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Biomedical subjects

R Nishimura

Publications and source records attributed to R Nishimura.

At least 55 records · Page 3Linked to original sources
PubMed2001

Results of hyperamplification of centrosomes in naturally developing tumors of dogs.

OBJECTIVE: To evaluate results of centrosome hyperamplification in naturally developing tumors of dogs. SAMPLE POPULATION: Tumor specimens from 9 dogs with tumors (rhabdomyosarcoma, osteosarcoma, chondrosarcoma, myxosarcoma, and mammary gland tumor) and 2 canine osteosarcoma cell lines. PROCEDURE: 3 antibodies for centrosome proteins (ie, anti-gamma-tubulin, anti-BRCA1, and anti-pericentrin) were used for immunohistochemical analysis. Double immunostaining for centrosomes was used to confirm the specificity of these antibodies for centrosomes. Mutational analysis of the canine p53 gene was carried out by polymerase chain reaction-single-strand conformation polymorphism analysis, and expression of canine MDM2 protein was evaluated by use of immunohistochemical analysis, using anti-MDM2 antibody. RESULTS: Immunohistochemical analysis of dog osteosarcoma cell lines with apparent aneuploidy revealed frequent hyperamplification of centrosomes in the osteosarcoma cell lines. Similar hyperamplified centrosomes were detected in the tumor tissues from all of the 9 tumors. The frequency of cells with hyperamplified centrosomes (3 to 20/cell) in each tumor tissue ranged from 9.50 to 48.1%, whereas centrosome hyperamplification was not observed in normal lymph nodes from these dogs. In 8 of the 9 tumors, mutation of p53 gene or overexpression of MDM2, or both, was detected. CONCLUSIONS AND CLINICAL RELEVANCE: Various types of naturally developing tumors in dogs often have hyperamplification of centrosomes associated with chromosome instability. Hyperamplification of centrosomes is a novel tumor marker for use in cytologic and histologic examinations of clinical specimens obtained from dogs.

Animals↗
PubMed2001

Telomere length and telomerase activity in canine mammary gland tumors.

OBJECTIVE: To measure telomere length and telomerase activity in naturally occurring canine mammary gland tumors. SAMPLE POPULATION: 27 mammary gland tumor specimens obtained during resection or necropsy and 12 mammary gland tissue specimens obtained from healthy (control) dogs. PROCEDURE: Telomere length in tissue specimens was measured by use of restriction endonuclease digestion and Southern blot analysis. Telomerase activity was measured by use of a telomeric repeat amplification protocol assay. RESULTS: Telomere length in mammary gland tumors ranged from 11.0 to 21.6 kilobase pairs (kbp; mean +/- SEM, 14.5+/-0.5 kbp) but did not differ among tumor types. Telomeres in mammary gland tumors were slightly shorter than in normal tissue specimens, but telomere length could not be directly compared between groups, because mean age of dogs was significantly different between groups. Age was negatively correlated with telomere length in control dogs but was not significantly correlated with length in affected dogs. Telomerase activity was detected in 26 of 27 mammary gland tumors and in 4 of 12 normal tissue specimens. However, telomerase activity and telomere length were not correlated in tumor specimens. CONCLUSIONS AND CLINICAL RELEVANCE: Telomere length is maintained in canine mammary gland tumors regardless of the age of the affected dog. Measurement of telomere length may be a useful tool for monitoring the in vivo effects of telomerase inhibitors in dogs with tumors.

Adenocarcinoma↗
PubMed2001

Effects of nitrous oxide on mask induction of anesthesia with sevoflurane or isoflurane in dogs.

