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Biomedical subjects

M Tsuchiya

Publications and source records attributed to M Tsuchiya.

At least 55 records · Page 3Linked to original sources
PubMed1999

Isothiazolone derivatives selectively inhibit telomerase from human and rat cancer cells in vitro.

The telomere hypothesis postulates stabilization of telomere length and telomerase activation as key events in cellular immortalization and carcinogeneses. Accordingly, telomerase has been suggested as a novel and highly selective target for design of antitumor drugs. Screening of a chemical library including 16 000 synthetic compounds yielded six that strongly inhibited telomerase activity in extracts of cultured human cells, including four isothiazolone derivatives and two unrelated compounds. The most potent inhibitor was 2-[3-(trifluoromethyl)phenyl]isothiazolin-3-one (TMPI), a concentration of 1.0 microM inhibited telomerase activity by 50% according to a telomere repeat amplification protocol (TRAP) assay. Analysis using partially purified telomerase from AH7974 rat hepatoma cells demonstrated noncompetitive inhibition with the telomere-repeat primer and mixed inhibition with the dNTPs; the inhibition constant was 2.5 microM. TMPI did not inhibit eukaryotic DNA polymerase alpha, beta, or human immunodeficiency virus reverse transcriptase (HIV RT). Thus, inhibition by TMPI was highly selective for telomerase. Inhibition by TMPI was quenched by 1 mM of dithiothreitol or glutathione, suggesting that TMPI inhibits telomerase by acting at a cysteine residue. TMPI inhibition of this enzyme may find application as an antineoplastic agent.

Animals↗
PubMed1999

A novel strategy for identifying differential gene expression: An improved method of differential display analysis.

We propose a novel alternative approach, an advanced method for recently developed strategies, for identifying differentially expressed genes. Firstly, double-stranded cDNAs were digested using Sau3AI and the 3'-end restriction fragments of the cDNA were ligated to a double-stranded adapter. Next, the restriction fragments were directly amplified using several combinations of adapter-specific primers and FITC-labeled oligo dT primers. The selected cDNA fragments were displayed on a polyacrylamide gel. Neither nested PCR nor purification of 3'-end fragments are necessary. We examined the validity of this approach by evaluating gene expression changes during granulocytic differentiation of HL-60 cells. This method can theoretically detect almost all gene expression changes more rapidly and through simpler manipulations than by any other approach.

Cell Differentiation↗
PubMed1999

Control of the expression of luteinizing hormone receptor by local factors in rat granulosa cells.

To identify the mechanisms underlying the hormone-dependent induction and maintenance of luteinizing hormone receptor (LH-R) in rat granulosa cells, the effect of follicle-stimulating hormone (FSH) and local factors on the LH-R mRNA levels were studied. LH-R mRNA levels of the cells incubated with FSH decreased rapidly after medium removal, and readdition of FSH with the fresh medium did not restore these levels. On the other hand, 8-bromoadenosine 3,5-cyclic monophosphate significantly enhanced the expression of LH-R mRNA after medium removal, while the level of LH-R mRNA was lower than that of the cells replaced by original medium including FSH. In addition, the incubation with 8-Br-cAMP produced dose-dependent responses for LH-R mRNAs and enhanced the activity of 1379 bp of the LH-R 5'-flanking region, while the level of LH-R mRNA decreased 3 days after medium removal. Further studies were undertaken to assess the role of factors in maintaining the LH receptor once induced by FSH. Since FSH and cAMP increase follistatin production in granulosa cells, we examined the effect of follistatin on LH-R induction in the presence of activin and FSH. Activin induced LH-R in the presence of FSH significantly, and follistatin antagonized this effect in a dose-dependent manner. However, insulinlike growth factor-I (IGF-I) induced LH-R mRNA in the presence of FSH even after medium change. IGF-I might be one of the important factors that act in the medium to maintain LH-R levels in granulosa cells.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1999

Humanized anti-HM1.24 antibody mediates myeloma cell cytotoxicity that is enhanced by cytokine stimulation of effector cells.

