Search PubMed⌕ Search

Biomedical subjects

N Watanabe

Publications and source records attributed to N Watanabe.

At least 163 records · Page 9Linked to original sources

Cell-type specificity of l-leucyl l-leucine methyl ester.

l-Leucyl l-leucine methyl ester (LeuLeuOMe) is a lysosomotropic agent which is converted to a membranolytic compound by dipeptidyl peptidase I and kills human leukocytes such as CD8+ T cells and monocytes but not B cells. The reagent has also been used in mice on the assumption that the cell-type specificity to murine leukocytes is the same as that to human leukocytes. During study on the effect of LeuLeuOMe on antigen-driven IL-2 production using murine splenocytes as antigen-presenting cells, however, we noticed that murine B cells were sensitive to LeuLeuOMe. We therefore examined the cell-type specificity using murine splenocytes and peritoneal macrophages. Flow cytometric analysis revealed that the most sensitive cells to LeuLeuOMe were CD8+ cells and that CD19+ cells (B cells) were as sensitive as CD3+ cells (T cells). Murine splenic B cells, which were either positively or negatively sorted with a cell sorter, were also sensitive to LeuLeuOMe, whereas human peripheral blood B cells, which were positively sorted, were not. Peritoneal macrophages were the most insensitive to LeuLeuOMe. Thus, this study demonstrated that the cell-type specificity to murine leukocytes is different from that to human leukocytes.

Animals↗

Disrupted transforming growth factor-beta signaling and deregulated growth in human biliary tract cancer cells.

Biliary tract carcinoma is a common neoplasm in Japan, and its treatment is difficult because it tends to promote fibrosis and easily invades surrounding tissues. To better characterize the biological features of this carcinoma, we investigated abnormalities in the transforming growth factor-beta (TGF-beta) signaling pathway in five human biliary tract cancer cell lines: RBE, KMBC, SK-ChA-1, Mz-ChA-1, and Mz-ChA-2. We stably transfected into these cells the luciferase reporter plasmid carrying promoter of the plasminogen activator inhibitor-1 gene, the expression of which is stimulated by TGF-beta1. Treating the KMBC and Mz-ChA-1 cells with TGF-beta1 neither inhibited cell growth nor stimulated luciferase activity. In contrast, the RBE and Mz-ChA-2 cells responded well to TGF-beta1 treatment. TGF-beta1-treated SK-ChA-1 cells exhibited attenuated luciferase activity and their growth was not inhibited. Smad4 mRNA was not detected in SK-ChA-1 and Mz-ChA-1 cells by Northern blot analysis. Genetic analysis disclosed a nonsense mutation in the Mad homologue 2a domain of the Smad4 gene in the SK-ChA-1 cells and a heterozygous deletion in the TGF-beta type II receptor gene in the KMBC cells. Expression of the exogenous Smad4 gene in the Mz-ChA-1 cells by transient transfection restored their luciferase activity. When these TGF-beta1-insensitive and less-TGF-beta1-sensitive cell lines were xenografted into nude mice, they developed tumors that had more prominent, intervening fibrosis (desmoplasia) than the tumors caused by TGF-beta1-sensitive cells. Thus, a tight correlation between disruption of the TGF-beta signaling pathway and deregulated growth of cancer cells has been demonstrated in biliary tract carcinoma. This seems to be a critical event in this carcinoma and may also be correlated with stromal cell reaction in cancer invasion.

Animals↗

The complete optical spectrum of liquid water measured by inelastic x-ray scattering.

Interaction of light with matter is of paramount importance in nature. The most fundamental property of a material in relation to light is its oscillator strength distribution, i.e., how strongly it absorbs light as a function of wavelength. Once the oscillator strength distribution is determined precisely for a wide enough energy range, the optical constants such as absorbance and reflectance as well as a number of other properties of the material, some of which are seemingly unrelated to photoabsorption, can be deduced. Most important of all is the fact that the interaction of matter with fast charged particles can be described by its complete optical spectra [Inokuti, M. (1986) Photochem. Photobiol. 44, 279-285]. Despite their importance, however, the complete optical spectra of volatile liquids including water have never been obtained accurately because of experimental difficulties inherent in vacuum UV spectroscopy. Inelastic x-ray scattering spectroscopy can provide quantitative data equivalent to those from vacuum UV absorption spectra. Herein, we show the complete optical spectrum of liquid water determined by making use of intense monochromatic x-rays supplied by the wiggler line X21 of the National Synchrotron Light Source.

Journal Article↗

An immuno-polymerase chain reaction assay for human interleukin-18.

