Immuno-PCR assay for homodimeric osteoprotegerin.
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Dimerization↗
Biomedical subjects
N Watanabe
Publications and source records attributed to N Watanabe.
[Telomerase, cell immortality and cancer].
Telomerase is an enzyme that replaces repetitive (TTAGGG)n sequences on the ends of chromosomes that would otherwise be lost during successive cell divisions. Telomerase activity is closely linked to attainment of cellular immortality, a step in carcinogenesis, while lack of such activity contributes to cellular senescence. Telomerase is activated in more than 85% of malignant tumors. However, with the exception of some self-renewing tissues with high regenerative potential, telomerase activity is usually repressed in normal somatic tissues. Based on these reports, we investigated telomerase activity in gastric mucosal tissues. Telomerase activity is highest in cancer, followed by intestinal metaplasia, chronic gastritis, and normal mucosa. In patients with intestinal-type gastric cancer, telomerase activity was higher in those with intestinal metaplasia and H. pylori infection than in patients without infection. Our results suggest that H. pylori infection may influence telomerase activity in cancer and noncancerous tissue. Genes encoding three major components of human telomerase have been recently cloned. They included those for human telomerase RNA component (hTR), human telomerase reverse transcriptase (hTERT), and telomerase-associated protein 1 (TEP1). More recently, two human telomeric repeat binding factors (TRFs) have also been cloned: TRF1, considered to inhibit the action of telomerase at the telomeric region, and TRF2, believed to prevent fusion of chromosome ends and, in vitro, to remodel linear telomeric DNA into large duplex loops. However, the details of mechanisms regulating telomerase activity are still poorly understood, and specific components or binding proteins that might represent suitable targets for cancer gene therapy have not yet been identified. Therefore, we established quantitative assays using a TaqMan RT-PCR for mRNAs encoding the telomerase components hTR, hTERT, and TEP1, as well as for those encoding TRF1 and TRF2. By using our quantitative assays, we found the following results: 1) Expression of TRF1 and TRF2 mRNA was greater in the normal cells than in human malignant hematopoietic cell lines or in patients with acute leukemia, 2) hTERT mRNA expression showed changes paralleling telomerase activity and became undetectable with HL60 cell differentiation, 3) initially low expression of TRF1 and TRF2 mRNA increased during differentiation. Our results suggest that not only hTERT but also TRF1 and 2 are important regulators of telomerase activity.
Cell Movement↗
Detection of anti-survivin antibody in gastrointestinal cancer patients.
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Antibodies, Neoplasm↗
[A case of lung cancer in which FDG-PET was useful for preoperative diagnosis].
The patient was 48-year-old woman. The patient was referred to our hospital due to detection of an abnormal shadow on a chest X-ray image during a health checkup. The results of a CT scan morphologically suggested a benign tumor, but a diagnosis could not be made even after bronchoscopic cytology. FDG-PET revealed accumulation at the tumor site suggestive of malignancy, and an operation was performed. Prompt pathological examination showed that the tumor was adenocarcinoma, and right lower lobectomy and lymphnode dissection were performed. FDG-PET is thought to be useful for diagnosis in cases of lung tumor in which a preoperative diagnosis can not be established.
Adenocarcinoma↗
[Reference intervals of urinary biochemical analysis in Japanese men].
To determine the reference intervals of urinary albumin, immunoglobulin G, transferrin, alpha 1 microglobulin, and N-acetyl-beta-D-glucosaminidase, we measured those components and creatinine in urine from men with normal blood pressure(systolic < or = 139 mmHg and diastolic < or = 89 mmHg), HbA1c < or = 5.8% and negative qualitative tests of urinary protein and hematuria. The subjects were 150 men in their thirties, 136 in their forties, and 135 in their fifties. Urines was collected in the morning after at least 12 hours fast and stored at -80 degrees C until assayed. The levels of creatinine decreased with age, whereas no change was observed in the levels of other urinary components. Consequently, the levels of those components expressed as g. creatinine increased with age. The reference intervals per g. creatinine were determined for each age-bracket using the iterative truncation method after logarithmical transformation of the values.
Acetylglucosaminidase↗
Vdelta1 and Vdelta2 gammadelta T cells express distinct surface markers and might be developmentally distinct lineages.
