Biomedical subjects
Publications and source records attributed to H Ito.
[reaction: see text] The enantioselective aldol reaction using a novel binuclear zinc catalyst of acetone with several aldehydes gave products in good yields (62-89%) with a high level of enantioselectivity (ee = 76-92%).
A polymer brush containing a diethylamino group as an anion-exchange group was appended onto a polymer substrate by radiation-induced graft polymerization and subsequent chemical modifications. Bovine serum albumin as a chiral ligand for L-tryptophan was bound to the polymer brush at a density ranging from 17 to 150 g BSA/l. For comparison, BSA was adsorbed onto the gel network containing a diethylaminoethyl group. The molar binding ratio of L-tryptophan to BSA on the polymer brush was 1.7-fold higher than that to BSA on the gel network.
BACKGROUND: Heat shock proteins (HSPs) are well known as cytoprotective proteins. Geranylgeranylacetone (GGA), a nontoxic anti-ulcer drug, was recently shown to have HSP-inducing capacity. In the present study, the activity of GGA was tested in a rat orthotopic liver transplantation (OLT) model to determine whether the compound has beneficial effects in warm ischemia-reperfusion injury. METHODS: Either GGA or a control vehicle was orally administered to donor rats before graft harvest. Harvested livers were subjected to 45-min warm ischemia (37 degrees C) followed by OLT. HSP mRNA expressions and HSP syntheses in the graft livers were evaluated by reverse transcriptase polymerase chain reaction and Western blot analysis, respectively. RESULTS: When the donors were treated with a vehicle, all recipients died of primary nonfunction within 2 days after OLT. In contrast, when the donors were treated with GGA (200 mg/kg per day) for 4 weeks, the 7-day survival rate of recipients was dramatically improved (90%). By giving a high dose of GGA (600 mg/kg per day) for 1 week, a similar improvement in recipient survival was seen (83.3%). GGA administration accumulated mRNA for both HSP72 and HSP90 in the livers even before warm ischemia and facilitated the syntheses of HSP72 and HSP90 after warm ischemia. In addition, GGA pretreatment also significantly reduced the serum levels of tumor necrosis factor-alpha (TNF-alpha) after reperfusion. CONCLUSIONS: These findings indicate that both the enhanced induction of HSPs and the suppression of a cytotoxic mediator (TNF-alpha) might be involved in the beneficial effects of GGA on ischemia-reperfusion injury. Thus, oral administration of GGA would be a useful tool for preventing primary nonfunction in liver transplantation.
We cloned a novel ankyrin repeat protein, Arpp, by immunoscreening a cDNA library constructed from a human esophageal carcinoma cell line, TE1, with an antibody directed to a hypothetical protein encoded by antisense p53 mRNA. Arpp protein is composed of 333 amino acids and contains four ankyrin-like repeat motifs in the middle portion of the protein, a PEST-like sequence and a lysine-rich sequence similar to a nuclear localization signal in the N-terminal region, and a proline-rich region containing consensus phosphorylation sites in the C-terminal region. Protein sequence analysis revealed that Arpp is homologous (52.7% identity) to Carp which is shown to be involved in the regulation of the transcription of the cardiac ventricular myosin light chain 2 gene. Arpp mRNA was found to be expressed in normal skeletal and cardiac muscle. Interestingly, Arpp expression was detectable in bilateral ventricles, but undetectable in bilateral atria and large vessels, suggesting that Arpp may play a specific function in cardiac ventricles as well as skeletal muscles.
We examined the effects of YM-31636 (2-(1H-imidazol-4-ylmethyl)-8H-indeno[1,2-d]thiazole monofumarate), a newly synthesized 5-HT(3) receptor agonist, on defecation in normal and constipated ferrets, and evaluated it as an agent against constipation. YM-31636 facilitated defecation without inducing diarrhea or emetic episodes. This effect occurred within 1 h after oral administration, mostly within 30 min, whereas sodium picosulfate, a widely used laxative, tended to increase the frequency of defecation for several hours with much lower peak incidence than that of YM-31636, and induced diarrhea. UK14304 (brimonidine), an alpha2 receptor agonist, and morphine reduced the frequency of defecation and YM-31636 restored it. These effects of YM-31636 were antagonized by ramosetron, a 5-HT(3) receptor antagonist. These results suggest that YM-31636 could be promising in the treatment of constipation. Because of an early and reliable onset of action compared with sodium picosulfate, YM-31636 could make it easier to control the time of defecation.