Conventional enzyme-linked immunosorbent assays (ELISA) are sufficient to measure normal and elevated serum interleukin (IL)-18 concentrations, but have limited sensitivity when measuring low concentrations of IL-18 such as in patients with the acquired immunodeficiency syndrome. We have developed a highly sensitive method for detecting human (h) IL-18 using an immuno-polymerase chain reaction (PCR). A mouse monoclonal anti-hIL-18 antibody and rabbit polyclonal anti-hIL-18 antibody was used for an indirect sandwich ELISA with a detection limit of 40 ng/l and a very low background. For immuno-PCR, biotinylated DNA was produced from the plasmid Bluescript by PCR amplification with biotinylated M13-20 primer and nonbiotinylated M13 reverse primer. Immuno-PCR for hIL-18 was performed for 40 cycles using 1 ng/l of biotinylated DNA. This immuno-PCR has a detection limit of 2.5 pg/l, 1.6x10(4) times lower than that of the ELISA. In addition, our system avoids sampling error caused by heat transfer from the ELISA plate to the PCR tube because all procedures from immobilization of the antibody to PCR amplification can be performed in the same tube. This immuno-PCR for hIL-18 is the most sensitive method for detecting hIL-18 reported to date.

Animals↗

Markedly different pathogenicity of four immunoglobulin G isotype-switch variants of an antierythrocyte autoantibody is based on their capacity to interact in vivo with the low-affinity Fcgamma receptor III.

Using three different Fcgamma receptor (FcgammaR)-deficient mouse strains, we examined the induction of autoimmune hemolytic anemia by each of the four immunoglobulin (Ig)G isotype-switch variants of a 4C8 IgM antierythrocyte autoantibody and its relation to the contributions of the two FcgammaR, FcgammaRI, and FcgammaRIII, operative in the phagocytosis of opsonized particles. We found that the four IgG isotypes of this antibody displayed striking differences in pathogenicity, which were related to their respective capacity to interact in vivo with the two phagocytic FcgammaRs, defined as follows: IgG2a > IgG2b > IgG3/IgG1 for FcgammaRI, and IgG2a > IgG1 > IgG2b > IgG3 for FcgammaRIII. Accordingly, the IgG2a autoantibody exhibited the highest pathogenicity, approximately 20-100-fold more potent than its IgG1 and IgG2b variants, respectively, while the IgG3 variant, which displays little interaction with these FcgammaRs, was not pathogenic at all. An unexpected critical role of the low-affinity FcgammaRIII was revealed by the use of two different IgG2a anti-red blood cell autoantibodies, which displayed a striking preferential utilization of FcgammaRIII, compared with the high-affinity FcgammaRI. This demonstration of the respective roles in vivo of four different IgG isotypes, and of two phagocytic FcgammaRs, in autoimmune hemolytic anemia highlights the major importance of the regulation of IgG isotype responses in autoantibody-mediated pathology and humoral immunity.

Anemia, Hemolytic, Autoimmune↗

The role of p38 mitogen-activated protein kinase in IL-6 and IL-8 production from the TNF-alpha- or IL-1beta-stimulated rheumatoid synovial fibroblasts.

We examined the role of p38 mitogen-activated protein (MAP) kinase in the tumor necrosis factor alpha (TNF-alpha)- or interleukin-1beta (IL-1beta)-induced production of interleukin-6 (IL-6) and interleukin-8 (IL-8) in fresh rheumatoid synovial fibroblast (RSF) cultures concomitantly with the induction of p38 MAP kinase activity. Pretreatment of RSF with a specific p38 MAP kinase inhibitor, SB203580, blocked the induction of IL-6 and IL-8 without affecting nuclear translocation of nuclear factor kappaB (NF-kappaB) or IL-6 and IL-8 mRNA levels. These findings suggest that p38 MAP kinase inhibitor may have synergistic, rather than additive, effect for the treatment of rheumatoid arthritis.

Arthritis, Rheumatoid↗

Establishment of quantitative reverse transcription--polymerase chain reaction assays for human telomerase-associated genes.

