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Biomedical subjects

A Tsujimura

Publications and source records attributed to A Tsujimura.

At least 19 recordsLinked to original sources
PubMed2002

Susceptibility locus for non-obstructive azoospermia is localized within the HLA-DR/DQ subregion: primary role of DQB1*0604.

Non-obstructive azoospermia is a male infertility characterized by no or little sperm in semen as a result of a congenital dysfunction in spermatogenesis. Previous studies have reported a higher prevalence of particular human leukocyte antigen (HLA) antigens in non-obstructive azoospermia. As the expression of the RING3 gene located in the HLA class II region was predominant in the testis, mainly around spermatids and pachytene spermatocytes, it is tempting to speculate that RING3 is one of the strong candidate genes responsible for the pathogenesis of the disease. In this study, the genetic polymorphism in the RING3 gene was investigated by the direct sequencing technique. As a result, a total of 14 single nucleotide polymorphisms were identified. Among them, six were localized in the coding region but none of them was accompanied by an amino-acid substitution. No significant difference in the allelic distribution at these 14 polymorphic sites was observed between the patients and healthy controls, suggesting that the susceptible gene for non-obstructive azoospermia is not the RING3 gene. Then, in order to map the susceptibility locus for non-obstructive azoospermia precisely within the HLA region, 11 polymorphic microsatellite markers distributed from the SACM2L gene just outside the HLA class II region (187 kb telomeric of the DPB1 gene) to the OTF3 gene in the HLA class I region were subjected to association analysis in the patients. Statistical analysis of distribution in the allelic frequency at each microsatellite locus demonstrated that the pathogenic gene for non-obstructive azoospermia is located within the HLA-DR/DQ subregion. In fact, DRB1*1302 and DQB1*0604 were found to be strongly associated with non-obstructive azoospermia by polymerase chain reaction-based DNA typing. Further, haplotype analysis suggested that the DQB1*0604 allele may play a decisive role in the pathogenesis of non-obstructive azoospermia.

Alleles↗
PubMed2002

Conventional multiple or microdissection testicular sperm extraction: a comparative study.

BACKGROUND: Testicular sperm extraction (TESE) with ICSI is becoming the first-line treatment for non-obstructive azoospermia (NOA). Recently, the sperm retrieval rate (SRR) by microdissection TESE was reported to be higher than by conventional TESE. However, a comprehensive comparison between multiple and microdissection TESE patients including histological findings has not been reported. METHODS: Patients with NOA who underwent microdissection TESE (n = 56) or multiple TESE (n = 37) were compared. Pre-operative characteristics were similar between groups. In addition, microscopic findings during microdissection TESE also were investigated. RESULTS: Operative time was significantly longer for microdissection TESE than for multiple TESE. Histological examination suggested that spermatogenesis was relatively more impaired in the microdissection TESE group than in the multiple TESE group. Despite this, SRR by microdissection TESE (42.9%) appeared higher than by conventional TESE (35.1%) although this observation failed to reach statistical significance. Seventeen of 26 patients (65.4%) with heterogeneous tubule were successful for sperm retrieval. No severe operative complications occurred in any patient in either group, and no patient required post-operative hormone replacement to treat hypogonadism. CONCLUSIONS: Microsurgical technique is safe and may improve SRR for TESE in a variety of patients with NOA, especially patients with heterogeneous testicular tubules.

Adult↗
PubMed2001

Withdrawal from nicotine facilitates diazepam binding inhibitor mRNA expression in mouse cerebral cortex.

Changes in diazepam binding inhibitor (DBI) mRNA expression after withdrawal from nicotine were examined. Withdrawal from nicotine Increased DBI mRNA expression in cerebral cortices derived from nicotine-dependent mice and in the neurons continuously exposed to nicotine (0.1 microM). These results indicate that withdrawal from nicotine after its long-term exposure induces steep increase of DBI mRNA expression as reported previously in ethanol- and morphine-dependent animals.

Animals↗
PubMed2001

NMDA receptor activation enhances diazepam binding inhibitor and its mRNA expressions in mouse cerebral cortical neurons.