To develop a new immunotherapy for multiple myeloma, we have generated a monoclonal antibody (MoAb) that detects a human plasma cell-specific antigen, HM1.24. Our previous study has shown that mouse anti-HM1.24 MoAb inhibits the proliferation of human myeloma cells implanted into severe combined immunodeficiency mice. In this report, we evaluated the antitumor activity of the humanized anti-HM1.24 MoAb (IgG1kappa), which was constructed by grafting the complementarity-determining regions. In contrast to the parent mouse MoAb, humanized anti-HM1.24 MoAb mediated antibody-dependent cellular cytotoxicity (ADCC) against both myeloma cell lines and myeloma cells from patients in the presence of human peripheral blood mononuclear cells (PBMCs). The PBMCs from untreated myeloma patients exhibited ADCC activity as efficiently as those of healthy donors. Although decreased ADCC activity of PBMCs was observed in patients who responded poorly to conventional chemotherapy, it could be significantly augmented by the stimulation with interleukin-2 (IL-2), IL-12, or IL-15. There was a strong correlation between the percentage of CD16(+) cells and ADCC activity in the PBMCs of myeloma patients. Moreover, peripheral blood stem cell collections from myeloma patients contained higher numbers of CD16(+) cells than PBMCs and exhibited ADCC activity that was enhanced by IL-2. These results indicate that humanized anti-HM1.24 MoAb has potential as a new therapeutic strategy in multiple myeloma and that treatment of effector cells with immunomodulating cytokines can restore the effect of humanized anti-HM1.24 MoAb in patients with diminished ADCC activity.

Adult↗
PubMed1999

Molecular cloning and characterization of a surface antigen preferentially overexpressed on multiple myeloma cells.

HM1.24 antigen has been identified as a surface molecule preferentially expressed on terminally differentiated B cells, and its overexpression is observed in multiple myeloma cells. The HM1.24 antigen is, therefore, expected as a most potent target molecule for antibody-based immunotherapy for multiple myeloma. Here, we have identified the cDNA for human HM1.24 antigen and also analyzed its gene structure including the promoter region. The HM1.24 antigen is a type II membrane glycoprotein, which has been reported as a bone marrow stromal cell surface antigen BST2, and may exist as a homodimer on myeloma cell surface. Although a reason for the overexpression in myeloma cells is not understood, very interestingly, the promoter region of the HM1.24 gene has a tandem repeat of three cis elements for a transcription factor, STAT3, which mediates interleukin-6 (IL-6) response gene expression. Since IL-6 is a differentiation factor for B cells, and known as a paracrine/autocrine growth factor for multiple myeloma cells, the expression of HM1.24 antigen may be regulated by the activation of STAT3. Importantly, a humanized anti-HM1.24 antibody effectively lysed the CHO transformants which expressed HM1.24 antigen as high as human multiple myeloma cells, but not the cells with lower antigen expression. This evaluation shows that ADCC heavily depends on the expression level of target antigens and, therefore, the immunotherapy targeting the HM1.24 antigen should have a promising potential in clinical use.

Amino Acid Sequence↗
PubMed1999

Pharmacokinetics of ezlopitant, a novel non-peptidic neurokinin-1 receptor antagonist in preclinical species and metabolite kinetics of the pharmacologically active metabolites.

The pharmacokinetics of ezlopitant were determined in the rat, gerbil, guinea pig, ferret, dog and monkey after intravenous and oral administration. In general, ezlopitant is marked by high clearance values that approach or exceed hepatic blood flow values, moderate to high values for steady-state volume of distribution (3. 9-28 L/kg), and terminal phase half-life values ranging from 0.6 h in the guinea pig to 7.7 h in the rat. Oral bioavailability ranged from <0.2% (guinea pig) to 28% (dog). Data from portal vein cannulated dogs suggested that 37% of an oral dose of ezlopitant enters the portal vein as an unchanged drug in this species. Ezlopitant is metabolized to two pharmacologically active metabolites, an alkene (CJ-12 458) and a benzyl alcohol (CJ-12 764). After administration of the parent compound, CJ-12 764 was found in greater abundance than CJ-12 458 in all species examined. Ezlopitant and CJ-12 458 were highly protein bound in plasma (or serum), whereas the protein binding of CJ-12 764 was somewhat lower. Measurement of the kinetics of ezlopitant, CJ-12 458 and CJ-12 764 in the cerebrospinal fluid (CSF) of dogs demonstrated that all three compounds can partition into the CSF, and thereby, be capable of contributing to centrally mediated pharmacological effects. Thus, these data suggest that the pharmacological activity exhibited by ezlopitant in preclinical species in vivo is likely a result of the parent compound plus the active metabolites. Furthermore, the contributions of ezlopitant and the active metabolites to pharmacological activity probably varies with the identity of the model species, as well as the dose and route of ezlopitant administration.