Telomerase is an enzyme that synthesizes and adds repetitive telomeric sequences of (TTAGGG)n to the ends of chromosomes. Recently, several telomerase-associated genes have been cloned, making it possible to study the expression of these genes. Quantitative comparisons of the expression of these genes and of telomerase activity might help clarify the regulation of telomerase activity. Therefore, we established the validity of a quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay for the human telomerase catalytic subunit (hTERT) mRNA and telomerase associated protein (TEP1) mRNA using the TaqMan fluorogenic detection system. Using this assay, we quantitated hTERT mRNA and TEP1 mRNA expression in two human pancreatic cancer cell lines, AsPC-1 and PANC-1. Our results indicated that the levels of hTERT mRNA and TEP1 mRNA expression in AsPC-1 were 1.50 and 2.31 times higher than in PANC-1 cells. This TaqMan RT-PCR assay appears to be useful in determining the quantities of hTERT and TEP1 mRNAs in clinical specimens. Taken together, our results indicate that it is possible to measure the expression of the major telomerase genes subunits. Furthermore it is possible to apply this technique to determine the amount of other types of mRNA.

Carrier Proteins↗

Mapping of a particular element using an absorption edge with an X-ray fluorescence imaging microscope.

An X-ray fluorescence imaging microscope with a Wolter-type objective mirror (magnification: 13) has been constructed at beamline 39XU of SPring-8. Monochromatic X-rays (DeltaE/E approximately 10(-4)) in the energy range 6-10 keV were used for X-ray fluorescence excitation of the specimens. Using two monochromatic X-rays above and below the absorption edge of a particular element, a two-dimensional image of the element could be obtained. As a result, two-dimensional element mapping of the test specimens (Cu, Co, Ni, Fe and Ti wires) and constituent minerals (Fe, Mn and Ti) of a rock specimen (a piemontite-quartz schist) became possible.

Journal Article↗

Transient infiltration of neutrophils into the thymus in association with apoptosis induced by whole-body X-irradiation.

Generally, the process of apoptosis does not cause leakage of noxious cytosolic contents and is therefore non-inflammatory. However, as previously shown, macrophages ingesting apoptotic CTLL-2 cells produced pro-inflammatory cytokines, particularly interleukin-8 (IL-8) and macrophage inflammatory protein-2 (MIP-2), a murine IL-8 homolog. This predicted that rapid and massive apoptosis may induce neutrophil accumulation in vivo. In this study, we tested this prediction by inducing apoptosis by whole-body X-irradiation in mice. After exposure to 4 Gy X-ray irradiation, mice exhibited considerable apoptosis of thymic cells, which was associated with transient infiltration of neutrophils as well as MIP-2 mRNA expression. In contrast, in p53-deficient mice in which irradiation-induced apoptosis was suppressed, as has been reported, infiltration of neutrophils into the thymus was less than that found in p53+/+ mice. Taken together, these results suggest that massive and rapid apoptosis can result in infiltration of neutrophils.

Animals↗

Increased uptake of 99mTc-HL91 in tumor cells exposed to X-ray radiation.

99mTc-HL91, a hypoxic marker, may be a predictor of tumor response to radiotherapy and an indicator of tumor oxygenation in the course of treatment. In this study, serial changes in 99mTc-HL91 uptake were observed in the normoxic condition in a human bladder cancer cell line exposed to a single dose or a fractionated dose of 10 Gy with an x-ray beam. The uptake per cell increased during cell growth retardation induced by the irradiation. This finding indicates that 99mTc-HL91 uptake is affected by injury to cells due to radiation; it may therefore be difficult to correctly assess the tissue oxygenation status during radiotherapy with 99mTc-HL91.

Cell Division↗

Detecting meningeal carcinomatosis from breast cancer with thallium-201 SPECT.

Thallium-201 (201Tl) scintigraphy is one of the imaging methods used in the detection of various tumors including brain metastasis. We evaluated a patient with meningeal carcinomatosis from breast cancer by using 201Tl single-photon emission computed tomography (SPECT). Meningeal spread of a tumor was noted on enhanced CT. SPECT revealed tumor localization in meningeal carcinomatosis. These results suggest that SPECT with 201Tl may be useful in detecting meningeal carcinomatosis from breast cancer.

Breast Neoplasms↗

Gliosarcoma with thallium-201 SPECT.

Thallium-201 (201Tl) chloride scintigraphy is the imaging method use for the detection of various tumors including glioblastoma, but only limited information on 201Tl uptake in gliosarcoma is available. We investigated a patient with gliosarcoma by means of 201Tl single-photon emission computed tomography (SPECT) and MRI. SPECT imaging revealed high 201Tl uptake in the tumor, which was closely correlated with contrast-enhancement on MRI. These results suggest that SPECT with 201Tl may be useful for detecting gliosarcoma and provide physiological information on this tumor.

Adult↗

[Morphological studies of the form of the cupula in the semicircular canal ampulla].

