Biomedical subjects
Publications and source records attributed to H Tsuda.
Gene rgpG is required for biosynthesis of rhamnose-glucose polysaccharide (RGP) in Streptococcus mutans. Its deduced amino acid sequence had similarity to WecA, which initiates syntheses of enterobacterial common antigen and some O antigens in Escherichia coli. Gene rgpG complemented a wecA mutation of E. coli, suggesting that rgpG may function similarly in RGP synthesis.
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We systematically screened for the aetiology of thrombophilia in 115 patients with venous, arterial and small vessel thromboses. Forty-one patients (36% of those we examined) suffering from a variety of thromboses, including deep vein thrombosis, pulmonary embolism, arterial occlusion, cerebral infarction, Moyamoya disease and ulcerative colitis, were characterized either with positive lupus anticoagulants or with decreased activities of protein S, protein C, antithrombin III and/or plasminogen. Eight mutation sites were confirmed in 11 thrombotic patients using gene analysis. Decreased protein S activity was found with a high incidence (23 out of 115) in Japanese patients who suffered from not only venous thrombosis but also arterial and small vessel thrombosis. We emphasize here the important role of protein S in the pathogenesis of thrombosis in the Japanese population.
Recently plasmapheresis has been performed for connective tissue disease. Double-filtration plasmapheresis (DFPP) has mainly been performed for rheumatoid arthritis (RA) and systemic lupus erythematosus (SEL) since 1980. In DFPP, autoantibodies and immune complex are removed by the second filter. The adaptation of plasmapheresis is commonly for cases (1) which are resistant to some medications, (2) which have a high titer of antibodies or immune complex, (3) in which the medication must be decreased or stopped because of side effects and complications, or (4) which are in the acute active phase. We suggest DFPP to be useful as a treatment for connective tissue disease.
Advances in identification of the risk factors for cancer development in different organs imply more effective screening for early malignancies. The associated increase in the predictive value, as well as the introduction of procedures with high sensitivity and specificity, provide promise that early intervention will result in a marked decrease in the mortality and morbidity due to a wide range of major cancers. Furthermore, a variety of methods are now available to detect lesions at a sufficiently early stage for curative surgery to be attempted. However, for the full promise to be realized, it is of paramount importance that both physicians and the public at large are fully aware of the potential benefits.
Two cases with HLA-B 27 negative, Chlamydia-induced reactive arthritis (ReA) were described. Case 1: A 30 y.o. male developed balanitis, urethritis, arthritis of both knees, elbows, shoulders and hip joints on May in 1997. Laboratory findings revealed CRP 2.7 mg/dl (normal range < 0.3), ESR 33 mm/h and negative rheumatoid factor (RF) test. Anti-Chlamydia trachomatis antibodies, IgG 2.22, IgA 3.33 were positive. HLA-typing revealed A 2, A 24 (9), B 39 (16), B 52 (5). He was diagnosed as ReA and arthritis subsided with treatment of minocycline and nonsteroidal antiinflammatory drugs (NSAIDs). Case 2: A 40 y.o. Iranian American male developed balanitis, urethritis, lumbago, arthritis of both elbows, knees and foot joints, iridocyclitis on August in 1995. Chlamydia trachomatis was detected in the urethral swab culture. He was diagnosed as ReA and treated with minocycline and NSAIDs. He was referred to our hospital on June in 1996. Arthritis at both knees and feet was detected. Laboratory findings revealed CRP 0.8 mg/dl, negative RF test was revealed. Antibodies to Chlamydia were positive (IgG 1.49, IgA 1.53) positive. HLA typing revealed A 1, A 2, B 37, B 55 (22). He was again treated with minocycline and NSAIDs and ReA ameliorated. Since HLA-B 22, B 37 and B 39 have been reported to cross-react or to have homology with B 27, B 22, B 37 and B 39 are likely to related to inducing ReA.