OBJECTIVE: To determine the effects of nitrous oxide (N2O) on the speed and quality of mask induction with sevoflurane or isoflurane in dogs. ANIMALS: 7 healthy Beagles. PROCEDURE: Anesthesia was induced with sevoflurane or isoflurane delivered in 100% oxygen or in a 2:1 mixture of N2O and oxygen via a face mask. Each dog received all treatments with at least 1 week between treatments. Initial vaporizer settings were 0.8% for sevoflurane and 0.5% for isoflurane (0.4 times the minimum alveolar concentration [MAC]). Vaporizer settings were increased by 0.4 MAC at 15-second intervals until settings were 4.8% for sevoflurane and 3.0% for isoflurane (2.4 MAC). Times to onset and cessation of involuntary movements, loss of the palpebral reflex, negative response to tail-clamp stimulation, and endotracheal intubation were recorded, and cardiopulmonary variables were measured. RESULTS: Administration of sevoflurane resulted in a more rapid induction, compared with isoflurane. However, N2O had no effect on induction time for either agent. Heart rate, mean arterial blood pressure, cardiac output, and respiratory rate significantly increased and tidal volume significantly decreased from baseline values immediately after onset of induction in all groups. Again, concomitant administration of N2O had no effect on cardiopulmonary variables. CONCLUSIONS AND CLINICAL RELEVANCE: Administration of N2O did not improve the rate or quality of mask induction with sevoflurane or isoflurane. The benefits provided by N2O attributable to concentrating and second gas effects appear minimal in healthy dogs when low solubility inhalation agents such as isoflurane and sevoflurane are used for mask induction.

Anesthesia, Inhalation↗
PubMed2001

Respiratory reflexes in spontaneously breathing anesthetized dogs in response to nasal administration of sevoflurane, isoflurane, or halothane.

OBJECTIVE: To characterize respiratory reflexes elicited by nasal administration of sevoflurane (Sevo), isoflurane (Iso), or halothane (Hal) in anesthetized dogs. ANIMALS: 8 healthy Beagles. PROCEDURE: A permanent tracheostomy was created in each dog. Two to 3 weeks later, dogs were anesthetized by IV administration of thiopental and alpha-chloralose. Nasal passages were isolated such that inhalant anesthetics could be administered to the nasal passages while the dogs were breathing 100% O2 via the tracheostomy. Respiratory reflexes in response to administration of each anesthetic at 1.2 and 2.4 times the minimum alveolar concentration (MAC) and the full vaporizer setting (5%) were recorded. Reflexes in response to administration of 5% of each anesthetic also were recorded following administration of lidocaine to the nasal passages. RESULTS: Nasal administration of Sevo, Iso, and Hal induced an immediate ventilatory response characterized by a dose-dependent increase in expiratory time and a resulting decrease in expired volume per unit of time. All anesthetics had a significant effect, but for Sevo, the changes were smaller in magnitude. Responses to administration of each anesthetic were attenuated by administration of lidocaine to the nasal passages. CONCLUSIONS AND CLINICAL RELEVANCE: Nasal administration of Sevo at concentrations generally used for mask induction of anesthesia induced milder reflex inhibition of breathing, presumably via afferent neurons in the nasal passages, than that of Iso or Hal. Respiratory reflexes attributable to stimulation of the nasal passages may contribute to speed of onset and could promote a smoother induction with Sevo, compared with Iso or Hal.

Administration, Intranasal↗
PubMed2001

Aberrations of the p53 tumor suppressor gene in various tumors in dogs.