Animals↗
PubMed1999

The humanized anti-HM1.24 antibody effectively kills multiple myeloma cells by human effector cell-mediated cytotoxicity.

A mouse monoclonal antibody, anti-HM1.24 (IgG2a/kappa), binds to a surface antigen preferentially overexpressed on multiple myeloma (MM) cells, and exhibits potent antitumor cell activity against MM cells by antibody-dependent cell-mediated cytotoxicity (ADCC). To develop an antibody-based immunotherapy against MM, a humanized anti-HM1.24 antibody, in which all FRs correspond to naturally processed human FRs, has been successfully constructed with the aid of both the hybrid variable region and two-step design methods. This humanized anti-HM1.24 antibody (IgG1/kappa) is able to effectively induce ADCC against human myeloma KPMM2 and ARH77 cells in the presence of human PBMCs as effectively as a chimeric anti-HM1.24 antibody. The humanized anti-HM1.24 antibody, therefore, could be expected as a potent immunotherapeutic agent for MM patients.

Amino Acid Sequence↗
PubMed1999

Mouse Rt6.1 is a thiol-dependent arginine-specific ADP-ribosyltransferase.

Mouse T-cell antigens Rt6.1 and Rt6.2 are glycosylphosphatidylinositol-anchored arginine-specific adenosine diphosphate (ADP)-ribosyltransferases. In the present study, we obtained evidence that an arginine-specific ADP-ribosyltransferase activity liberated from BALB/c mouse splenocytes by phosphatidylinositol-specific phospholipase C increased fivefold in the presence of dithiothreitol and that the activity was immunoprecipitated by polyclonal antibodies generated against recombinant rat RT6.1. When mouse Rt6.1 was expressed as a recombinant protein, the transferase activity of Rt6.1 was stimulated by dithiothreitol, and inhibited by N-ethylmaleimide, while activities of recombinant mouse Rt6.2 and the Glu-207 mutant of rat RT6.1 [Hara, N., Tsuchiya, M., and Shimoyama, M. (1996) J. Biol. Chem. 271, 29552-29555] were unaffected by either agent. In addition to four cysteine residues conserved among mouse Rt6 and rat RT6 antigens, Rt6.1 has two extra cysteine residues at positions 80 and 201. To investigate a contribution of these extra cysteines in mouse Rt6.1 to thiol dependency of Rt6.1 transferase activity, Cys-80 and Cys-201 of Rt6.1 were replaced with serine and phenylalanine, respectively, the corresponding residues of mouse Rt6. 2 and rat RT6.1. Transferase activity of the Phe-201 mutant of Rt6.1 lost thiol dependency while that of the Ser-80 mutant remained thiol-dependent. Thus, we conclude that mouse Rt6.1 is a thiol-dependent arginine-specific ADP-ribosyltransferase, and that Cys-201 confers thiol dependency on Rt6.1 transferase. Our study indicates that arginine-specific ADP-ribosyltransferase activity detected on BALB/c mouse splenocytes is attributed to Rt6.1 and that Rt6.1 differs from Rt6.2 in enzymatic property of the transferase and perhaps in immunoregulatory functions.

ADP Ribose Transferases↗
PubMed1999

Introduction of NAD decreases fMLP-induced actin polymerization in chicken polymorphonuclear leukocytes--the role of intracellular ADP-ribosylation of actin for cytoskeletal organization.