BACKGROUND AND OBJECTIVE: The cupula in the ampulla of the semicircular canal, like the tectorial membrane in the cochlea, forms the essential link in transferring endolymph fluid movement stimuli to the hair cells. Clear understanding of physiological cupula movement only started evolving towards the end of the 1970s. Currently, the cupula is assumed to adhere firmly to the ampulla wall, with diaphragm-like displacement in the central section and at the base. PATIENTS/METHODS: In contrast to detailed functional descriptions, there have been very few investigations on cupula morphology. These have proved difficult due to the extremely hydrous structure, which inevitably results in shape distortion during fixation. This difficulty applies particularly to electron microscopy. RESULTS: The present morphological investigations were performed in artificial endolymph on fresh preparations in the salmon (Salmo salar). After staining and isolating the cupula from the ampulla, a precise morphological investigation was performed. A relatively uniform basic structure was observed, with considerable interindividual variability in cupula shape. CONCLUSIONS: Such variability suggests that impairment of cupular form can occur and could be associated with altered peripheral vestibular function.

Animals↗

Helicobacter pylori infection: augmentation of telomerase activity in cancer and noncancerous tissues.

Telomerase adds hexameric repeats of 5'-TTAGGG-3' to the ends of chromosomal DNA (telomere) and has been implicated in cell immortalization and cellular senescence. The aim of this study was to measure quantitatively the telomerase activity and human telomerase RNA component (hTR) content in gastric cancer and to examine the relation between these values and histologic factors including Helicobacter pylori as a risk factor for gastric cancer. Telomerase activity was measured by a modified telomeric repeat amplification protocol in cancerous and noncancerous tissues (intestinal metaplasia, chronic gastritis, normal mucosa) from 27 gastric cancer patients; hTR expression was examined by the quantitative reverse transcriptase-polymerase chain reaction using fluorescent probes. Telomerase activity was higher in cancers (total product generated: 33.7) than in noncancerous tissues. Telomerase activity was higher in intestinal metaplasia (16.7) and chronic gastritis (10.6) than in normal mucosa (3.5). In patients with intestinal-type gastric cancer, telomerase activity was higher in intestinal metaplasia with H. pylori infection than in that without infection. hTR expression was not correlated with telomerase activity. H. pylori infection may influence telomerase activity in cancer and noncancerous tissues.

Aged↗

Analysis of initial fouling process in coastal environment: effects of settlement, attachment, and metamorphosis promoters.

Effects of lumichrome, L-tryptophan, and curcumin on fouling organisms were examined on panels immersed in a near-shore aquatic environment. These products showed effective action in aquariums in preliminary screening tests for promoting different steps of fouling. Lumichrome showed metamorphosis-inducing activity for ascidian larvae (Halocynthia), L-tryptophan was a settlement-inducer for larvae of barnacles (Balanus), and curcumin showed attachment-promoting activity on the blue mussel (Mytilus). In order to establish that these tests are helpful in screening actual fouling or antifouling compounds, we examined the action of these three compounds in a coastal environment by following the first steps of biofouling, that is, by studying the quantity of chlorophyll, the number of bacterial cells, and the larvae settled. These experiments on the seashore indicated that these compounds did not act as promoters for the target organisms; however, they did show promoting effects on some nontarget organisms.

Journal Article↗

Limitations of urinary telomerase activity measurement in urothelial cancer.

The reported frequency of detectable telomerase activity in spontaneously voided urine samples from patients with urothelial cancer varied from 0 to 85%. We examined stasis in the bladder and specimen storage as interfering conditions in this assay. Telomerase activity in exfoliated cells was measured by a polymerase-chain-reaction-based assay in spontaneously voided urine from urothelial cancer patients. Effects of retention in the bladder and specimen storage from voiding to measurement of telomerase activity were modeled by suspending 10(6) cells from the cancer-derived T24 line in normal urine (pH 6.5) at 37 degrees C and 25 degrees C, respectively. Hematuria was modeled by adding hemoglobin. In T24 cells suspended in urine at 37 degrees C, telomerase activity had decreased to approximately 20% of preincubation activity after 1 h, and had disappeared after 3 h. In urine at 25 degrees C, telomerase activity in T24 cells had decreased to approximately 40% of preincubation activity at 1 h and to <10% at 6 h. When we examined telomerase activity in exfoliated cells in spontaneously voided urine from urothelial cancer patients (excluding first-voided morning specimens), telomerase activity was detected in only 21% of samples (four of 19) despite measurement with 1 h of voiding and steps to avoid hemoglobin interference. Measurement of telomerase activity in spontaneously voided urine is insufficiently sensitive and reliable for the diagnosis of urothelial cancer.

Carcinoma, Transitional Cell↗