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Biomedical subjects

R Nakashima

Publications and source records attributed to R Nakashima.

At least 19 recordsLinked to original sources
PubMed2004

HMG-CoA reductase inhibitors prevent bone loss in patients with Type 2 diabetes mellitus.

AIMS: It has been reported that 3-hydroxy-3-methylglutaryl co-enzyme A (HMG-CoA) reductase inhibitors increase bone mineral density (BMD) in vivo. We investigated the effect of HMG-CoA reductase inhibitors on BMD in patients with Type 2 diabetes mellitus. PATIENTS AND METHODS: We selected 122 patients with Type 2 diabetes, who were not taking active vitamin D preparations. Their mean age was 67.3 +/- 9.2 years. They were divided into a control group (n=63) without HMG-CoA reductase inhibitor therapy and an HMG-CoA group (n=59) who were treated with these drugs. The BMD of the distal one-third of the radius was measured by dual-energy X-ray adsorptiometry at baseline and after 2 years. RESULTS: There were no significant differences between the control and HMG-CoA groups at baseline with respect to age, gender, body mass index, duration of diabetes, haemoglobin A1c, fasting plasma glucose, adjusted calcium, serum phosphorus, alkaline phosphatase, albumin excretion rate and radial BMD. However, there was a significantly smaller annual decrease of the radial BMD in the HMG-CoA group. Multiple regression analysis with a forward elimination procedure revealed a positive correlation of the radial BMD Z-score with body mass index, while there was a negative correlation with alkaline phosphatase and albumin excretion rate. In addition, the annual rate of change of the radial BMD showed a positive correlation with HMG-CoA reductase inhibitor therapy. CONCLUSIONS: These findings suggest that HMG-CoA reductase inhibitors may prevent bone loss in patients with Type 2 diabetes.

Aged↗
PubMed2001

Mesenteric panniculitis of the colon with obstruction of the inferior mesenteric vein: report of a case.

Mesenteric panniculitis is a rare disease characterized by nonspecific inflammation of the fat tissue of the mesentery. We present an extremely rare case of mesenteric panniculitis of the sigmoid colon, complicated by occlusion of the inferior mesenteric vein. A 75-year-old male presented with a one-month history of abdominal distention and abdominal mass without pain. Physical examination revealed a firm mass in the lower abdomen. Barium enema study demonstrated rugged mucosa and a serrated contour in the rectosigmoid colon. Computed tomography showed that the mass arose from the mesentery, which surrounded the mesenteric vessels. The density of the mass was slightly higher than that of fatty tissue. Based on these radiologic findings, the patient was diagnosed as having mesenteric panniculitis of the rectosigmoid colon. Colonoscopy showed narrowing with edematous mucosa in the rectosigmoid colon, whereas marked dilated vessels were noted in the proximal portion of the sigmoid colon. Angiography showed occlusion of the inferior mesenteric vein, with venous flow returning via a collateral vein. The patient was observed without medication because his condition was satisfactory. His symptoms subsequently disappeared during a period of several weeks. The mass in the lower abdomen gradually diminished in size, disappearing three months later. Computed tomography and barium enema showed improvement of the lesion. The favorable outcome of the present case was probably because of formation of a collateral vein. The present case suggests that aggressive therapy for mesenteric panniculitis should be avoided, because the outcome of this disorder is good, even when there is obstruction of vessels.

Abdominal Pain↗
PubMed2000

Highly potent nociceptin analog containing the Arg-Lys triple repeat.