We calculated microvessel counts (MVCs) by analyzing CD31-stained sections in three tumor zones (central, intermediate, and peripheral) in 147 cases of invasive ductal carcinoma (IDC). The purpose of the study was to discover whether there is a difference in MVC in the different zones of tumor, which zone contains the highest MVC within the tumor, from which zone the MVCs best correlate with tumor recurrence or tumor death, and which histologic factors correlate with the MVC of the tumor. Sections were scanned to assess the highest number of microvessels in any single 200 x field (0.384 mm2). In all of our cases, the average MVCs of the central, intermediate, and peripheral zones of the IDCs were 34.4, 39.4, and 51.5 per 200x field, respectively. The MVC significantly increased from the central to the peripheral zones (P < .001). In the univariate analysis, in at least one tumor zone, the MVC was correlated with T classification, tumor necrosis, fibrotic focus (a scar-like area within IDCs), and c-erbB-2 protein expression. The only factor significantly correlated with a higher MVC in all of the three zones was fibrotic focus. Moreover, in the multivariate analysis, tumors having high MVCs in the peripheral zone were significantly associated with higher hazard ratios for tumor recurrence (P < .05). This study showed that the MVC of an IDC significantly increases from the central to the peripheral zones, and it showed that angiogenesis in the peripheral zone is associated with prognosis. Therefore, estimation of angiogenesis should be performed in the peripheral zone for reliable prediction of outcome in breast cancer patients. As a surrogate for angiogenesis, fibrotic focus seems to be a useful marker for malignant potential in breast cancer.
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A 45-year-old man was admitted complaining of chest pain and pain and edema in the left lower extremity. Ultrasonography and venography results yielded a diagnosis of left femoral vein thrombosis, and pulmonary embolism was diagnosed later. Intravenous heparin therapy (10,000 IU/day) improved the patient's clinical signs. During this therapy, however, pain and edema of the right lower extremity developed, leading to a diagnosis of right femoral vein thrombosis. The patient was admitted to our hospital. At that time, coagulation studies showed an FDP level of 44.7 micrograms/ml and an FDP-DD level of 24.5 micrograms/ml. We surmised that the bilateral deep vein thrombosis had been caused by hyperhomocysteinemia (17.8 mumol/l). Genetic and other acquired risk factors for thrombophilia were ruled out. The patient's clinical signs again improved as a result of intravenous heparin therapy (15,000 IU/day), and FDP and FDP-DD levels returned to normal. We concluded that hyperhomocysteinemia is a risk factor for thrombosis and that it can generate thrombosis in other locations even during heparin therapy.
In an attempt to explore the molecular mechanisms for protein S deficiency, a patient with such a deficiency was examined at the DNA, RNA and protein levels. Nucleotide analyses revealed that the proband, the mother and the grandmother had a G-->C substitution in the invariant AG dinucleotide at the splicing acceptor site of intron A/exon 2. This patient was heterozygous for this substitution and the mutant allele was inherited from the proband's mother and grandmother. Reverse transcription-polymerase chain reaction analysis demonstrated several kinds of splicing abnormalities such as exon skipping and cryptic splicing, in addition to correct splicing. Semiquantitation of mRNA for the protein S gene revealed that the amount of the proband's mRNA was reduced to 60% of normal. Thus, this mutation impaired the normal processing of mRNA for the protein S gene, resulting in the subject's severe protein S deficiency.
We performed a multicenter, an early phase II clinical trial to evaluate the feasibility, safety and efficacy of myeloablative therapy supported by autologous peripheral blood stem cell transplantation (auto-PBSCT) for the treatment of acute myelogenous leukemia (AML) in first remission. A total of 105 patients were enrolled in the study, and 56 patients in first complete remission received auto-PBSCT. The median age was 44 years. Of the 56 patients, 34 (60.7%) had M2 or M3 AML by the French-American-British Classification system. The median concentration of infused CD34+ cells was 2.3 x 10(6)/kg by recipient body weight. Median days to reach an absolute neutrophil count > 500/microliter and a platelet count > 20000/microliter were 14 and 16, respectively. The median disease-free survival rate was estimated to be 62.0% at a median follow-up time of 534 days. Although the study enrolled a small number of patients and the follow-up period was relatively short, the preliminary results were encouraging and indicated that myeloablative chemotherapy with auto-PBSCT is feasible and can be performed safely as a post-remission therapy for AML. A prospective randomized clinical trial of auto-PBSCT versus standard chemotherapy alone will be necessary to assess the efficacy of high-dose therapy facilitated by auto-PBSCT as a post-remission therapy for AML.
