Biomedical subjects

K Tsuruda

Publications and source records attributed to K Tsuruda.

At least 19 recordsLinked to original sources

PubMed2005

Sensitivity of adult T-cell leukaemia lymphoma cells to tumour necrosis factor-related apoptosis-inducing ligand.

Tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) induces apoptosis in many transformed cells, but not in normal cells, and hence TRAIL has recently emerged as a novel anti-cancer agent. Adult T-cell leukaemia lymphoma (ATLL) is a neoplasm of T-lymphocyte origin aetiologically associated with human T-lymphotropic virus type 1 (HTLV-I), and is resistant to standard anti-cancer therapy. We thus characterized the sensitivity of ATLL cells to TRAIL in this study. Although most primary ATLL cells and cell lines expressed TRAIL death receptors on their surface, they showed only restricted sensitivity to TRAIL. Among the 10 ATLL cell lines examined, one was sensitive, but two had insufficient death-receptor expression, two had an unknown resistant mechanism with abrogation of the death signal upstream of caspase-8, and the remaining five showed attenuation of the signal in both extrinsic and intrinsic pathways by X-linked inhibitor of apoptosis and Bcl-2/Bcl-xL respectively. Furthermore, the level of HTLV-I tax expression was significantly correlated to TRAIL resistance. Interestingly, ATLL cells themselves expressed TRAIL on the cell surface. Constitutive production of TRAIL may offer resistance, thus allowing the development of TRAIL-resistant ATLL cells. Consequently, the resistant mechanism in ATLL cells against TRAIL was assigned to multiple factors and was not explained by a definitive single agent.

Antineoplastic Agents↗

PubMed2005

Proviral status of HTLV-1 integrated into the host genomic DNA of adult T-cell leukemia cells.

Human T-cell leukemia virus type-1 (HTLV-1) is the etiological agent of adult T-cell leukemia (ATL), and leukemic cells always carry the proviral genome monoclonally integrated into their host genomes at the same sequence site, designated as the monoclonal integration. Using Southern blot hybridization (SBH) and sequenced tagged site polymerase chain reaction assays, we examined the proviral status in 558 clinical specimens from 350 patients who are suspected to have ATL. A total of 321 specimens (57.5%) from 241 patients showed positive results for the monoclonal integration according to SBH, using EcoR1 and Pst1. The 241 patients consisted of 136 patients (56.4%) with the complete provirus (C-type), 62 patients (25.7%) with a defective provirus (D-type), and 43 patients (17.8%) with multibands (M-type). The incidence of the D- and M-types were in the order of smoldering, chronic, and acute subtypes of ATL, suggesting that such an aberrant proviral status is generated on the way to multistep carcinogenesis and is subsequently clinically important for the malignant behavior of the disease. Moreover, our data showed that the partial deletion of the proviral genome is initiated first at the site of the gag region and spreads into the sites of the pol and env regions, whereas the long terminal repeats and pX regions are almost always conserved. These results suggest that analysis of the proviral status provides useful diagnostic and virologic-oncological information about ATL and HTLV-1 pathology, especially the important role of pX gene in tumorigenesis.

Adult↗

PubMed2001

Occurrence of tetrodotoxin in the skin of a rhacophoridid frog Polypedates sp. from Bangladesh.

Twenty-three specimens of a tree-frog Polypedates sp. were collected from two locations (Mymensingh and Barisal) of Bangladesh in 1999, and assayed for their toxicity scores and toxin principle. Among the tissues, only the skin of the Mymensingh specimens was found to be toxic in mouse test, with the toxicity scores of 31-923MU/g. The toxin isolated from the skin was analyzed by high-performance liquid chromatography, electrospray ionization-time of flight mass spectrometry and proton nuclear magnetic resonance, and characterized as tetrodotoxin, a toxin principle.

Animals↗

PubMed2001

Actinobacillus actinomycetemcomitans induces lethal effects on the macrophage-like human cell line U937.