One of the structural characteristics of a neuropeptide nociceptin is the existence of Arg-Lys (RK) residues at positions 8-9 and 12-13; both RKs have been suggested to bind to the acidic amino acid cluster in the second extracellular loop of the seven transmembrane domain receptor ORL1. With a design strategy of attempting to obtain an analog that binds more strongly to the receptor's acidic cluster, we synthesized a series of nociceptin analogs in which the RK dipeptide unit was placed at positions 6-7, 10-11, or 14-15 adjacent to the parent RKs. Among these nociceptin analogs containing the RK triple repeat, [Arg-Lys(6-7)]- and [Arg-Lys(10-11)]nociceptins exhibited weak activities (6-9 and 60-90% of nociceptin, respectively) both in the receptor binding assay and in the [(35)S]GTPgammaS binding functional assay. In contrast, [Arg-Lys(14-15)]nociceptin was found to be very potent in both assays (3-fold in binding and 17-fold in GTPgammaS functional assay). [Arg-Lys(14-15)]nociceptin was the first peptide analog found to be stronger than the parent nociceptin, and structure-activity studies have suggested that the incorporated Arg-Lys(14-15) interacts with either the receptor acidic amino acid cluster or the receptor aromatic amino acid residues.

Amino Acid Sequence↗
PubMed2000

FHIT alterations in cancerous and non-cancerous cervical epithelium.

We have reported a significant frequency of an alteration of the fragile histidine triad (fhit) gene in squamous-cell carcinoma of the uterine cervix (series 1). To further define the role of fhit alteration in the development of cervical carcinoma, we surveyed 36 normal cervical epithelium, 22 cervical intra-epithelial neoplasias (CINs) and 20 additional cases of invasive cervical carcinomas (series 2). fhit transcripts were analyzed using reverse-transcription-polymerase-chain-reaction amplification and sequencing. Loss of expression of fhit was observed in 14 of 48 (29%) invasive carcinomas (8/28, series 1; 6/20, series 2) but not in any normal squamous epithelia or CINs analyzed. Abnormal fhit transcripts, including deletions and/or insertions, were observed in 12 of 48 (25%) invasive carcinomas (9/28, series 1; 3/20, series 2), 6 of 22 (27%) CINs, and 10 of 40 (25%) normal squamous epithelia (0/4, series 1; 10/36, series 2). Point mutation was detected in 9 of 48 (19%) cervical carcinomas (8/28, series 1; 1/20, series 2). Inactivation in both alleles was observed in 18 of 48 cervical carcinomas (38%), but not in any of 22 CINs or 40 normal squamous epithelia. Loss or impaired expression of the fhit-gene product was detected in 13 of 30 (43%) cervical carcinomas by immunohistochemistry, whereas all 6 normal cervical epithelia, or 22 CINs, expressed fhit protein. There was a strong association of impaired fhit protein expression with the disruption of normal fhit transcript in cervical carcinoma. No apparent correlation was observed between fhit inactivation and HPV infection. Our results suggest that fhit-gene inactivation occurs, not as an initiating event, but rather as a later event in cervical carcinogenesis, when the cervical tumor has acquired an invasive character.

Acid Anhydride Hydrolases↗
PubMed2000

X-ray structure of azide-bound fully oxidized cytochrome c oxidase from bovine heart at 2.9 A resolution.

Two azide ions were identified, one between the Fe and Cu atoms in the O(2)-reduction site and the other at the transmembrane surface of the enzyme, in the crystal structure of the azide-bound form of bovine heart cytochrome c oxidase at 2.9 A resolution. Two geometries, a mu-1,3 type geometry between the Fe and Cu atoms and a terminal geometry on the Fe atom, are equally possible for an azide ion in the O(2)--reduction site. The other azide molecule was hydrogen bonded to an amide group of an asparagine and a hydroxyl group of tyrosine in a mu-1,1 type geometry. The antisymmetric infrared bands arising from these azide ions, which show essentially identical intensity [Yoshikawa & Caughey (1992), J. Biol. Chem. 267, 9757-9766], strongly suggest terminal binding of the azide to Fe. The electron density of all three imidazole ligands to Cu(B) was clearly seen in the electron-density map of the azide-bound form of bovine heart enzyme, in contrast to the crystal structure of the azide-bound form of the bacterial enzyme [Iwata et al. (1995), Nature (London), 376, 660-669], which lacks one of the three imidazole ligands to Cu(B).

Animals↗
PubMed1999

Structure analysis of bovine heart cytochrome c oxidase at 2.8 A resolution.