In assessing the present status of rodent models for the analysis and prevention of carcinogenesis, the discussion emphasized that models exist for very few of the major cancers occurring in the United States and Japan. Almost without exception, those that do exist were invented for etiological and mechanistic studies of chemical and physical carcinogenesis. Applicability to cancer chemoprevention was not a primary objective in their development. However, they have been adapted and used for identifying and characterizing chemopreventive agents out of necessity. It is therefore not surprising that they generally fail to fulfill most or all of the requisites of "ideal" models enumerated above, and consequently, the validity of data produced through their use has been questioned. Evolution of future mechanistic models may similarly lead to their eventual adaptation to chemoprevention, but advances will be fragmentary, and the rate of progress seems likely to be slow. Animal models discussed at this workshop were designed specifically for this purpose and promise to expedite the accumulation of valuable new information. Nonetheless, recurrent discussion identified the urgent need for institution of a major, dedicated research initiative with the expressed objective of developing rodent models for organ-specific chemoprevention based on current understanding of underlying genetic and cellular processes. No concerted programs with this objective presently exist either in the United States or Japan. In addition to research specifically designed to meet this need, efforts should be made to involve investigators developing mechanistic animal models in the validation of their models through modulating cancer development by chemopreventive agents. Support for such research initiatives will be essential to continued progress toward the overall objective of identifying safe and effective cancer chemopreventive agents.
A significant positive association has been reported between p16 expression and clinical outcome for epithelial ovarian cancer patients. However, there is a reciprocal correlation between genetic alterations of single members of the p16-cyclin D1/CDK4-pRb pathway (G1 pathway). Simultaneous evaluation of these four elements may produce a better prognostic factor than p16 alone. We studied the prognostic significance of the G1 pathway in 59 epithelial ovarian cancer patients undergoing surgery and platinum-based chemotherapy by immunohistochemical technique. Abnormal expression of p16 or pRb was defined by negative nuclei staining, and that of CDK4 and cyclin D1 was defined by 50% nuclear staining. An abnormal G1 pathway was indicated in cases that have at least one abnormality among these four elements. Abnormal expression of p16, pRb, and cyclin D1/CDK4 was observed in 33.9, 3.4, and 15.3% of studied cases, respectively. Abnormal G1 pathway was detected in 49.2% (29 of 59) of all cases. The patients with normal G1 pathway tended to achieve a higher complete response rate (81.0%) to chemotherapy, compared with patients with abnormal G1 pathway (55.0%); however, there was no significant difference (P = 0.1001) between the two groups. Univariate analyses identified advanced stage [hazards ratio (HR), 3.665; P = 0.0218], histological low grade (HR, 3.625; P = 0.0066), and abnormal G1 pathway (HR, 2.935; P = 0.03) as prognostic factors for overall survival. The G1 pathway might help as a prognostic factor to select high-risk patients.
Ultrastructural studies of granulocytes were performed on two unrelated patients with hereditary thrombocytopenia, giant platelets, and inclusion bodies in granulocytes. Each patient had been diagnosed with May-Hegglin anomaly. In both cases, inclusion bodies in granulocytes consisted of clusters of ribosomes and small segments of rough endoplasmic reticulum. Additional clinical features suggesting Alport syndrome were lacking in these propositi and their family members. These observations imply that the patients were affected not with May-Hegglin anomaly but with Sebastian platelet syndrome. They would thus represent the seventh and eighth families known to carry this hereditary disease.
Chemopreventive effects of bovine lactoferrin (bLF), previously shown to strongly inhibit intestinal carcinogenesis in rats (K. Sekine, E. Watanabe, J. Nakamura, N. Takasuka, D.J. Kim, M. Asamoto, V. Krutovskikh, T.H. Baba, T. Ota, M.A. Moore, M. Masuda, H. Sugimoto, H. Nishino, T. Kakizoe, H. Tsuda, Inhibition of azoxymethane-initiated colon tumor by bovine lactoferrin administration in F344 rats, Jpn. J. Cancer Res. 88 (1997) 523-526; K. Sekine, Y. Ushida, T. Kuhara, M. Iigo, H. Baba-Toriyama, M.A. Moore, M. Murakoshi, Y. Satomi, H. Nishino, T. Kakizoe, H. Tsuda, Inhibition of initiation and early stage development of aberrant crypt foci and enhanced natural killer activity in male rats administered bovine lactoferrin concomitantly with azoxymethane, Cancer Lett. 121 (1997) 211-216), on spontaneous intestinal polyp development were assessed in the ApcMin mouse, a model for both familial adenomatous polyposis and sporadic colon cancers. In the experiment, 54 mice at 6 weeks of age were given 2% bLF (15 mice), 0.2% bLF (15 mice) and AIN-93G (24 mice) as basal diet ad libitum for 8 weeks. An overall tendency for a reduction in the total number of polyps in the small intestine was evident in the bLF-treated animals, along with significant suppression in the jejunum at the 2% dose (P < 0.05, 68% of the control). In addition, body growth suppression, presumed to be due to anemia and/or intussusception as a consequence of numerous polyps in the intestine, was alleviated. No toxic effects were observed in the intestinal epithelium. Although not as obvious as observed for the rat case, the data suggest that bLF may be a chemopreventor of intestinal polyposis.