We examined the cytotoxicity in culture medium of Actinobacillus actinomycetemcomitans against the human monocyte-macrophage-like cell line U937 using the trypan blue exclusion test and WST-1 test. We found that A. actinomycetemcomitans Y4 showed the highest cytotoxic activity among the three different serotype strains and the cytotoxic effects of both bacterial cells and culture supernatants in A. actinomycetemcomitans Y4 were stronger on phorbol-12-myristate 13-acetate (PMA)-induced U937 cells than uninduced U937 cells. Morphological changes in PMA-induced U937 cells treated with culture supernatants differed from those treated with leukotoxin, and a difference in the susceptibility to 56 degrees C heat treatment was found between culture supernatants and leukotoxin. The cytotoxic activity by WST-1 was determined more rapidly and strongly than that by trypan blue assay. These findings suggested that the cytotoxic effect of A. actinomycetemcomitans was influenced by the differentiation of U937 cells and may be more potent on the respiratory chain than the cell membrane.

Aggregatibacter actinomycetemcomitans↗

PubMed2001

Simultaneous expression of guinea pig UDP-glucuronosyltransferase 2B21 and 2B22 in COS-7 cells enhances UDP-glucuronosyltransferase 2B21-catalyzed morphine-6-glucuronide formation.

Although UDP-glucuronosyltransferases (UGTs) act as an important detoxification system for many endogenous and exogenous compounds, they are also involved in the metabolic activation of morphine to form morphine-6-glucuronide (M-6-G). The cDNAs encoding guinea pig liver UGT2B21 and UGT2B22, which are intimately involved in M-6-G formation, have been cloned and characterized. Although some evidence suggests that UGTs may function as oligomers, it is not known whether hetero-oligomer formation leads to differences in substrate specificity. In this work, evidence for a functional hetero-oligomer between UGT2B21 and UGT2B22 is provided by studies on the glucuronidation of morphine in transfected COS-7 cells. Cells transfected with UGT2B21 cDNA catalyzed mainly morphine-3-glucuronide formation although M-6-G was also formed to some extent. In contrast, cells transfected with UGT2B22 cDNA did not show any significant activity toward morphine. When UGT2B21 and UGT2B22 were expressed simultaneously in different ratios in COS-7 cells, extensive M-6-G formation was observed. This stimulation of M-6-G formation was not observed, however, when microsomes containing UGT2B21were mixed with those containing UGT2B22 in the presence of detergent. Furthermore, this effect was not very marked when human UGT1A1 and UGT2B21 were coexpressed in COS-7 cells. This is the first report suggesting that UGT hetero-oligomer formation leads to altered substrate specificity.

Amino Acid Sequence↗

PubMed2001

Toxicity and toxin profiles of the newt, Cynops pyrrhogaster from western Japan.

A total of 382 specimens of a Japanese newt, Cynops pyrrhogaster, were collected from western Japan during 1996 to 1999, and assayed for their individual, geographical, sexual, seasonal variations, and anatomical distribution of toxicity by mouse. Most of the specimens tested showed toxicity scores ranging from 5 to 370 MU/g, where no seasonal, but large individual, sexual, and regional variations of toxicity were clearly recognized. Among the parts, skin and muscle showed higher toxicity scores (56 MU/g) than liver, stomach, intestine and gonad, whose toxicity ranged from less than 2 to 33 MU/g. The C. pyrrhogaster toxin was purified by several steps of column chromatography and was shown to consist of tetrodotoxin (TTX) and 6-epiTTX as main components, and 4-epiTTX, 4,9-anhydro-6-epiTTX, and 4,9-anhydroTTX as minor ones by means of HPLC and 1H-NMR analyses.

Animals↗

PubMed2000

Antibacterial effect of silver-zeolite on oral bacteria under anaerobic conditions.