The crystal structure of bovine heart cytochrome c oxidase has been determined at 2.8 A resolution by the multiple isomorphous replacement (MIR) method with three heavy-atom derivatives. An asymmetric unit of the crystal has a molecular weight of 422 kDa. Eight heavy atoms as main sites of a CH3HgCl derivative were clearly located by solving the difference Patterson function. The electron density obtained by the MIR method was refined by density modification, consisting of solvent flattening, histogram matching and non-crystallographic symmetry averaging. The enzyme exhibits a dimeric structure in the crystal. Out of 3606 amino-acid residues in 26 subunits in the dimer, 3560 residues were located in the electron-density map. The structure was refined by X-PLOR. The final R factor and the free R factor were 0.199 and 0.252 at 2.8 A resolution, respectively. One monomer in the dimeric structure with a stronger packing interaction has a lower averaged temperature factor than the other, by 16 A2. The region +/-12 A from the centre of the transmembrane part is almost 100% alpha-helix, despite the glycine residue content being as high as 7.1% in the transmembrane region. The residues around haem a of animals have evolved away from those of bacteria in contrast with the residues of the haem a3. The hierarchy of the structural organization of the enzyme complex has been proposed on the basis of intersubunit interactions.

Animals↗
PubMed1999

Alteration of p16 and p15 genes in human uterine tumours.

The roles of the p16 and p15 inhibitor of cyclin-dependent kinase tumour suppressor genes were examined in human uterine cervical and endometrial cancers. p16 mRNA, examined by reverse transcription polymerase chain reaction (RT-PCR), was significantly reduced in five of 19 (26%) cervical and four of 25 (16%) endometrial tumours. Reduced expression of p16 protein, detected by immunohistochemistry, occurred even more frequently, in nine of 33 (27%) cervical and seven of 37 (19%) endometrial tumours. Hypermethylation of a site within the 5'-CpG island of the p16 gene was detected in only one of 32 (3%) cervical tumours and none of 26 endometrial tumours. Homozygous p16 gene deletion, evaluated by differential PCR analysis, was found in four of 40 (10%) cervical tumours and one of 38 (3%) endometrial tumours. Homozygous deletion of p15 was found in three of 40 (8%) cervical tumours and one of 38 (3%) endometrial tumours. PCR-SSCP (single-strand conformation polymorphism) analysis detected point mutations in the p16 gene in six (8%) of 78 uterine tumours (four of 40 (10%) cervical tumours and two of 38 (5%) endometrial tumours). Three were mis-sense mutations, one in codon 74 (CTG-->ATG) and one in codon 129 (ACC-->ATC), both in cervical carcinomas, and the other was in codon 127 (GGG-->GAG) in an endometrial carcinoma. There was one non-sense mutation, in codon 50 (CGA-->TGA), in an endometrial carcinoma. The remaining two were silent somatic cell mutations, both in cervical carcinomas, resulting in no amino acid change. These observations suggest that inactivation of the p16 gene, either by homologous deletion, mutation or loss of expression, occurs in a subset of uterine tumours.

Animals↗
PubMed1999

Genetic alterations in the transforming growth factor receptor complex in sporadic endometrial carcinoma.

Cellular responses to the transforming growth factor beta (TGFbeta) ligand, including inhibition of cell proliferation, are mediated by a heteromeric receptor complex composed of TGFbeta types I and II receptors (TbetaR-I and TbetaR-II). Loss of responsiveness to TGFbeta, attributed to inactivation of the TbetaR complex, has been implicated in the development of tumors in a number of human epithelial and lymphoid tissues. To gain a better understanding of TGFbeta signal transduction pathways in endometrial carcinogenesis, we have investigated the role of the TbetaR complex by evaluating the TbetaR-I and TbetaR-II genes for mutations throughout the entire coding region in human sporadic endometrial tumors. Using reverse transcription-PCR, "Cold" single-strand conformation polymorphism analysis, and direct DNA sequencing, it was found that 1 of 39 (2.6%) and 7 of 42 samples (17%) contained code-altering changes in the kinase domain of TbetaR-I and TbetaR-II, respectively. In 7betaR-I, a 3-bp deletion was found resulting in replacement of Arg and Glu at codon 237 and 238 by Lys. With TbetaR-II, mutations were found in the kinase, the extracellular, and the C-terminal domains. No frameshift mutations were detected; however, a silent population polymorphism (AAC-->AAT at codon 389) in TbetaR-II was found in 19 of 42 (44%) tumor samples. These results suggest that alteration in TbetaR-II, but not TbetaR-I, has an important role in the development of endometrial carcinoma.