We report here that the two major types of gammadelta T cells found in human blood, Vdelta1 and Vdelta2, were found to have markedly different phenotypes. Vdelta2 cells had a phenotype typical of most alphabeta T cells in blood; i.e., they were CD5(+), CD28(+), and CD57(-). In contrast, Vdelta1 cells tended to be CD5(-/dull), CD28(-), and CD57(+). Furthermore, although Vdelta1 T cells appeared to be "naive" in that they were CD45RA(+), they were CD62L(-) and on stimulation uniformly produced interferon-gamma, indicating that they are in fact memory/effector cells. This phenotype for Vdelta1 cells was similar to that of intestinal intraepithelial lymphocytes, a subset that can develop in the absence of the thymus. We suggest that the Vdelta1 and Vdelta2 T cell subsets represent distinct lineages with different developmental pathways. The disruption of the supply of normal, thymus-derived T cells in HIV-infected individuals might be responsible for the shift in the Vdelta2/Vdelta1 ratio that occurs in the blood of individuals with HIV disease.
Antigens, Differentiation, T-Lymphocyte↗
[Development of transgenic animal models for Alzheimer's disease].
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Alzheimer Disease↗
[Clinical significance of measurement of circulating tumor necrosis factor alpha].
Tumor necrosis factor alpha (TNF alpha) is an important mediator of inflammatory and autoimmune diseases. The analysis of its pathophysiologic roles has been difficult because low concentrations of TNF alpha, including those in healthy controls, cannot be measured by existing methods. We developed a sensitive Immuno-PCR assay for the detection of TNF alpha in human serum. The DNA label was generated by PCR amplification using biotinylated primer and was bound by streptavidin to the biotinylated third antibody. TNF alpha sandwiched by antibodies was detected by amplification of the DNA label using PCR. The limit of detection of the assay was 0.001 pg/ml, an approximately 5 x 10(4)-fold improvement compared with conventional ELISA. The mean serum TNF alpha concentration in Duchenne muscular dystrophy patients (27.8 pg/ml) was approximately 1,000 times higher than that in healthy subjects (0.027 pg/ml). The mean value in inflammatory bowel disease patients was approximately 2,000 times that in healthy subjects (mean excess for UC, 1,100 times; for CD, 7,700 times). These findings suggest that measuring the serum concentration using a highly sensitive Immuno-PCR assay may be useful for analyzing the clinical significance of TNF alpha in various diseases.
Biomarkers↗
[An autopsy case of an infant born with a caul who died due to asphyxia after live-birth].
We reported an autopsy case of a child born with a caul. The infant was a female and seemed to be full-term. No remarkable pathological findings for disease or anomalies were observed. The floating test of lungs was partially positive. And on the gastro-intestinal floating test, only stomach floated. In histological examination, the alveoli in the lungs were partially slightly open. We thought that those were due to the cardio-pulmonary resuscitation. Pulmonary surfactant was positive immunohistochemically. And the stable microbubble test on the liquid in her stomach was very strong. Even if a child is born alive, he or she will soon die due to asphyxia but for the production of surfactant in the lungs. We can evaluate more precisely the potential respiratory ability of a neonate died due to asphyxia by means of stable microbubble test on newborn gastric contents and immunohistochemical staining of surfactant in lungs.
Asphyxia Neonatorum↗
Migration and differentiation of autoreactive B-1 cells induced by activated gamma/delta T cells in antierythrocyte immunoglobulin transgenic mice.
Using normal and transgenic (Tg) mice, we have shown that peritoneal B-1 cells are activated by administration of cytokines or lipopolysaccharide and migrate to other lymphoid organs where they differentiate into antibody-secreting cells. However, little is known about the process of B-1 cell migration and differentiation in vivo. We developed a mouse line by crossing the antierythrocyte antibody Tg mice (HL mice) with TCR-gamma/delta Tg mice specific for a self-thymus leukemia (TL) antigen in the recombination activating gene (RAG)2(-/-) background. In the presence of the self-antigen, Tg gamma/delta T cells increased in number and manifested activated phenotypes. Peritoneal B-1 cells in these mice migrated into mesenteric lymph nodes and differentiated into autoantibody-secreting cells, resulting in strong autoimmune hemolytic anemia. Furthermore, transfer of RAG2(-/-) x HL bone marrow or peritoneal cells into the peritoneal cavity of RAG2(-/-) x TCR-gamma/delta Tg mice gave rise to donor-derived B-1 cells in mesenteric lymph nodes, and these cells produced the autoantibody. Thus, this study demonstrates that the migration of B-1 cells and differentiation into the antibody-secreting cells can be induced by noncognate T cell help and implies the possibility that gamma/delta T cells may induce B-1 cell differentiation in vivo.
Anemia↗
The quantity of TCR signal determines positive selection and lineage commitment of T cells.
