin in ischemic neuronal death of the monkey hippocampal cornu ammonis (CA) 1 sector and also to clarify whether its selective inhibitor epoxysuccinyl peptides such as CA-074 and E-64c can inhibit the neuronal death or not. In the preceding reports, we demonstrated mu-calpain activation and subsequent rupturing of the lysosomal membrane of postischemic CA1 neurons and also increase of enzyme activity of cathepsins B and L in monkeys undergoing a complete 20-min whole brain ischemia. Here, morphological, immunohistochemical and enzymatical analyses were performed to examine the efficacy of two selective cathepsin inhibitors in the postictal blockade of delayed neuronal death in the monkey hippocampus. Both inhibitors could significantly decrease enzyme activities of cathepsins B and L in all hippocampal sectors. When CA-074 was intravenously administered immediately after the ischemic insult, approximately 67% of CA1 neurons were saved from delayed neuronal death on day 5 after ischemia. In contrast, when E-64c was similarly administered, approximately 84% of CA1 neurons were saved from delayed neuronal death on day 5. The surviving neurons showed mild central chromatolysis and negligible immunoreactivity for cathepsins B and L. These observations indicate that the use of cathepsin inhibitors may become novel strategy for prevention of ischemic delayed neuronal death in the primate hippocampus.

Animals↗
PubMed1999

Purification, crystallization, and preliminary X-ray crystallographic analysis of thermus thermophilus V(1)-ATPase B subunit.

The gene of V(1)-ATPase B subunit from the thermophilic eubacterium Thermus thermophilus has been cloned and the protein overproduced in Escherichia coli. The purified protein, with a molecular weight of 53.2 kDa, was crystallized from 10% (w/v) polyethylene glycol 1000, 120 mM magnesium chloride, and 100 mM Na-tricine, pH 8.0, by the vapor diffusion method. The crystals diffracted X-rays beyond 3.5 A on a synchrotron radiation source. The crystals belong to the monoclinic space group C2, with unit cell dimensions of a = 153.1 A, b = 129.6 A, c = 92.7 A, and beta = 100.3 degrees. Assuming that three or four molecules are contained in an asymmetric unit, the V(M) value is calculated as 2.8 or 2.1 A (3)/Da, respectively.

Crystallization↗
PubMed1999

Mild hypothermia for temporary brain ischemia during cardiopulmonary support systems: report of three cases.

Recovery without residual neurological damage after cardiac arrest with temporary cerebral ischemia is rare. Therefore, it is most important that every effort is made to prevent brain damage occurring immediately after successful cardiopulmonary resuscitation. We report herein the cases of three patients who suffered either cardiogenic or hypovolemic shock and were resuscitated by a cardiopulmonary support system followed by mild hypothermia. All three patients recovered completely without any neurologic damage. The outcomes of these three patients demonstrated that mild hypothermia may be important for cerebral preservation after cardiopulmonary resuscitation.

Aged↗
PubMed1999

Transabdominal inguinal hernioplasty combined with abdominal aortic aneurysm repair.

The number of patients being encountered with abdominal aortic aneurysm (AAA) and inguinal hernia is increasing. We describe herein a technique of performing a concomitant one-stage operation for both disorder. After conventional transperitoneal AAA repair, transabdominal preperitoneal hernia repair is carried out through the same incision using a prosthesis made from the same material as the graft used for AAA. The maneuver is similar to that of laparoscopic hernioplasty. We employed this technique in the treatment of four patients, none of whom developed any complications such as infection or recurrence of the inguinal hernia. Thus, we conclude that this one-stage operation for AAA and inguinal hernia may bring physical and economic benefits to patients who have both diseases concomitantly.

Aged↗