Activin Receptors, Type I↗
PubMed1998

Mutational analysis of the transforming growth factor beta receptor type II gene in human ovarian carcinoma.

In the present study, we evaluated a series of sporadic ovarian carcinomas for mutations within the entire coding region of TbetaR-II. Using reverse transcription-PCR and "Cold" single-strand conformational polymorphism analysis, 6 of 24 samples (25%) were found to contain code-altering mutations in TbetaR-II: (a) four mutations resulting in amino acid substitutions in the highly conserved serine/threonine kinase domain; (b) one mutation resulting in a conservative amino acid change in the transmembrane domain; and (c) a 1-bp insertion in the polyadenylic acid microsatellite region resulting in a reading frameshift. In addition, six cases (25%) exhibited a common bp substitution (C-->T at nucleotide 1322) in both tumor and patient-matched normal tissues. This is the first report of such TbetaR-II mutations in primary human ovarian carcinomas. Immunohistochemical analysis demonstrated a loss of expression of TbetaR-II in 5 of 22 available tumors (23%; 4 of which also had mutations in the coding region) and decreased expression of TbetaR-II in 10 of 22 available tumors (44%; 1 of which had a mutation in the coding region). Thus, the loss or decreased expression of TbetaR-II seems to be a common event in sporadic ovarian carcinomas, and mutational inactivation, due to either frameshift mutations in the polyadenylic acid microsatellite region or point mutations in conserved functional domains, is one mechanism by which this occurs.

Adult↗
PubMed1998

Redox-coupled crystal structural changes in bovine heart cytochrome c oxidase.

Crystal structures of bovine heart cytochrome c oxidase in the fully oxidized, fully reduced, azide-bound, and carbon monoxide-bound states were determined at 2.30, 2.35, 2.9, and 2.8 angstrom resolution, respectively. An aspartate residue apart from the O2 reduction site exchanges its effective accessibility to the matrix aqueous phase for one to the cytosolic phase concomitantly with a significant decrease in the pK of its carboxyl group, on reduction of the metal sites. The movement indicates the aspartate as the proton pumping site. A tyrosine acidified by a covalently linked imidazole nitrogen is a possible proton donor for the O2 reduction by the enzyme.

Animals↗
PubMed1998

Aberrant FHIT transcripts in squamous cell carcinoma of the uterine cervix.

The fragile histidine triad (FHIT) tumor suppressor gene at 3p14.2 has abnormalities in several types of human cancers. To investigate the potential role of FHIT in cervical cancer, which exhibits frequent loss of heterozygosity of 3p, we have examined primary cervical cancer samples from 28 patients for alterations of the FHIT gene. Abnormal FHIT transcripts were detected using reverse transcription-polymerase chain reaction (PCR) and subsequently by sequencing. Of 28 primary cervical carcinomas analyzed, 12 tumors (43%) showed abnormal FHIT transcripts, including deletion, insertion and point mutation. Loss of a FHIT transcript was observed in 2 cases (7%). Allelic loss of the FHIT gene was detected in 16 of 27 informative cases (59%). Oncogenic human papillomavirus (HPV) type 16, 18, 33, 35, 58 and 59 were not only present but were expressed in 24 of 28 cases (85%) by consensus PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) analysis for the HPV E6 and E7 genes. Our data indicate that alteration of the FHIT gene is an important genetic event associated with cervical cancer and oncogenic

Acid Anhydride Hydrolases↗
PubMed1998

Differential production of monoclonal antibodies to carbohydrate moiety or peptides moiety of glycoproteins by different routes of immunization.