Effects of N-methyl-D-aspartate (NMDA) on diazepam binding inhibitor (DBI) and its mRNA expression in mouse cerebral cortical neurons were examined. A significant increase in DBI mRNA expression was observed 1 day after the exposure to 0.1 microM NMDA and the maximal expression occurred 2 days after the exposure, whereas transient exposure to 0.1 microM NMDA for 15 min, 1 and 3 h produced no changes in the expression. Similarly, no changes in the expression were found by the concomitant exposure to NMDA and MK-801, a NMDA receptor antagonist, for 72 h subsequent to the incubation with NMDA alone for 3 h. Such NMDA-induced increases in DBI mRNA expression were dose-dependently inhibited by MK-801. Moreover, neuronal DBI content significantly increased by treatment with NMDA, which was completely abolished by MK-801. These results indicate that continuous activation of NMDA receptors is an essential factor for increasing DBI expression in the neurons.

Animals↗
PubMed2001

Alterations in cerebral diazepam binding inhibitor expression in drug dependence: a possible biochemical alteration common to drug dependence.

Mechanisms for formation of drug dependence and expression of withdrawal syndrome have not fully clarified despite of huge accumulation of experimental and clinical data at present. Several clinical features of withdrawal syndrome are considered to be common among patients with drug dependence induced by different drugs of abuse. One of them is anxiety. Recent investigations have revealed that diazepam binding inhibitor (DBI), a peptide consisting of 87 amino acids with molecular weight of about 10 kDa, serves as an inverse agonist for benzodiazepine (BZD) receptors with endogenously anxiogenic potential. These lines of data suggest that cerebral DBI expression in brain may participates in formation of drug dependence and/or emergence of withdrawal syndrome. Based on this working hypothesis, we have examined DBI expression in the brain derived from mice depended on alcohol (ethanol), nicotine, and morphine to investigate functional relationship between cerebral DBI expression and drug dependence. Cerebral DBI expression significantly increases in animals with drug dependence induced by these drugs, and in the cases of nicotine- and morphine-dependent mice concomitant administration of antagonists for nicotinic acetylcholine and opioid receptors, respectively, abolished the increase. Abrupt cessation of administration of drugs facilitated further increase in DBI expression. Therefore, these alterations in DBI expression have close relationship with formation of drug dependence and/or emergence of withdrawal syndrome, and are considered to be a common biochemical process in drug dependence induced by different drugs of abuse. Finding and elucidation of mechanisms for common biochemical alterations among drug dependence may provide a clue to clarify mechanisms for formation of drug dependence and/or emergence of withdrawal syndrome.

Alcoholism↗
PubMed2001

Ultrastructural comparison of uterine leiomyoma cells from the same myoma nodule before and after gonadotropin-releasing hormone agonist treatment.

OBJECTIVE: To compare ultrastructural features of leiomyoma cells from the same uterine myoma nodule before and after GnRH agonist (GnRH-a) treatment and to examine the relation between these ultrastructural changes and the extent of myoma volume reduction with GnRH-a treatment. DESIGN: Prospective clinical study. SETTING: University teaching hospital. PATIENT(S): Twenty women with uterine leiomyomas who were scheduled for surgery. INTERVENTION(S): Transcervical needle biopsy of uterine myoma, s.c. leuprolide acetate injection (3.75 mg) at least three times every 4 weeks before surgery, and hysterectomy or myomectomy. MAIN OUTCOME MEASURE(S): The changes in ultrastructural features of uterine leiomyoma cells and the percentage decrease in the volume of the largest myoma at 12 weeks of GnRH-a treatment measured by magnetic resonance imaging. RESULT(S): A decrease in myofilaments, mitochondrial swelling, and emergence of the lysosomal body were observed in relation to GnRH-a treatment. Furthermore, a positive correlation was observed between the decrease in myofilaments and myoma shrinkage. CONCLUSION(S): Cellular atrophy due to a decrease in myofilaments plays a major role in the reversible myoma shrinkage resulting from GnRH-a treatment.