OBJECTIVE: To evaluate aberrations of the p53 tumor suppressor gene in naturally developing tumors in dogs. SAMPLE POPULATION: Tumor specimens from 15 dogs with various tumors, including malignant lymphoma (7 dogs), monocytic leukemia (1), mammary gland adenoma (1), mammary gland benign mixed tumor (1), rhabdomyosarcoma (1), colon cancer (1), and osteosarcoma (3). PROCEDURE: Aberrations of the p53 gene in these tumor tissues were examined by reverse transcriptase-polymerase chain reaction and single-strand conformation polymorphism analysis, using 3 fragments that covered the entire open reading frame of the canine p53 gene, followed by nucleotide sequencing of the abnormal bands. RESULTS: Point mutations, deletions, and insertions resulting in a number of amino acid substitutions of wild-type p53 were detected in 7 of the 15 tumor specimens from dogs with malignant lymphoma, monocytic leukemia, rhabdomyosarcoma, colon cancer, and osteosarcoma. Of these 7 dogs, 2 had aberrations of the p53 gene on both alleles, whereas 5 had aberrations of the p53 gene on 1 allele and concurrently lacked the wild-type p53 transcript. Many of the aberrations of the p53 gene detected in these tumors were located in the transactivation, DNA binding, and oligomerization domains. CONCLUSIONS AND CLINICAL RELEVANCE: Various naturally developing tumors in dogs often have inactivation of the p53 tumor suppressor gene, which may be 1 of the multiple step-wise genetic changes during tumorigenesis. This study indicates that p53 gene can be a target for gene therapy for tumors in dogs.

Animals↗
PubMed2001

A novel single-nucleotide polymorphism in the 3'-untranslated region of the human dihydrofolate reductase gene with enhanced expression.

A novel single-nucleotide polymorphism (SNP), 829C-->T in the 3'-untranslated region of the human dihydrofolate reductase (DHFR) gene transcript, was identified in the study population of 37 patients with childhood leukemias/lymphomas and 83 healthy Japanese children. Frequencies of the DHFR 829C/C, 829C/T, and 829T/T genotypes were 83.8, 10.8, and 5.4%, respectively, in the cases and 74.7, 19.3, and 6.0% in the controls, showing no significant difference in genotype frequencies between the cases and controls. When determined by real-time quantitative reverse transcription-PCR analysis, the highest expression of the DHFR transcript was demonstrated in the samples with a DHFR 829T/T polymorphism (P < 0.001). Direct association of the presence of the SNP with methotrexate-related adverse events in each patient was not demonstrated in this limited analysis. These data suggest that the novel DHFR 829 polymorphism is associated with a positive role in gene expression and provide evidence of a functional SNP in the 3' regulatory region of the gene.

3' Untranslated Regions↗
PubMed2001

[A phase I study of docetaxel (TXT) and doxifluridine (5'-DFUR) combination therapy in patients with advanced and recurrent breast cancer].

We investigated efficacy and tolerance of chemotherapy with doxifluridine (5'-DFUR) and docetaxel (TXT) in advanced/recurrent breast cancer. Subjects were enrolled by central registration. The regimen included 5'-DFUR orally for 14 consecutive days, and TXT intravenously on day 8. It was repeated every 3 weeks, as long as possible, including dosage levels of 5 scheduled steps. Patient registration was started in August 1999 and 5 patients given level 1 regimen (5'-DFUR, 800 mg/day; TXT, 50 mg/m2) were evaluated. Although the results revealed neutropenia of grade 3 in 4/5 patients and leukocytopenia in 2/5 patients, no other side effects were observed. Taking into consideration the toxicity profiles of each drug in level 1, a level 2b regimen (5'-DFUR, 800 mg/day; TXT, 60 mg/m2) was accepted. Seven patients were registered for level 2b dosage and were examined for the safety of the regimen. Two patients discontinued the level 2b regimen due to percutaneous adverse reactions (DLT) and 6/7 patients developed neutropenia of grade 4. Clinical effects in level 1 group included: 1 CR, 2 PR, 1 long NC (NC longer than 24 weeks), and 1 NE, for a response rate of 60.0% (3/5 patients). Those in level 2b included: 2 CR, 2 PR, 1 NC, and 2 NE, for a response rate of 57.1% (4/7 patients). Based on the safety and efficacy of the combined therapy, the recommended dosage of this regimen is 5'-DFUR, 800 mg/day, combined with TXT, 60 mg/m2. A Phase II study is being conducted using this dosage.

Adult↗
PubMed2000

Transforming growth factor-beta up-regulates the beta 5 integrin subunit expression via Sp1 and Smad signaling.