We reported previously that the arginine-specific ADP-ribosyltransferase in chicken polymorphonuclear leukocytes specifically modified actin, thereby inhibiting actin polymerization in vitro. In the present study, we investigated the effect of ADP-ribosylation on actin polymerization in situ. In the leukocytes, the introduction of NAD inhibited the increase in filamentous actin contents induced by a chemotactic peptide formyl-methionyl-leucyl-phenylalanine, while introduction of NAD together with novobiocin, a specific inhibitor for ADP-ribosyltransferase, did not. These results suggest that ADP-ribosylation regulates the formation of filamentous actin by the covalent modification of the protein in vivo.

Actins↗
PubMed1999

A technique for reconstruction of the medial patellofemoral ligament.

Additional medial patellofemoral ligament reconstruction was performed successfully on six consecutive patients with recurrent dislocation of the patella because of residual patellar instability after medial transfer of the tibial tubercle. A technique for medial patellofemoral ligament reconstruction is described, and complications and postoperative management are discussed. The reconstruction was performed using a double strand hamstring tendon graft in five patients and iliotibial allograft in one. Good stabilization of the patella was achieved in all six patients, resulting in improved confidence in higher levels of activity. The satisfactory outcome of additional medial patellofemoral ligament reconstruction suggests the possibility that the procedure may be part of the optional procedure in proximal realignment for recurrent dislocation of the patella.

Adolescent↗
PubMed1999

[Autoimmune fatigue syndrome and fibromyalgia syndrome].

We have encounted two patients with fibromyalgia (FM) initially diagnosed as having autoimmune fatigue syndrome (AIFS). To investigate the relationship between AIFS and FM, the distribution of the tender points in patients with AIFS was assessed according to the ACR criteria for FM. It was revealed that AIFS patients had 5.6 tender points on averages. Patients with headaches, digestive problems, or difficulty going to school had more tender points than patients without. Patients with ANA titers < 1: 160 had more tender points than patients with ANA > or = 1: 160. Anti-Sa negative patients had more tender points than positive patients. These results suggest a relationship between AIFS and FM in terms of the pathophysiologic mechanisms of the numerous tender points. In other words, ANA-positive FM patients could be one form of AIFS, as well as ANA-positive chronic fatigue syndrome patients. Thus, autoimmunity could explain the controversial disease entities of FM and/or CFS.

Adolescent↗
PubMed1999

Amelioration of acromegaly after pituitary infarction due to gastrointestinal hemorrhage from gastric ulcer.

We report a rare case of acromegaly in which pituitary infarction possibly developed in a GH-producing pituitary adenoma following gastrointestinal bleeding from peptic ulcer. In this case, pituitary infarction resulted in spontaneous remission of acromegaly associated with diabetes mellitus. In addition, detailed histological investigation revealed that clinically silent pituitary apoplexy was mainly an acute ischemic event which occurred recently in a GH-producing adenoma. This event led to massive coagulation necrosis of the tumor and endocrinological improvement.

Acromegaly↗
PubMed1999

Guinea pig D-amino-acid oxidase cDNA and phylogenetic position.

The nucleotide sequence of cDNA that encodes guinea pig D-amino-acid oxidase (DAO) was determined. The cDNA consisted of 1,399 nucleotides and a poly(A) tail. The cDNA encodes 347 amino acid residues. In contrast to the hamster, rat, and mouse DAOs, guinea pig DAO had the 25th amino acid residue. The homology in amino acid sequences between the guinea pig DAO and the rodent DAOs was not high in comparison to the homology in amino acid sequences between the guinea pig DAO and DAOs of humans, pigs and rabbits. The phylogenetic position of the guinea pig varied depending on the source of sequences (amino acids or nucleotides) and the methods of phylogenetic tree construction. These results suggest that the guinea pig is not a simple rodent.

Amino Acid Sequence↗
PubMed1999

ADP-ribosylation of tubulin by chicken NAD-arginine ADP-ribosyltransferase suppresses microtubule formation.

We obtained evidence that tubulin and actin, two major cytoskeletal proteins, are preferentially ADP-ribosylated in the bovine brain cytosol by NAD-arginine ADP-ribosyltransferase purified from chicken polymorphonuclear leukocytes. ADP-ribosylation of tubulin almost completely blocked self-assembly of the protein. The stoichiometry of ADP-ribose incorporation into unassembled and assembled tubulin was 6 and 2 mol/mol of tubulin, respectively. These findings suggest that sites of ADP-ribosylation in the unassembled tubulin molecule are crucial for tubulin assembly, and that covalently attached ADP-ribose moieties interfere with tubulin interaction by steric hindrance or conformational change. Thus, ADP-ribosylation may be involved in cytoskeletal organization in the brain via the modification of tubulin.