Adult↗
PubMed2001

Effects of hepatocyte growth factor on E-cadherin-mediated cell-cell adhesion in DU145 prostate cancer cells.

OBJECTIVES: To examine how hepatocyte growth factor (HGF) affects cell-cell adhesion junctions on scattering in prostate cancer cells. HGF is known to induce scattering (dispersion of clustered cells into single cells) in various epithelial cells, including prostate cancer cells, but the mechanisms surrounding this action are not fully understood. Cell-cell adhesion junctions are composed of E-cadherin and its associated intracellular catenins and play important roles in the maintenance of cell integrity. METHODS: The human prostate cancer cell line DU145 was used in this study. The associations and changes of various adhesion molecules with HGF treatment were investigated by inhibition assays, Western blot analysis, and immunofluorescence staining. RESULTS: In the inhibition assay, anti-E-cadherin neutralizing monoclonal antibody caused the dissociation of DU145 cells similar to the scattering with HGF treatment. The expression of E-cadherin decreased with HGF, and the expression of alpha-catenin and beta-catenin did not change by Western blot analysis. In immunofluorescence staining, HGF caused the translocation of E-cadherin from cell-cell adhesion junctions to the cytoplasm. CONCLUSIONS: These results indicate that HGF induces scattering by decreasing the expression of E-cadherin and causes its translocation to the cytoplasm of DU145 cells.

Cadherins↗
PubMed2001

Three soluble form messages of murine CD46 are produced through alternative mRNA splicing.

Murine CD46 (mCD46) is a type 1 membrane protein expressed predominantly in testicular germ cells, the distribution profile of which is in contrast to that of human CD46 showing a ubiquitous tissue distribution. We have identified an additional message of mCD46 that encodes a putative secretory form [Nomura et al. (1999) Immunogenetics 50, 245-254]. Here, we cloned three cDNAs encoding putative soluble CD46 from murine testis. These soluble form messages were yielded on insertion of unidentified nucleotide sequences, 77, 179, and 73 ntds, into the junctions between the SCR3 and SCR4 (variant 2), ST(c) and UK (variant 3), and SCR4 and ST(c) (variant 1) domains, respectively, the last one corresponding to the reported soluble form. The exons corresponding to these three inserts were identified in the murine CD46 genome, indicating that the alternative splicing of mRNA participates in the generation of these various CD46 messages. In normal mouse sera and cell lines, however, virtually no soluble CD46 was detected on immunoblotting. On Northern blotting analysis with specific probes, on the other hand, variant 1 was found to be predominantly expressed in the liver and heart. In addition, all variant messages were detected on PCR in all organs examined. When a rabbit cell line, RK13 cells, was transfected with cDNA of variant 1, protein synthesis was detected on immunoblotting. Although the mCD46 protein production was inefficient, this variant 1 exhibited factor I-cofactor activity as to inhibition of the complement cascade. Since the mCD46 protein was reported to be markedly up-regulated on infection of murine cells with mCMV, the soluble mCD46 proteins may act as a complement regulator that controls the systemic complement system under the conditions of a viral infection.

Alternative Splicing↗
PubMed2001

Genomic analysis of idiopathic infertile patients with sperm-specific depletion of CD46.

Three infertile subjects with no expression of CD46 (membrane cofactor protein of complement) on their spermatozoa were found when screening 542 idiopathic male infertile patients. The sperm CD46 isoform was reported to be associated with the sperm-egg interaction, yet a ubiquitous expression of CD46 confers resistance to complement-mediated injury on host cells. All three patients expressed normal CD46 isoforms on their lymphocytes and granulocytes. Thus, the loss of CD46 is sperm-specific, probably due to testicular germ cell-specific regulation of CD46 production. Recently, a mechanism of the gene regulation of human CD46 was elucidated in which the silencer element of the 3' UT and the promoter region of human CD46 gene partly participate. Here, we analyzed these regions of the CD46 gene in our 3 patients. We found no abnormality in 3' and 5' regions of the CD46 genome in the 3 patients. Thus, in these infertile patients sperm-specific depletion of CD46 is not governed by the so for identified regulators in the CD46 gene. Other unknown factors outside the known regulatory regions would play a role in the regulation of sperm-specific CD46 expression.