OBJECTIVES: The purpose of this study was to evaluate the antibacterial effect of silver-zeolite (SZ) against oral bacteria under anaerobic conditions. METHODS: The antibacterial activity of SZ was evaluated by determining the minimum inhibitory concentrations (MICs) using two-fold serial dilutions of SZ in Brain Heart Infusion broth. Release of Ag+ into the broth was measured by an atomic absorption technique. RESULTS: SZ inhibited the growth of the bacteria tested under anaerobic conditions. The MIC of SZ ranged between 256 and 2048 micrograms/ml, which corresponded to a range of 4.8-38.4 micrograms/ml of Ag+. All strains grew in broth containing 16,384 micrograms/ml of type-A zeolite. SIGNIFICANCE: These results suggested that SZ may be a useful vehicle to provide antibacterial activity to dental materials used even under anaerobic conditions such as deep in the periodontal pocket.

Anaerobiosis↗

PubMed2000

Qualitative and quantitative characterization of Fas (CD95) expression and its role in primary human acute leukemia cells.

Fas antigen, a cell surface molecule, directly mediates apoptosis, and is expressed on a limited number of human tissues. Blood or bone marrow samples from patients with acute myelogenous leukemia (AML), acute lymphoblastic leukemia (ALL) and mixed leukemia were examined qualitatively and quantitatively for the expression of Fas as well as its function using flow cytometry and the annexin V staining method. Fas expression was flow cytometrically unimodal with heterogeneous density, and showed quantitatively characteristic features in different diseases: undetectable in mixed leukemia, faint to weak in ALL, low in M0 and M1, and variable (low to strong) in M2, M3, M4, and M5. Both the full-length and the alternatively spliced truncated mRNAs were detected constitutively even in acute leukemia cells with qualitatively negative and quantitatively faint Fas, and the band density of the former transcripts detected by RT-PCR was correlated with the level of expression of the Fas protein. Short-term culturing of freshly isolated leukemia cells gave rise to an increase of Fas density. In acute leukemia cells, the apoptosis induced by anti-Fas MoAb was compared with that induced by etoposide (a topoisomerase II inhibitor). We found that fresh ALL and AML cells were resistant to the anti-Fas IgM antibody, while etoposide could trigger apoptosis in all types of leukemia tested. The combined effects of the anti-Fas MoAb and etoposide were not always synergistic. These results suggest that Fas is a biological marker for characterizing ALL and AML cells, and provide insight into creating a new therapeutic modality using cytotoxic drugs and cytokines together with modulation of Fas.

Acute Disease↗

PubMed2000

Lactacystin activates FLICE (caspase 8) protease and induces apoptosis in Fas-resistant adult T-cell leukemia cell lines.

Lactacystin (LC) is a specific inhibitor of the proteasome, and has recently been shown to induce apoptosis in certain cell lines. In the present study, we established Fas-resistant adult T-cell leukemia (ATL) cell subclones RSO4 and RST1 from their parental Fas-sensitive cell lines SO4 and ST1, and examined whether LC can overcome Fas resistance. LC completely inhibited proteasome function as determined by a peptidyl-MCA substrate (LLVY-MCA and LLE-MCA), and induced apoptosis in these cell lines irrespective of Fas sensitivity at low concentrations (approximately 10 microM). LC induced the activation of caspase 3 (CPP32/Yama) and caspase 6 proteases in an identical manner to Fas-mediated apoptosis. Moreover, LC induced the activation of caspase 8 (FLICE) protease, which is the initiator of the Fas-mediated apoptotic cascade. Synthesized proteasome inhibitory peptide MG-115 (ZLLnV-CHO) also induced apoptosis in these cell lines. These results indicated that proteasome inhibitors overcome Fas-resistance by bypassing the proximal part of the Fas signal. Inhibition of the proteasome function may be a new strategy for the treatment of ATL.

Acetylcysteine↗

PubMed1999

Fas-resistance in ATL cell lines not associated with HTLV-I or FAP-1 production.