ADP Ribose Transferases↗
PubMed1999

Association of angiotensin-converting enzyme gene polymorphism and renal pathology in Japanese children with IgA nephropathy.

Polymorphism of the gene that codes for angiotensin I-converting enzyme (ACE) is associated with increased severity of immunoglobulin A (IgA) nephropathy in adult patients. We evaluated the relationship between the polymorphism of ACE genotypes and the pathological and clinical findings in Japanese children with IgA nephropathy. Patients with moderate/diffuse mesangial proliferation, glomerular sclerosis and tubulointerstitial damage showed a significant increase of the D/D type compared to those who had mild/focal mesangial proliferation, without glomerular sclerosis or tubulointerstitial damage (p < 0.05). Proteinuria at the first renal biopsy was significantly higher in the former group compared with the latter group except glomerular sclerosis (p < 0.01). IgA nephropathy patients with tubulointerstitial damage also showed an increased serum creatinine level compared to patients without the damage (p < 0.03). We conclude that ACE gene polymorphism may be correlated with the prognosis of IgA nephropathy in Japanese children.

Biopsy↗
PubMed1999

Phylogenic analysis of echovirus type 30 isolated from a large epidemic of aseptic meningitis in Japan during 1997-1998.

During 1997 to 1998, a nationwide epidemic of aseptic meningitis occurred in Japan. More than 4,500 isolates from patients with aseptic meningitis were identified as echovirus type 30. To investigate the character of these isolates, we examined the nucleotide sequences of thirty-seven geographical representatives and compared them with 50 strains isolated during the past 20 years. The phylogenic analysis used partial sequences from either the VP1 or VP4-VP2 region of the viral capsid. This analysis revealed that the isolates were divided into six genomic groups. All isolates identified during 1997-1998 belonged to only two genomic groups; these two groups are thought to be the causative viral agents involved in the recent epidemic.

Enterovirus B, Human↗
PubMed1999

Job search and employment-related issues in athletic training education programs.

OBJECTIVE: To investigate the amount of classroom instruction appropriated for employment-related issues and how career service centers were used by students in National Athletic Trainers' Association (NATA)-approved/Commission on Accreditation of Allied Health Education Programs (CAAHEP)-accredited undergraduate athletic training curriculums. DESIGN AND SETTING: We developed a job placement survey and mailed it to subjects. The study was conducted through the School of Physical Education at West Virginia University. SUBJECTS: The subjects were all undergraduate athletic training curriculum directors employed in an NATA-approved or CAAHEP-accredited program. MEASUREMENTS: We developed a survey instrument based on our experiences with instructional and educational issues in athletic training. The survey instrument consisted of items that investigated the amount of classroom time and education devoted to different aspects of preparing athletic training students for the job market. RESULTS: A total of 74 surveys were returned, for an 88% return rate. Eighty-six percent of the curriculum directors spent an average of 116 minutes (mean = 116 +/- 142.09; range, 5 to 780) instructing students about employment issues. Curriculum directors spent 63 minutes (mean = 63 +/- 47.86; range, 10 to 270) instructing students on how to develop a résumé. Most curriculum directors (74%) spent time with students discussing how to prepare for an interview (mean = 51 +/- 47.07 minutes; range, 1 to 270). Nearly all curriculum directors (97%) responded that students learned about recognizing allied health postgraduate career options other than athletic training. College or university career service centers were available to 96% of students, but only 53% sought instructional assistance from them. Curriculum directors spent an average of 38 +/- 49.31 minutes (range, 15 to 120) discussing programs available at career service centers. CONCLUSIONS: Athletic training curriculum directors were providing basic employment preparation skills to students. However, a wide variance existed for time allotted to employment- related issues. Additional research is needed to compare employment instructional methods with program success rates for job placement within athletic training.

Journal Article↗