3' Untranslated Regions↗
PubMed2001

Association of the shrinkage of uterine leiomyoma treated with GnRH agonist and deletion of long arm of chromosome 7.

Specific chromosomal abnormalities, such as del(7q), t(12;14), 12 trisomy, or the rearrangement of 6p, are seen in approximately 30% of uterine leiomyomas despite their benign status. We investigated the association between the shrinkage of uterine leiomyomas treated with a GnRH agonist and the interstitial deletion of chromosome 7q, which is one of the most common chromosomal abnormalities in uterine leiomyomas. This study covered 29 women with uterine leiomyomas who were treated with a GnRH agonist before surgery. The volume of the largest myoma nodule was measured by means of MRI before and at 12 weeks after the beginning of GnRH agonist treatment, and the percentage of the reduction in volume was calculated. Genomic DNA was extracted from leiomyoma tissue and peripheral blood, and amplified by PCR using fluorescently-tagged oligonucleotide primers of twelve microsatellite loci on chromosome 7. The PCR products were analyzed for loss of heterozygosity (LOH) using an automated fluorescent DNA sequencer. Of the 29 informative tumors, five (17%) showed LOH with deletion of the common region, D7S491. The mean percentages of the reduction in volume of the largest myomas with LOH or without LOH were 32+/-13 and 18+/-58%, respectively (not significant). One tumor showing interstitial deletion of both alleles (homo-deletion) reduced in volume by 19%. Another tumor showing an extraband increased in volume by 13%. Although tumor specific chromosomal deletion suggested the existence of tumor suppressor genes in this region, there was no significant association between the shrinkage of uterine leiomyomas treated with a GnRH agonist and the interstitial deletion of chromosome 7q.

Adult↗
PubMed2000

Identification and characterization of a silencer regulatory element in the 3'-flanking region of the murine CD46 gene.

The murine membrane cofactor protein (CD46) gene is expressed exclusively in testis, in contrast to human CD46, which is expressed ubiquitously. To elucidate the mechanism of differential CD46 gene expression among species, we cloned entire murine CD46 genomic DNA and possible regulatory regions were placed in the flanking region of the luciferase reporter gene. The reporter gene assay revealed a silencing activity not in the promoter, but in the 3'-flanking region of the gene and the silencer-like element was identified within a 0.2-kb region between 0.6 and 0.8 kb downstream of the stop codon. This silencer-like element was highly similar to that of the pig MHC class-I gene. The introduction of a mutation into this putative silencer element of murine CD46 resulted in an abrogation of the silencing effect. Electrophoretic mobility-shift assay indicated the presence of the binding molecule(s) for this silencer sequence in murine cell lines and tissues. A size difference of the protein-silencer-element complex was observed depending upon the solubilizers used for preparation of the nuclear extracts. A mutated silencer sequence failed to interact with the binding molecules. The level of the binding factor was lower in the testicular germ cells compared with other organs. Thus the silencer element and its binding factor may play a role in transcriptional regulation of murine CD46 gene expression. These results imply that the effects of the CD46 silencer element encompass the innate immune and reproductive systems, and in mice may determine the testicular germ-cell-dominant expression of CD46.

3T3 Cells↗
PubMed2000

Mechanism for increase in expression of cerebral diazepam binding inhibitor mRNA by nicotine: involvement of L-type voltage-dependent calcium channels.