BACKGROUND: Although polyneuropathy related to chronic alcoholism has been reported frequently, its clinical features and pathogenesis remain to be clarified. OBJECTIVE: To determine the clinicopathologic features and pathogenesis of alcoholic polyneuropathy associated with pain in patients with normal thiamine status, particularly in comparison to beriberi neuropathy. PATIENTS AND METHODS: Clinical, electrophysiologic, and histopathologic findings were assessed in 18 patients with painful alcoholic polyneuropathy and normal thiamine status. RESULTS: Symmetric sensory-dominant polyneuropathy predominantly involving the lower limbs was the major clinical pattern. Painful sensations with or without burning quality represented the initial and major symptom. Progression of symptoms usually was gradual, continuing over months or years. Electrophysiologic and pathologic findings mainly indicated an axonal neuropathy. Densities of small myelinated fibers and unmyelinated fibers were more severely reduced than the density of large myelinated fibers, except in patients with a long history of neuropathic symptoms and marked axonal sprouting. CONCLUSIONS: The clinicopathologic features of painful symptoms and small axon loss are distinct from those of beriberi neuropathy. Sensory-dominant involvement with prominent neuropathic pain is characteristic of alcoholic neuropathy when thiamine deficiency is not involved, supporting the view of direct neurotoxic effect by alcohol or its metabolites.
The interaction of high power microwave with collisionless unmagnetized plasma is studied. Investigation on the generation of superthermal electrons near the critical layer, by the resonance absorption phenomenon, is extended to very high microwave power levels (eta=E(2)(0)/4 pi n(e)kT(e) approximately 0.3). Here E0, n(e), and T(e) are the vacuum electric field, electron density, and electron temperature, respectively. Successive generation of electron bunches having maximum energy of about 2 keV, due to nonlinear wave breaking, is observed. The electron energy epsilon scales as a function of the incident microwave power P, according to epsilon proportional to P0.5 up to 250 kW. The two-dimensional spatial distribution of high energy electrons reveals that they are generated near the critical layer. However, the lower energy component is again produced in the subcritical density region indicating the possibility of other electron heating mechanisms.
Levels of the small heat-shock proteins (sHSPs) HSP27 and alphaB-crystallin during differentiation of mouse C2C12 cells were determined using specific immunoassays. Increases of these proteins were about 3-fold and 10-fold, respectively. Under the same conditions, however, the level of HSP70 in C2C12 cells barely increased, indicating selective accumulation of HSP27 and alphaB-crystallin with differentiation. While expression of mRNA for alphaB-crystallin was also markedly increased and that for HSP27 was but to a lesser extent, mRNA for HSP70 could barely be detected during differentiation. Activation of the heat-shock factor was not observed, in contrast to the case with heat-stressed undifferentiated cells. Various inhibitors of protein kinases affected the differentiation and the associated increase of sHSPs. Rapamycin, an inhibitor of p70 S6 kinase, completely inhibited the differentiation and suppressed the accumulation of HSP27 and alphaB-crystallin. SB203580, an inhibitor of p38 MAP kinase, also inhibited differentiation, but the accumulation of alphaB-crystallin was rather enhanced. PD98059, an inhibitor of MAP kinase kinase, significantly increased expression of a differentiation marker for muscle cells, creatine kinase M isozyme, as well as accumulation of alphaB-crystallin. These results suggest that accumulation of sHSPs during differentiation of C2C12 cells is regulated in a complex manner.
beta-estradiol 3-benzoate (E(2)B) (10, 16, 20, 40, 80 and 160 microg/kg body weight) was administered daily to experimental groups of adult mice for the following periods; 2, 3 days, 1, 2, 4, and 8 weeks. Morphological changes in the testes were observed by both light and electron microscopy. Exfoliation of the germ cells was observed in the lumen of the seminiferous tubule. The spermatogenic cycle, especially stage XII, was disordered. Spermatids older than step 6 were severely affected. Detected abnormalities in the spermatids were deformation of the nucleus and acrosome. Partial deletion in the Sertoli-spermatid ectoplasmic specialization was also observed. Germ cells younger than step 7 spermatids were not affected morphologically. These abnormalities were not detected in the mice treated with the chemical at less than 16 microg/kg body weight. It is concluded that the chemical seems to affect round spermatids metabolically, but the morphological effect can be detected only from the spermatids older than step 6. The effects of the chemical on adult mice were reversible.