It is generally accepted that the avidity of TCR for self Ag/MHC determines the fate of immature thymocytes. However, the contribution of the quantity of TCR signal to T cell selection has not been well established, particularly in vivo. To address this issue, we analyzed DO-TCR transgenic CD3zeta-deficient (DO-Tg/zetaKO) mice in which T cells have a reduced TCR on the cell surface. In DO-Tg/zetaKO mice, very few CD4 single positive (SP) thymocytes developed, indicating that the decrease in TCR signaling resulted in a failure of positive selection of DO-Tg thymocytes. Administration of the peptide Ag to DO-Tg/zetaKO mice resulted in the generation of functional CD4 SP mature thymocytes in a dose-dependent manner, and, unexpectedly, DO-Tg CD8 SP cells emerged at lower doses of Ag. TCR signal-dependent, sequential commitment from CD8(+) SP to CD4(+) SP was also shown in a class I-restricted TCR-Tg system. These in vivo analyses demonstrate that the quantity of TCR signal directly determines positive and negative selection, and further suggest that weak signal directs positively selected T cells to CD8 lineage and stronger signal to CD4 lineage.
Amino Acid Sequence↗
Regulated expression of the BTEB2 transcription factor in vascular smooth muscle cells: analysis of developmental and pathological expression profiles shows implications as a predictive factor for restenosis.
BACKGROUND: We have previously shown BTEB2, a Krüppel-like zinc finger transcription factor, to regulate expression of the SMemb/NMHC-B gene, which has been implicated in phenotypic modulation of smooth muscle cells (SMCs). The present study was done to assess the developmental and pathological expression profiles of BTEB2 and to further evaluate the clinical relevance of BTEB2 expression in human coronary artery disease. METHODS AND RESULTS: Immunohistochemistry showed developmentally regulated expression of BTEB2 with abundant expression in fetal but not in adult aortic SMCs of humans and rabbits. In balloon-injured aortas, predominant expression of BTEB2 was seen in neointimal SMCs. Atherectomy specimens obtained from primary and restenotic lesions showed predominant expression of BTEB2 to stellate SMCs. The incidence of restenosis in primary lesions was significantly higher in lesions containing BTEB2-positive cells than in lesions without (55.6% versus 25.0%, P:=0.01). CONCLUSIONS: The present study shows that BTEB2 expression is developmentally and pathologically regulated. BTEB2 is preferentially expressed in dedifferentiated or activated SMCs. Examination of human coronary artery specimens suggests that primary lesions containing BTEB2-positive cells are associated with higher risk of restenosis than BTEB2-negative lesions. These results suggest that BTEB2 can serve as a molecular marker for phenotypic modulation of vascular SMCs.
Adult↗
Roles of P-selectin in inflammation, neointimal formation, and vascular remodeling in balloon-injured rat carotid arteries.
BACKGROUND: P-selectin plays key roles in mediating inflammation through promoting adherence of leukocytes to activated platelets and endothelium. This process is one of the initial events in atherosclerosis and restenosis after coronary angioplasty. METHODS AND RESULTS: Using a rat balloon-injury model, we examined the role of P-selectin in vascular inflammatory processes. In the acute phase, immunohistochemistry revealed that P-selectin was intensely expressed on both activated platelets covering the denuded segment and endothelial cells of the inflamed adventitial small vessels. Treatment with an anti-P-selectin monoclonal antibody (MAb) for 8 consecutive days significantly inhibited neointimal formation at day 14 (42% inhibition; P:<0.05), and this effect persisted at day 56 (40% inhibition; P:<0.01) compared with the control group. Vascular shrinking accompanying adventitial fibrosis was also attenuated at day 56. Inhibition of both neointimal formation and vascular shrinking resulted in the lumen area of the anti-P-selectin treatment group being approximately 3 times larger at day 56 than that of the control group. Accumulation of CD45-positive leukocytes in the developing neointima, media, and adventitia at day 8 was significantly inhibited by treatment with the anti-P-selectin MAb. Scanning electron microscopy demonstrated that anti-P-selectin treatment resulted in a less thrombogenic surface of the arterial intima, which featured a pseudoendothelial appearance at day 14 after injury. CONCLUSIONS: These results suggest that inhibition of P-selectin-mediated leukocyte recruitment prevents the development of neointimal formation, adventitial inflammation, and vascular shrinking and promotes pseudoendothelialization by luminal smooth muscle cells. This treatment thus beneficially affects vascular remodeling after balloon injury in rats.
Angioplasty, Balloon↗
Role of common cytokine receptor gamma chain (gamma(c))- and Jak3-dependent signaling in the proliferation and survival of murine mast cells.