We investigated the mechanisms underlying the increase in diazepam binding inhibitor (DBI) and its mRNA expression induced by nicotine (0.1 microM) exposure for 24 h using mouse cerebral cortical neurons in primary culture. Nicotine-induced (0.1 microM) increases in DBI mRNA expression were abolished by hexamethonium, a nicotinic acetylcholine (nACh) receptor antagonist. Agents that stabilize the neuronal membrane, including tetrodotoxin (TTX), procainamide (a Na(+) channel inhibitor), and local anesthetics (dibucaine and lidocaine), dose-dependently inhibited the increased expression of DBI mRNA by nicotine. The nicotine-induced increase in DBI mRNA expression was inhibited by L-type voltage-dependent Ca(2+) channel (VDCC) inhibitors such as verapamil, calmodulin antagonist (W-7), and Ca(2+)/calmodulin-dependent protein kinase II (CAM II kinase) inhibitor (KN-62), whereas P/Q- and N-type VDCC inhibitors showed no effects. In addition, nicotine exposure for 24 h induced [3H]nicotine binding to the particulate fractions of the neurons with an increased B(max) value and no changes in K(d). Under these conditions, the 30 mM KCl- and nicotine-induced 45Ca(2+) influx into the nicotine-treated neurons was significantly higher than those into non-treated neurons. These results suggest that the nicotine-stimulated increase in DBI mRNA expression is mediated by CAM II kinase activation resulting from the increase in intracellular Ca(2+) through L-type VDCCs subsequent to the neuronal membrane depolarization associated with nACh receptor activation.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed2000

A set of temperature sensitive-replication/-segregation and temperature resistant plasmid vectors with different copy numbers and in an isogenic background (chloramphenicol, kanamycin, lacZ, repA, par, polA).

A set of plasmid vectors conferring chloramphenicol resistance (Cm(R)), 3064bp in size, or kanamycin resistance (Km(R)), 2972bp in size, were developed, having multiple cloning sites in lacZ' genes for alpha-complementation. pTH18cs1, pTH19cs1, pTH18ks1 and pTH19ks1 are temperature-sensitive (ts) in DNA replication (ts-Rep); pTH18cs5, pTH19cs5, pTH18ks5 and pTH19ks5 are ts in plasmid segregation (ts-Seg); and pTH18cr, pTH19cr, pTH18kr and pTH19kr are temperature resistant (tr) in both. They are based on the pSC101 replicon consisting merely of the replication origin and repA gene, compatible with ColE1/pMB1/p15-derived plasmids, and thus do not require polA function of host cells. The copy numbers of the ts-Rep, tr and ts-Seg plasmids were 14, 5 and 1 per chromosome at 30 degrees C, respectively. These plasmids are fairly stable when inherited at 30 degrees C, but not above 37 degrees C or 41.5 degrees C, depending on the repA mutations and host strains. They are isogenic apart from the ts mutations in the repA gene, and thus provide with useful tools for having appropriate controls in various experiments including bacterial gene-targeting, transposon mutagenesis, toxic gene expression, differential substitution on host functions, gene dosage analysis and so on.

Bacterial Proteins↗
PubMed2000

Generation of active TGF-beta by prostatic cell cocultures using novel basal and luminal prostatic epithelial cell lines.

Two prostatic epithelial lines, one of basal origin and one of luminal origin, were established from the dorsolateral prostates of p53 null mice. The cell lines are nontumorigenic when inoculated subcutaneously under the renal capsule or intraprostatically in syngeneic mice. The luminal cell line (PE-L-1) expresses cytokeratins 8 and 18 and the basal cell line (PE-B-1) expresses cytokeratins 5 and 14. The basal cells require serum for growth, whereas the luminal cells grow only in serum-free medium. Both cell lines require the presence of growth factors for optimal growth in culture, with EGF and FGF-2 having the greatest effect on the growth rate. Both lines express androgen receptor (AR) mRNA and protein. Androgen stimulates growth of the basal cell line, indicating that the ARs are functional, whereas growth of the luminal cells is unaffected by androgens. The luminal line is significantly inhibited by exogenous TGF-beta and produces low levels of endogenous TGF-beta. In contrast, the basal cell line produces significant amounts of TGF-beta and its growth is not influenced by this cytokine. Coculture of luminal cells with prostatic smooth muscle cells results in the generation of increased levels of biologically active TGF-beta, indicating a paracrine mechanism of TGF-beta activation that may be involved in the maintenance of normal prostatic function. To our knowledge this is the first report describing both basal and luminal prostatic cell lines from a single inbred animal species and the first indication that prostatic epithelial and stromal cells interact to generate the biologically active form of TGF-beta. These lines will provide an important model for determining basal/luminal interactions in both in vitro and in vivo assays.