Integrin-mediated cell-matrix interactions play important roles in regulating cell function. Since transforming growth factor-beta (TGF-beta) modulates many osteoblast activities, we hypothesized that the growth factor acts in part by modulating integrin expression. TGF-beta increased cell adhesion to vitronectin and up-regulated the surface level of alpha(v)beta(5) via increasing beta(5) protein synthesis by a transcriptional mechanism. Promoter activity analysis demonstrated that a TGF-beta-responsive element resides between nucleotides -63 and -44. Electrophoretic mobility shift assay and immunoprecipitation/Western studies indicated that the nuclear complex formed using the -66/-42 oligonucleotide contained both Sp1/Sp3 and Smad proteins. Since nuclear Sp1/Sp3 levels were not altered, whereas Smad levels were increased by TGF-beta, we investigated the roles of Smad proteins in the up-regulation of beta(5) gene activation. Co-transfection of cells with beta(5) promoter reporter construct and expression vectors for Smad3, Smad4, and Sp1 increased the stimulatory effect of TGF-beta. Furthermore, expression of dominant negative Smad3 or Smad4 in cells decreased or abolished the stimulation of beta(5) promoter activity by TGF-beta. Smad4 mutant also inhibited the up-regulation of surface beta(5) level by TGF-beta. Thus, TGF-beta increases expression of the integrin beta(5) gene by mechanisms involving Sp1/Sp3 and Smad transcription factors.

Animals↗
PubMed2000

Novel protein transfection of primary rat cortical neurons using an antibody that penetrates living cells.

An Ab-based system to deliver functional proteins into neurons was developed using the murine mAb, mAb 3E10. This was achieved by covalently conjugating catalase to the Ab so that the conjugate retained high activity for the degradation of hydrogen peroxide. Three-dimensional fluorescence microscopy was used to demonstrate penetration of the Ab into the nucleus of living primary cortical neurons. The Ab conjugate localized in both the cytoplasm and nucleus. Retention of catalase activity after penetration and distribution of conjugate was demonstrated by reduction in cell death following exposure of treated neurons to hydrogen peroxide. These studies illustrate the potential of this method for the intracellular delivery of therapeutic proteins.

Animals↗
PubMed2000

Visceral mast cell tumors in dogs: 10 cases (1982-1997).

OBJECTIVE: To characterize the clinical features of visceral mast cell tumors (MCT) without associated cutaneous involvement in dogs. DESIGN: Retrospective study. ANIMALS: 10 dogs with histologically confirmed MCT without associated cutaneous lesions. PROCEDURE: Information on signalment, clinical signs, laboratory examinations, and time from first admission to death was obtained from the medical record of each dog. RESULTS: Purebred male dogs of miniature breeds appeared to have a higher prevalence of visceral MCT. Clinical signs included anorexia, lethargy, vomiting, and diarrhea. Anemia (n = 7), hypoproteinemia (5), and mastocythemia (5) were detected. Treatments, including glucocorticoids, were not successful. Primary sites of tumors were the gastrointestinal tract (n = 6) and the spleen or liver (1); the primary site was not confirmed in the remaining 3 dogs. In 7 dogs, tumors were categorized as grade II or III, on the basis of histologic findings. The prognoses were poor, and all dogs died within 2 months after first admission. CONCLUSIONS AND CLINICAL RELEVANCE: Visceral MCT is uncommon in dogs, and the prognosis is extremely poor. Biological behavior and drug susceptibility of visceral MCT may be different from cutaneous MCT. The lack of specific clinical signs may result in delay of a definitive diagnosis. The rapid progression of clinical signs and difficulty in diagnosis contributes to a short survival time.