A preventive role for human T-cell leukemia virus type-I (HTLV-I) and Fas-associated phosphatase-1 (FAP-1) in Fas-mediated apoptosis has been reported in HTLV-I-infected cells. In the present study, we examined whether these molecules increased during the acquisition of Fas-resistance in adult T-cell leukemia (ATL) cell lines. SO4, ST1 and KK1 are Fas-sensitive ATL cell lines, and produce small amounts of HTLV-I in vitro. Although their subclones RSO4 and RST1 are completely Fas-resistant, they produced an equivalent amount of HTLV-I to SO4 and ST1. Moreover, FAP-1 mRNA was not detected in these cell lines irrespective of Fas sensitivity. Thus, Fas resistance in ATL cells was not directly associated with the increased production of HTLV-I or FAP-1.

Antibodies, Monoclonal↗

PubMed1999

Fas gene mutation in the progression of adult T cell leukemia.

Fas antigen (Apo-1/CD95) is an apoptosis-signaling cell surface receptor belonging to the tumor necrosis factor receptor superfamily. Adult T cell leukemia (ATL) cells express Fas antigen and show apoptosis after treatment with an anti-Fas monoclonal antibody. We established the ATL cell line KOB, which showed resistance to Fas-mediated apoptosis, and found that KOB expressed two forms of Fas mRNA, the normal form and a truncated form. The truncated transcript lacked 20 base pairs at exon 9, resulting in a frame shift and the generation of a premature stop codon at amino acid 239. The same mutation was detected in primary ascitic cells and peripheral blood cells. The mutation was not detected in lymph node cells, however, although all of the primary ATL cells were of the same clonal origin. A retroviral-mediated gene transfer of the truncated Fas to Jurkat cells rendered the cells resistant to Fas-mediated apoptosis, suggesting a dominant negative interference mechanism. These results indicate that an ATL subclone acquires a Fas mutation in the lymph nodes, enabling the subclone to escape from apoptosis mediated by the Fas/Fas ligand system and proliferate in the body. Mutation of the Fas gene may be one of the mechanisms underlying the progression of ATL.

Aged↗

PubMed1999

Qualitative and quantitative characterization of Fas (APO-1/CD95) on leukemic cells derived from patients with B-cell neoplasms.

Expression density and function of Fas (APO-1/CD95) on malignant B-cells, an antigen thought responsible for abnormal tumor biology, remains to be fully understood. Fifty-five cases with B-cell neoplasms of acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), B-cell malignant lymphoma (ML), and myeloma (MM) were studied for qualitative and quantitative expression and function of Fas using flow cytometry and annexin-V staining methods. Fas expression was flow cytometrically unimodal with heterogeneous density and showed quantitatively characteristic features among different diseases; weak in ALL, faint in CLL, moderate in HCL, and strong in ML, respectively. Not only full-length but also alternatively spliced truncated mRNAs were detected even in leukemic B-cells with qualitatively faint or negative Fas, and then band density of the former transcripts by RT-PCR was correlated to the Fas protein expression level. Short-term culture of freshly isolated cells gave rise to increases of Fas density and susceptibility for apoptosis, suggesting that the mRNA and inducible Fas are functional at least in vitro. These results show that Fas is a biological marker for characterizing B-cell neoplasms reflecting various stages of B-cell ontogeny and may have clinical utility as a therapeutic strategy.

Apoptosis↗

PubMed1999

Discrepant expression of membrane and soluble isoforms of Fas (CD95/APO-1) in adult T-cell leukaemia: soluble Fas isoform is an independent risk factor for prognosis.