Animals↗
PubMed2000

Transforming growth factor-beta is an autocrine mitogen for a novel androgen-responsive murine prostatic smooth muscle cell line, PSMC1.

A prostatic smooth muscle cell line (PSMC1) was established from the dorsolateral prostate of p53 null mice. The cell line is nontumorigenic when inoculated subcutaneously, under the renal capsule or intraprostatically in syngeneic mice. These cells express alpha-smooth muscle actin (alpha-SMA), indicating their smooth muscle origin, and TGF-beta significantly enhances expression of alpha-SMA. The cells express both androgen receptor (AR) mRNA and protein, and respond mitogenically to physiological concentrations of androgens. PSMC1 cells produce significant amounts of TGF-beta, which stimulates growth by an autocrine mechanism. Dihydrotestosterone (DHT) increases proliferation of PSMC1 cells by promoting TGF-beta secretion. Considering the significant inhibitory effect of TGF-beta on prostatic epithelial cells and its stimulatory effect on the PSMC1 cells, we postulate that TGF-beta produced by prostatic smooth muscle cells may have a paracrine effect on the prostatic epithelium. We also postulate that TGF-beta may be involved in the etiology of benign prostatic hyperplasia (BPH) by stimulating excessive stromal proliferation. Line PSMC1 is the first reported androgen-responsive murine smooth muscle cell line. It will be useful for in vivo and in vitro experiments to study the mechanisms of androgen action on prostatic stroma and for delineating the interactions that occur between prostatic smooth muscle and epithelium that may lead to prostatic diseases such as BPH.

Androgens↗
PubMed2000

Neutropenia accompanying parvovirus B19 infection after gynecologic surgery.

The hematologic data and symptoms of 7 patients seropositive for parvovirus B19 IgM antibody after gynecologic surgery were analysed. Parvovirus may have been transmitted by fibrin glue prepared from heat-treated human plasma and used for hemostasis during surgery. The peripheral blood neutrophil count decreased to below 1 x 10(9)/l between postoperative day (POD) 10 and 18, but recovered spontaneously to within the normal range. G-CSF injection was effective in preventing neutropenia or obtaining a prompt recovery. The reticulocyte count fell below 10 x 10(9)/l between POD 13 and 19, and also recovered spontaneously. A slapped-cheek rash was not observed in any of the 7 patients.

Adolescent↗
PubMed2000

[Role of hepatocyte growth factor in invasion of prostate cancer cell lines through tumor-stromal interaction].

We examined how prostate stromal cell-derived hepatocyte growth factor (HGF) affects invasion of prostate cancer cells through tumor-stromal interaction. The effects of HGF, various growth factors [transforming growth factor (TGF)-alpha, TGF-beta 1, basic fibroblast growth factor, keratinocyte growth factor, and platelet-derived growth factor], and conditioned medium (CM) from prostate stromal cells (PrSC) on prostate cancer cells (LNCaP, PC-3 and DU145) were determined by collagen gel invesion assay. DU145 cells and PrSC were co-cultured for matrigel invasion chamber assay. LNCaP and PC-3 cells did not respond to any of the factors examined. Invasion of DU145 cells into the collagen gel matrix was induced by HGF and TGF-beta 1, but not by any of the other factors tested. When DU145 cells were cultured in CM from PrSC or co-cultured with PrSC, the cells acquired invasive potential, and this invasion was inhibited by an antibody against HGF, but not against TGF-beta 1. Induction activity of CM from cancer cells to stimulate HGF production by PrSC was studied by ELISA method and Western blotting. Native type HGF production in PrSC was enhanced by some unknown inducer(s) produced by cancer cells. In summary, PrSC-derived HGF enhanced invasive activity of the prostate cancer cell line DU145 through tumor-stromal interaction wherein DU145 cells secreted some HGF-inducer(s) for PrSC.

Cell Communication↗