Steroid hormones, especially testosterone, play important roles in the carcinogenesis of prostate cancer, and several studies have reported changes in risk with polymorphisms of genes involved in steroid metabolism. One example is the CYP17 gene, which has a polymorphic T-to-C substitution in the 5'-untranslated region giving rise to A1 (T) and A2 (C) alleles. Steroid 5alpha-reductase type II (SRD5A2), which converts testosterone to the metabolically more active dihydrotestosterone, exhibits 2 polymorphisms: V89L, which substitutes leucine for valine at codon 89, and A49T, which substitutes threonine for alanine at codon 49. We therefore designed a case-control study of 105 prostate-cancer patients and 210 controls with benign prostatic hyperplasia for the purpose of investigating the association between prostate-cancer risk and polymorphisms in the SRD5A2 and CYP17 genes among the Japanese. The frequency of the CYP17 A2/A2 genotype in cases (18.8%) was higher than in controls (14.5%). Compared with the A1/A1 genotype, the odds ratio for the A2/A2 genotype was 2.39 (95% confidence interval 1.04-5.46, p = 0.04). The frequency of the SRD5A2 LL genotype in cases (29.3%) was also slightly higher than in controls (24.6%), but this was not significant. Regarding the A49T polymorphism of SRD5A2, we could not detect the T allele in any of the examined samples. These data suggest a significant association between the CYP17 polymorphism and prostate-cancer risk among the Japanese.
Genomic imprinting is the phenomenon by which the two alleles of certain genes are differentially expressed according to their parental origin. Extensive analysis of allelic expression at multiple imprinted loci in a normal population has not performed so far. In the present study, we examined the allelic expression pattern of three imprinted genes in a panel of 262 Japanese normal individuals. We observed differences in the extent of maintenance of allele-specific expression of the three genes. The allelic expression of small nuclear ribonucleoprotein N (SNRPN) was stringently regulated while that of multimembrane-spanning polyspecific transporter-like gene 1 (IMPT1) showed a large degree of variation. Significant biallelic expression of insulin-like growth factor II (IGF2) was observed in about 10% of normal individuals. Our findings add to the accumulating evidence for variable allelic expression at multiple loci in a normal human population. This epigenetic heterogeneity can be a stable trait and potentially influence individual phenotypes.
Matrix metalloprotease-2 (MMP-2) plays a pivotal role in cancer invasion and metastasis. Invasive human rhabdomyosarcoma cells (RD) secrete proMMP-2. We recently reported that anti-alpha3 integrin antibody induced the activated form of MMP-2 and enhanced proMMP-2 secretion by RD cells with concomitant enhancement of RD cell invasion. Since recent studies showed that calreticulin interacts with integrin alpha subunit, we hypothesized that calreticulin may be involved in signal transduction of anti-alpha3 integrin antibody-mediated MMP-2 secretion and activation. Here we demonstrate that anti-alpha3 integrin antibody induced a transient enhanced interaction of calreticulin with alpha3 integrin. Transfection of antisense oligonucleotides of calreticulin in RD cells abrogated the interaction between calreticulin and alpha3 integrin, and completely suppressed activation of MMP-2 and enhanced secretion of proMMP-2 induced by anti-alpha3 integrin antibody. Transient overexpression of calreticulin cDNA in RD cells significantly increased secretion of proMMP-2. The results demonstrate for the first time that calreticulin is directly involved in MMP-2 secretion and activation.
BACKGROUND: p73 and p51 are genes possessing amino-acid similarities to p53. We previously found no mutation in p73 in prostatic carcinoma, but did find abnormal expression of the gene. Involvement of these genes in prostatic carcinogenesis is still poorly understood. METHODS: Mutation analysis of the p51 gene and allelotyping of 3q28, on which p51 lies, were performed. Expression of p53, p73, and p51 was examined using reverse transcription-polymerase chain reaction, and expression levels were compared. RESULTS: No mutation in p51 was found (0/55 cases). Loss of heterozygosity at 3q28 was detected in 6 of 28 cases (21.8%). By expression analysis we found that in p53, 4 of 38 cases (10.5%) showed downregulation. No cases showed upregulation of p53. In contrast, p73 and p51 were downregulated in 42.1 and 39.5% of cases, respectively, and upregulated in 31.5 and 34.2% of cases, respectively. Expression levels of p51 corresponded with those of p73 in 25 of 38 cases (65.8%). CONCLUSIONS: Somatic mutations in p73 and p51 are not important in prostatic carcinogenesis. These genes may be associated with tumors by expression levels and may have roles in addition to tumor suppression.