In the course of structural studies on sulfated oligosaccharides isolated from porcine intestinal heparin after extensive digestion with Flavobacterium heparinase, we isolated several heparitinase-resistant unsaturated oligosaccharides. Amino sugar analysis of these oligosaccharides indicated that they contained galactosamine residues but no glucosamine residues. They were sensitive to chondroitinase ABC but resistant to chondroitinase AC-II, and therefore derived from dermatan sulfate, which was presumably contained as a minor component in the starting heparin preparation. The structures of these oligosaccharides were characterized by enzymatic digestions in conjunction with HPLC analysis of the digests and by one-dimensional and two-dimensional 500-MHz 1H-NMR spectroscopy. Structures of two tetrasaccharides and two hexasaccharides were determined as deltaHexAalpha1-3GalNAc(4S)beta1-4IdoAalpha1-3GalNAc(4S), deltaHexAalpha1-3GalNAc(4S,6S)]beta1-4IdoAalpha1-3GalNAc(4S) , deltaHexAalpha1-3GalNAc(4S)beta1-4IdoAalpha1-3GalNAc(4S)beta 1-4IdoAalpha1-3GalNAc(4S), and deltaHexAalpha1-3GalNAc(4S)beta1-4IdoAalpha1-3GalNAc(4S,6S)b eta1-4IdoAalpha1-3GalNAc(4S), where deltaHexA, IdoA, GalNAc, 4S and 6S represent 4-deoxy-alpha-L-threo-hex-4-enepyranosyluronic acid, L-iduronic acid, N-acetyl-D-galactosamine, 4-O-sulfate and 6-O-sulfate, respectively. The latter three compounds have never been reported as discrete structures. Since the four isolated oligosaccharides contained an unsaturated uronic acid residue at the nonreducing terminus, they appear to have been generated by eliminative cleavage by the action of Flavobacterium chondroitinase that was probably present as a minor contaminant in the Flavobacterium heparinase preparation used. Two out of the four oligosaccharides shared the rare disulfated disaccharide sequence, -3GalNAc(4S,6S)beta1-4IdoAalpha1-. These oligosaccharides will be useful as authentic reference compounds for microanalyzing biologically active domains of dermatan sulfate.
Beta-catenin forms complexes with Tcf and Lef-1 and functions as a transcriptional activator downstream of the Wnt signaling pathway. Activation of the pathway by stabilization of beta-catenin has been shown to be important in the development of colorectal carcinoma, which is mainly caused by inactivating mutations of the adenomatous polyposis coli tumor suppressor gene or by activating mutations in exon 3 of the beta-catenin gene. Here, we analyzed mutations in exon 3 of the beta-catenin gene in endometrial carcinoma cases in which loss of heterozygosity at the adenomatous polyposis coli tumor suppressor gene locus has been rarely reported. We found that 10 of 76 cases had beta-catenin gene mutations. All mutations identified were single-base missense mutations on serine/threonine residues (codons 33, 37, 41, and 45), altering the glycogen synthase kinase-3beta phosphorylation consensus motif, which participates in the degradation of beta-catenin. To determine whether these beta-catenin mutations actually led to stabilization of this protein, expression of beta-catenin was analyzed immunohistochemically, and 9 of 10 cases with the beta-catenin mutation and 20 of 66 cases without it showed accumulation of beta-catenin in the cytoplasm and/or nucleus. In total, 38% of cases showed accumulation of beta-catenin. These data indicate that stabilization of beta-catenin due to mutations in exon 3 of the beta-catenin gene and other mechanisms may have an important role in development of endometrial carcinomas.