Animals↗
PubMed2000

Quantitation of minimal residual disease in t(8;21)-positive acute myelogenous leukemia patients using real-time quantitative RT-PCR.

t(8;21) is one of the common chromosomal translocations in acute myelogenous leukemia (AML). Using a recently developed real-time quantitative polymerase chain reaction (PCR) system, we analyzed the minimal residual disease (MRD) in bone marrow samples from seven AML patients with t(8;21) at different time points during the clinical courses of their disease. Four of these patients received chemotherapy and allogenic bone marrow transplantation (allo-BMT), and the other three were treated with chemotherapy alone. Two of the patients that received allo-BMT suffered a relapse. In these patients, the levels of AML1-MTG8 mRNA expression were shown to quantitatively increase. After re-induction chemotherapy and donor lymphocyte infusion therapy, AML went into remission and the expression levels decreased. In the other two patients receiving allo-BMT, the disease went into remission and the level of AML1-MTG8 mRNA expression remained under the detectable range. The other three patients received several courses of chemotherapy, without allo-BMT, and all of them clinically reached the hematological and cytogenetic remission state. However, there were low but detectable levels of MRD in their bone marrow samples. These results suggest that the real-time quantitative PCR assay is very useful for the monitoring of MRD and detecting an early relapse. This assay may also be useful in determining the quantitative difference in myelo-ablative activity between the chemotherapy alone and chemotherapy in conjunction with allo-BMT.

Adult↗
PubMed2000

Ovarian thecoma with ascites and high serum levels of CA125.

We report a 34-year-old woman with an ovarian thecoma and ascites who exhibited high serum levels of CA125. Measuring serum tumor markers and imaging are two important diagnostic tools for malignant ovarian tumors. In the present case, a preoperative diagnosis of benign ovarian tumor could not be made due to the elevation of CA125 (895 U/ml) and nonspecific MRI findings.

Adult↗
PubMed2000

Trophoblastic cells expressing human chorionic gonadotropin genes in peripheral blood of patients with trophoblastic disease.

We attempted to identify the cells expressing alpha and beta subunits of human chorionic gonadotropin (hCG) in the peripheral blood of patients with trophoblastic disease and normal pregnant women by using reverse transcriptase polymerase chain reaction (RT-PCR) and Southern blot. By this method, the mRNAs of hCG alpha and hCG beta were detected in the peripheral blood mononulear cells (PBMNC) from 3 of 7 hydatidiform mole (mole) and 1 of 4 choriocarcinoma patients as well as from normal pregnant women during the first trimester. None of the mRNAs of hCG subunits was detected in the PBMNC from healthy male and nonpregnant healthy women examined. The expression of hCG alpha and hCG beta in patients with trophoblastic disease and normal pregnant women almost correlated with their plasma levels of intact hCG. The present study indicates that the cells expressing hCG alpha and hCG beta, which virtually represent trophoblasts, are circulating in the peripheral blood of patients with trophoblastic disease as well as of normal pregnant women.

Blotting, Southern↗
PubMed2000

The molar vesicle fluid contains the beta-core fragment of human chorionic gonadotropin.

The human chorionic gonadotropin (hCG) beta-core fragment (beta-CF) is a major molecular form of hCG beta subunit (hCGbeta) immunoreactivity in the urine of pregnant women and patients with trophoblastic disease. The majority of evidence supports the fact that the beta-CF is a degradative product of intact hCG and free hCGbeta in the kidneys. We found a beta-CF-like substance in the fluid of molar vesicles from a patient with complete hydatidiform mole. The molar fluid beta-CF (mbeta-CF) was indistinguishable from the beta-CF in the patient's urine (ubeta-CF) by immunoreactivity and by elution profile on gel chromatography. The binding study to lectins, however, showed that mbeta-CF contains a carbohydrate moiety that differs from that of ubeta-CF. Immunohistochemistry with anti-beta-CF antibody demonstrated a strong immunoreactivity in a large number of macrophages in the molar villous stroma. In vitro incubation of intact hCG with peritoneal macrophages showed a slow increase of intact hCG in the cell cytosol with the appearance of beta-CF-like substance in the cell supernatant. In conclusion, the source of beta-CF in molar fluid is likely to be macrophages existing in the villous stroma. Thus macrophages may ingest intact hCG and act as a local regulator of gonadotropic hormones.