The Fas signalling system probably plays a critical role in the natural and chemotherapeutic cell death machinery, suggesting that aberrant Fas expression is involved in growth control of tumours. The membrane isoform (mFas) is a 45 kD cell surface protein containing a single transmembrane region, and induces apoptosis in normal or tumour cells, whereas the soluble isoform (sFas) lacks the transmembrane domain due to alternative splicing of the transcript and is thought to block Fas-mediated apoptosis. To clarify the clinical roles of expression of these two Fas isoforms in adult T-cell leukaemia (ATL), we investigated the levels of the Fas isoforms in 81 patients with ATL. The expression patterns of the Fas isoforms were heterogenous, and there was no significant correlation between mFas and sFas levels: 10/81 cases were negative for mFas and had high serum sFas levels, whereas the remaining 71 cases were positive for mFas and had various levels of expression of the two Fas isoforms. Irrespective of the status of mFas expression in leukaemic cells, the mRNAs encoding these isoforms were always detectable, indicating the potential for protein translation. Although mFas expressed on freshly isolated ATL cells could iduce apoptosis in vitro, positive versus negative mFas status was not associated with any clinical aspects of ATL, whereas the sFas level was strongly correlated with clinical parameters such as serum LDH activity, tumour burden, serum soluble IL-2R level, hypercalcaemia and prognosis. These results suggest that the ratio of Fas isoforms varies, and high expression of the sFas protein and message reflects the malignant behaviour of ATL and is an independent risk factor for the prognosis.

Female↗

PubMed1999

Clonal analysis of B-cell leukemias and lymphomas using the polymerase chain reaction for the third complementarity determining region of the IgH gene: a study of 75 cases from Nagasaki Japan.

Using semi-nested polymerase chain reaction (PCR), we examined 75 Japanese cases of hematologic malignancies with B-cell antigens including 25 common acute lymphoblastic leukemia (ALL), 13 chronic lymphocytic leukemia (CLL), 28 B-cell malignant lymphoma (B-ML), 2 hairy cell leukemia (HCL), 7 acute myelogenous leukemia with B-cell antigens (AML-B), and 23 controls. When amplified products were analysed by a standard polyacrylamide gel electrophoresis, the sensitivity for detection of clonal IgH rearrangements in each group of ALL, CLL, B-ML, HCL, and AML-B was 88%, 92.3%, 71.4%, 100%, and 57.1%, respectively, with an overall sensitivity of 80.0%. There were no false positive results in any of the control samples. Single strand conformation polymorphism (SSCP) analysis of the amplified products gave rise to a much greater sensitivity, up to 84% overall. The false negative samples were mainly encountered in B-ML with SmIgG and non-Ig, suggesting miss-annealing between the primers used and the template DNA because of somatic hypermutation of IgH genes in such clones. This indicates that PCR analysis is very useful in detecting the clonal IgH rearrangements in B-cell malignancies, especially in ALL and CLL, but not in B-ML corresponding to neoplasms originating from pre-germinal center naive B-cells.

Clone Cells↗

PubMed1999

[Monoclonal analysis in B-cell neoplasms by the semi-nested polymerase chain reaction using consensus primers].

Monoclonality on B-cells is well known to be determined on the basis of presence of a rearranged-IgH gene, which is detected by Southern blot hybridization (SBH) remaining to be elucidated in respects of not only time-consumed, labour and cost benefit and also the use of much DNA samples. Alternative to this SBH, we examined the clinical usefulness of monoclonal analysis by the polymerase chain reaction technique which amplifies rearranged-CDR III region of IgH gene (IgH-PCR). The detective sensitivity of the IgH-PCR was different dependently upon each analysis for amplified products, namely 10(-2) per mononuclear cells in agarose gel analysis and 10(-3) in polyacrylamide gel and single strand conformation polymorphism analysis (PAGE and SSCP). Then, using the IgH-PCR and PAGE/SSCP analysis, 75 Japanese patients with B-neoplasm and 23 with T-cell neoplasms were examined for clonal IgH rearrangements. The diagnostic sensitivity in each group of B-ALL, B-CLL, B-lymphoma, HCL, AML with B-cell antigens, and non-T cell neoplasms was 88%, 92.3%, 71.4%, 100%, 57.1%, and 0%, respectively, with an overall sensitivity and specificity of 88% and 100%. This indicates that PCR analysis is very useful in detecting the clonal rearrangement of IgH genes on B-cell neoplasms, especially on ALL and CLL corresponding to neoplasms counterparting to naive B-cells.