The regulatory roles of the common cytokine receptor gamma chain (gamma(c))- and Jak3-dependent signaling in the proliferation and survival of mast cells were determined using gamma(c)-deficient (gamma(c)(-)) and Jak3-deficient (Jak3(-)) mice. Although the mast cells in gamma(c)(-) and Jak3(-) mice were morphologically indistinguishable from those in wild-type mice, the number of peritoneal mast cells was decreased in gamma(c)(-) and Jak3(-) mice as compared with that in wild-type mice. Among gamma(c)-related cytokines, interleukin (IL)-4 and IL-9, but not IL-2, IL-7, or IL-15, enhanced the proliferation and survival of bone marrow-derived mast cells (BMMCs) from wild-type mice. However, the effects of IL-4 and IL-9 were absent in BMMCs from gamma(c)(-) and Jak3(-) mice. In addition, IL-4Ralpha, gamma(c), and Jak3, but not IL-2Rbeta or IL-7Ralpha, were expressed in BMMCs. In contrast, IL-13 did not significantly induce the proliferation and survival of BMMCs even from wild-type mice, and IL-13Ralpha1 was not expressed in BMMCs. Furthermore, IL-4 phosphorylated the 65-kd isoform of Stat6 in BMMCs from wild-type mice but not from gamma(c)(-) and Jak3(-) mice. These results indicate that gamma(c)- and Jak3-dependent signaling is essential for IL-4- and IL-9-induced proliferation and survival of murine mast cells, that the effects of IL-4 are mediated by type I IL-4R and that type II IL-4R is absent on mast cells, and that IL-4 phosphorylates the 65-kd isoform of Stat6 in mast cells in a gamma(c)- and Jak3-dependent manner.
Animals↗
An automatic analyzer for catecholamines and their 3-O-methyl metabolites using a micro coulometric flow cell as a postcolumn reactor for fluorogenic reaction.
A coulometric flow cell for a miniaturized LC system was developed. The cell was examined, as 3-O-methyl catecholamines were converted to their relative omicron-quinones for subsequent fluorometric and chemiluminescence detection. Its performance was evaluated in comparison with commercially available amperometric and coulometric detectors in terms of specification of the low dead volume and high conversion efficiency. The fully automated small-bore LC analyzer for simultaneous determination of catecholamines and their 3-O-methyl metabolites included precolumn pretreatment, column switching, column separation, postcolumn oxidative conversion, fluorometric derivatization, and chemiluminescence detection. The detection limits were 0.3-2.0 fmol for catecholamines and their 3-O-methyl metabolites. Because of the high sensitivity, the required volume of rat plasma sample was only 15 microL.
Animals↗
Induction of M-phase arrest and apoptosis after HIV-1 Vpr expression through uncoupling of nuclear and centrosomal cycle in HeLa cells.
The human immunodeficiency virus type 1 (HIV-1) accessory protein Vpr induces cell cycle arrest in the G2 phase of the cell cycle followed by apoptosis. The mechanism of the arrest is unknown but the arrest is believed to facilitate viral replication. In the present study, we have established cell lines that allow conditional expression of Vpr, and have examined the mechanism of cell death following Vpr expression. We found that cells expressing Vpr enter M phase after long G2 arrest but formed aberrant multipolar spindles that were incapable of completing karyokinesis or cytokinesis. This abnormality provided the basis for apoptosis, which always followed in these cells. The multipolar spindles formed in response to abnormal centrosomal duplication that occurred during the G2 arrest but did not occur in cells arrested in G2 by irradiation. Thus, the expression of Vpr appears to be responsible for abnormal centrosome duplication, which in turn contributes in part to the rapid cell death following HIV-1 infection.
Apoptosis↗
Tetracuspid aortic valve in a patient with 22q11.2 microdeletion.
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Aortic Valve↗
4-(3-Chloro-4-methoxybenzyl)aminophthalazines: synthesis and inhibitory activity toward phosphodiesterase 5.
We synthesized various 4-(3-chloro-4-methoxybenzyl)aminophthalazines substituted at the 1- and 6-positions and evaluated their inhibitory activity toward phosphodiesterase 5 (PDE5) and their vasorelaxant activity in isolated porcine coronary arteries precontracted with prostaglandin F2alpha (10(-5) M). The preferred substituents at the 1-position of the phthalazine were 4-hydroxypiperidino, 4-hydroxymethylpiperidino, 4-(2-hydroxyethyl)piperidino, and 4-oxopiperidino. Among these compounds, [4-(3-chloro-4-methoxybenzyl)amino-1-(4-hydroxy)piperidino]-6-phthala zinecarbonitrile monohydrochloride (13) exhibited potent PDE5 inhibitory activity (IC(50) = 0.56 nM) with >1700-fold high selectivity over other PDE isozymes (PDE1-4). Compound 13 exhibited the most potent vasorelaxant action (EC(50) = 13 nM) in this series of compounds. Compound 13 reduced mean pulmonary arterial pressure by 29.9 +/- 3.1% when administered intravenously at 30 microg/kg to the chronically hypoxic rats and had an apparent oral bioavailability of about 19.5% in rats and was selected for further biological evaluation.
3',5'-Cyclic-GMP Phosphodiesterases↗