Body Fluids↗
PubMed2000

Differentiation induction of canine osteosarcoma cell lines by retinoids.

The effect of two retinoids, all- trans and 9- cis retinoic acid, on the differentiation of three canine osteosarcoma cells (OOS, HOS, and POS) was examined using markers specifically expressed by phenotypic osteoblasts. Both retinoids induced morphologic differentiation in all the canine osteosarcoma cells. Retinoids enhanced cell flattening and spreading, as well as reduction in cell overlapping. Alkaline phosphatase (ALP) activity and ALP staining was enhanced in OOS, and HOS cells, but decreased in POS cells. These results may suggest that OOS and HOS cells have immature osteoblastic properties and POS cells have mature osteoblastic properties. Retinoids decreased osteocalcin production in all the osteosarcoma cells. They induced an increase in production of type I collagen in HOS and POS cells, but a decrease in OOS cells. These results indicate that retinoids induce differentiation of canine osteosarcoma cells, resulting in an altered expression of their malignant phenotype.

Alitretinoin↗
PubMed2000

Quantitation of Fas and Fas ligand gene expression in human ovarian, cervical and endometrial carcinomas using real-time quantitative RT-PCR.

Alterations in the expression of Fas (CD95/APO-1) and its ligand (FasL) have been demonstrated in various types of cancers as a mechanism for tumour cell to escape from the immune system. In the present study, we evaluated the expression of the Fas and FasL genes in a wide range of primary gynaecological carcinomas. These included 31 ovarian, 29 cervical and 25 endometrial carcinoma tissues as well as four ovarian and three cervical carcinoma cell lines. Our real-time quantitative reverse transcription polymerase chain reaction analysis revealed that down-regulation of Fas expression is more prominent than the up-regulation of FasL expression in all types of gynaecological cancer studied. This down-regulation of Fas expression was also true for the seven carcinoma cell lines. Only one cervical carcinoma cell line, DoT, exhibited a high level of FasL expression. These results indicated that down-regulation of Fas expression is a common abnormality in many types of cancers including gynaecological cancers, whereas an increase in FasL expression is not a common phenomenon in these cancers.

Cervix Uteri↗
PubMed2000

Human villous macrophage-conditioned media enhance human trophoblast growth and differentiation in vitro.

In human chorionic villi, numerous macrophages, so-called Hofbauer cells, are located adjacent to trophoblasts. To determine the role of the macrophages in the proliferation and differentiation of trophoblasts, cytotrophoblast cells were cultured in serum-free culture-conditioned media of villous macrophages (VMCM), peritoneal macrophages (PMCM), and villous fibroblasts (VFCM). In VMCM, proliferation of cytotrophoblast cells was detected at 24 h by immunocytochemistry with Ki-67-antibody. A large number (P < 0.001) of multinucleated syncytia was formed in VMCM. In VMCM, cytotrophoblast cell fusion was completed by 96 h, which coincided with the peak of hCG secretion and initiation of human placental lactogen (hPL) release. Levels of hCG (P < 0.001) and hPL (P < 0. 001) secretion from syncytial cells were significantly higher in VMCM than in PMCM or in VFCM. Concentrations of macrophage colony-stimulating factor (M-CSF) and vascular endothelial growth factor (VEGF) analyzed by ELISA were greater in VMCM than in PMCM or in VFCM, whereas monocyte chemoattractant protein-1 (MCP-1) concentration was high in PMCM. The expression patterns of M-CSF, VEGF, and MCP-1 in villous macrophages and peritoneal macrophages by reverse transcriptase-polymerase chain reaction were similar to their secretion patterns. Thus, villous macrophages have a greater ability to stimulate hCG and hPL secretion than do peritoneal macrophages. This study suggests that macrophages within the villous stroma may stimulate the growth and differentiation of trophoblasts through their secreted substances.

Cell Differentiation↗