Clone Cells↗

PubMed1998

Pilot study on the effect of a mouthrinse containing silver zeolite on plaque formation.

A double-blind cross-over study was performed to evaluate the inhibitory activity of silver zeolite (SZ) mouthrinse on plaque formation. Eleven dental students participated in this study. SZ mouthrinse was prepared by suspending SZ powder into phosphate-buffered saline (PBS) at a concentration of 3% (w/w). Type-A zeolite was used as a placebo. Before the experiment, the subjects were rendered plaque-free by professional prophylaxis. They then suspended any oral hygiene for five days, during which time they rinsed with either SZ or type-A zeolite mouthrinse twice a day. SZ significantly reduced plaque formation compared to the placebo (p < 0.05), suggesting that silver ions released from the SZ inhibited plaque formation.

Adult↗

PubMed1997

Soluble and membrane isoforms of Fas/CD95 in fresh adult T-cell leukemia (ATL) cells and ATL-cell lines.

Fas, also designated as Apo-1 and CD95, is a cell membrane receptor (mFas) involved in apoptotic cell death. A soluble form (sFas) lacking the transmembrane domain due to alternative splicing has been isolated. Abnormal expression of sFas and mFas is likely to be involved in lymphoproliferative disorders and auto-immune diseases. Adult T-cell leukemia (ATL) caused by human T-cell-leukemia virus type-1 (HTLV-1) is well known to be a T-cell neoplasm with strong mFas expression, suggesting a role of Fas in the pathology of the disease. We examined protein and mRNA expression of the 2 isoforms of Fas in fresh ATL cells and ATL cell lines. In general, mFas was strongly expressed in ATL cells, and sFas levels in sera were high, especially in malignant ATL. However, expression of the isoforms in some cases of ATL varied; there was no mFas expression on the cell surface and sFas levels were high in serum. In contrast, all ATL cell lines examined showed strong mFas expression and scarce production of sFas in the supernatant, corresponding to strong expression of full-length Fas mRNA and weak to negative expression of alternatively spliced mRNA lacking the transmembrane domain. Our findings indicate that the mode of expression of Fas isoforms in ATL cells is not always homogenous and that Fas may play a role in the malignant behavior and oncogenesis of ATL.

Flow Cytometry↗

PubMed1997

Quantitative characterization and potential function of membrane Fas/APO-1 (CD95) receptors on leukaemic cells from chronic B and T lymphoid leukaemias.

The expression and function of the Fas-receptor (Fas-R) were examined in chronic lymphocytic leukaemia (CLL), hairy cell leukaemia-variant (HCL-v) and adult T-cell leukaemia (ATL). The expression of Fas-R in freshly isolated leukaemic cells was qualitatively and quantitatively different between each disease; faint in B-CLL, moderate in HCL-v and strong in ATL. Both full-length and alternatively spliced truncated forms of Fas mRNA were detected even in CLL B cells with faint to negative Fas-R, and Fas mRNA was also shown to be capable of increasing in vitro expression, i.e. the message was functional. In contrast, Fas-R expression on ATL cells was heterogenous and usually intense with a mean density approximately 3-fold higher than that of normal T cells. Fas-R was confirmed to have the potential function for anti-Fas monoclonal antibody-mediated cell death in vitro in Fas-R+ ATL cells. The expression level of Fas-R on the cells was higher in chronic than acute ATL (10,360 v 6260 antibody-binding capacity per cell, mFasABC; P<0.05) and was inversely correlated with serum LDH activity, suggesting that the strong Fas-R accounts for the slow progression of chronic ATL and the negative Fas-R protects from Fas-mediated cell death. These results show that Fas-R expression on leukaemic cells is valuable in their characterization and perhaps their function, and may contribute to the progression and immune evasion of malignant clones.

Apoptosis↗