Information about M6P/IGF2R and p53 genes in hepatocarcinogenesis is limited and controversial. We tested the loss of heterozygosity (LOH) of M6P/IGF2R and p53 genes in cirrhotic and neoplastic foci in surgically resected livers of 30 patients with hepatocellular carcinoma (HCC). The DNAs extracted from microdissected specimens were used for polymerase-chain-reaction (PCR)-based assay. LOH of the M6P/IGF2R gene in the primary HCCs was detected in 10 of 22 informative cases (45%). In five of these 10 cases (50%), LOH was detected in cirrhotic lesions adjacent to the HCCs. The allelic loss patterns of M6P/IGF2R in liver cirrhosis (LC) were identical to those in the corresponding HCC, suggesting that HCC could develop from one of the cells in which M6P/IGF2R had been lost. Furthermore, LOH of the p53 gene in HCC was detected in 10 (43%) of 23 informative cases, and p53 loss in cirrhotic foci adjacent to HCC was shown in one of the 10 cases (10%). The pattern of allelic loss of the p53 gene in the cirrhotic foci was identical with that in the corresponding tumor. The LOH of the M6P/IGF2R and p53 genes occurred independently in HCCs. LOH of the M6P/IGF2R locus was a relatively frequent and possibly early event in hepatocarcinogenesis, and LOH of the M6P/IGF2R gene and LOH of the p53 gene occurred independently.
Associations between genetic polymorphisms of CYP1A1, CYP1A2, CYP2E1, GSTM1 and GSTT1 and prostate cancer (PCa) were analyzed in a case-control study of 315 individuals. The frequency of valine (Val)/valine (Val) genotypes for CYP1A1 was 11.3% in cases compared with 5.5% in controls, this polymorphism thus being associated with a significantly increased risk of PCa (odds ratio=2.4, 95% confidence interval (CI)=1.01-5.57). No links were detected between PCa and polymorphisms in other enzymes. However, the combination of CYP1A1 (Ile/Val and/or Val/Val) polymorphisms with the GSTM1 null type resulted in an OR of 2.2 (CI=1.10-4.57, 1.12-4.20, respectively). This study suggests that the CYP1A1 polymorphism and its combination with GSTM1 may be associated with PCa susceptibility in the Japanese population.
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We cloned a cDNA encoding a novel lysyl oxidase-related protein, named LOXC, by suppression subtractive hybridization between differentiated and calcified ATDC5 cells, a clonal mouse chondrogenic EC cell line. The deduced amino acid sequence of mouse LOXC consists of 757 amino acids and shows 50% identity with that of mouse lysyl oxidase. Northern blot analysis showed a distinct hybridization band of 5.4 kilobases, and Western blot analysis showed an immunoreactive band at 82 kilodaltons. Expression of LOXC mRNA was detected in osteoblastic MC3T3-E1 cells and embryonic fibroblast C3H10T1/2 cells, whereas none of NIH3T3 fibroblasts and myoblastic C2C12 cells expressed LOXC mRNA in vitro. Moreover, the LOXC mRNA and protein levels dramatically increased throughout a process of chondrogenic differentiation in ATDC5 cells. In vivo, LOXC gene expression was localized in hypertrophic and calcified chondrocytes of growth plates in adult mice. The conditioned media of COS-7 cells transfected with the full-length LOXC cDNA showed the lysyl oxidase activity in both type I and type II collagens derived from chick embryos, and these activities of LOXC were inhibited by beta-aminopropionitrile, a specific inhibitor of lysyl oxidase. Our data indicate that LOXC is expressed in cartilage in vivo and modulates the formation of a collagenous extracellular matrix.
Tissue responses around implanted polymethylmethacrylate (PMMA) particles were analyzed by in situ hybridization with digoxigenin-labeled procollagen alpha1(I) (COL), osteonectin, osteocalcin, and osteopontin (OPN) mRNA probes. PMMA particles (150-300 microm in diameter) were implanted into rat tibiae, and specimens were collected at 3, 5, 7, and 10 days after operation. New bone was formed centripetally, and bone-forming osteoblasts expressed all four kinds of mRNAs. A COL signal was expressed most strongly and widely. In the early stage, COL-positive cells were detected on and among particles sporadically. A COL signal was rarely detected in cells on the surfaces of the particles, suggesting that PMMA particles may suppress osteoblast differentiation. Osteonectin and osteocalcin mRNAs were expressed in bone-forming osteoblasts in a similar pattern by day 7. By contrast, an OPN signal was detected mainly on the particles, not only in COL-positive osteoblasts but also in COL-negative round cells. The latter cells had acid phosphatase activity, suggesting that they might be macrophages responding to a foreign body. At day 10, an OPN signal was detected continuously in multinucleated cells on PMMA particles, whereas new bone was formed away from particles. Our approach helped us to understand the initial cellular reaction to materials, which may determine their biocompatibility.