The administration of ABO blood group active glycoproteins via the rear footpads of BALB/c mice with subsequent fusion of popliteal lymph node lymphocytes produced hybridomas differentially secreting antibodies to the peptide moiety of antigens. On the other hand, conventional intraperitoneal immunization of the same antigens and intraperitoneal boost followed by splenic lymphocyte fusion produced hybridomas differentially secreting antibodies to the carbohydrate moiety of antigens. Similar results were obtained in the production of monoclonal antibodies to erythrocyte membranes. Almost all hybridomas obtained by rear foodpad immunization secreted antibodies to the peptide moiety of erythrocyte membrane proteins, and those obtained by intraperitoneal immunization secreted antibodies to both peptide and carbohydrate moieties of erythrocyte membrane components. These results suggest the possibility of differential production of monoclonal antibodies to the carbohydrate moiety and the peptide moiety of some glycoproteins by different immunization routes.

Animals↗
PubMed1997

Molecular evidence that most but not all carcinosarcomas of the uterus are combination tumors.

The pathogenesis of carcinosarcoma is still a subject of controversy. In the present study, molecular techniques were applied to determine the pathogenesis of uterine carcinosarcomas. The patterns of chromosome X inactivation were analyzed, targeting a portion of exon 1 of the human androgen receptor (HUMARA) in malignant epithelial and mesenchymal components. The presence of p53 and K-ras mutations were also analyzed. H&E-stained sections of paraffin-embedded, formalin-fixed tissues were microdissected to obtain both epithelial and nonepithelial lesions from 25 carcinosarcomas, and DNAs were extracted by proteinase K digestion. Following treatment with methylation-sensitive restriction endonuclease (HhaI or HpaII), PCR amplification was performed using nested primers targeted to the HUMARA locus. Mutations in the p53 gene and K-ras gene were found in eight (32%) and six (24%) tumors, respectively. The patterns of chromosome X inactivation were different between the carcinomatous and sarcomatous components of three carcinosarcomas, indicating that these three tumors represent collision tumors. By contrast, the patterns of chromosome X inactivation, K-ras sequence, and p53 sequence were identical in both carcinomatous and sarcomatous components in 21 carcinosarcomas, indicating that these 21 tumors represent combination tumors. One case produced equivocal results that precluded determination of whether it represented a collision or combination tumor. These observations show that although most carcinosarcomas are combination tumors, some develop as collision tumors. The determination of histogenesis in individual cases of carcinosarcoma using molecular markers may be worthwhile, because the result could help predict the prognosis of individual cases and help guide clinical management.

Adult↗
PubMed1997

Clonal analysis of high-grade squamous intra-epithelial lesions of the uterine cervix.

We previously reported that invasive squamous cell carcinomas of the uterine cervix are of monoclonal composition. In the current study, we extended our previous work to determine the clonal composition of cases of high-grade squamous intra-epithelial lesion (HSIL). Clonal analysis targeting the HUMARA locus was performed on cervical tissue from 9 cases, 8 showing heterozygosity at the HUMARA locus and being, therefore, informative for clonality analysis. Uterine cervices were cut into 12 blocks, fixed with formalin and embedded in paraffin, and DNA was extracted from targeted lesions of each block. A total of 30 samples of cervical intra-epithelial neoplasia 3 (CIN3) (14 samples of carcinoma in situ and 16 samples of severe dysplasia) and 1 sample of CIN2 (moderate dysplasia) were analyzed. Monoclonal composition of the lesions was demonstrated in 30/30 cases of CIN3. Polyclonal composition was seen in the single case of CIN2. In 6 uterine cervices, in which dysplastic lesions were present in more than 3 blocks, the pattern of X-chromosome inactivation was the same in all lesions, suggesting that these individual lesions were derived from a single cell, with intraepithelial extension within the cervical mucosa. By contrast, one uterus contained 2 discontinuous dysplastic foci with different patterns of X-chromosome inactivation, indicating that the 2 lesions developed independently from each other. Our results demonstrate that (i) lesions of CIN3 (severe dysplasia and carcinoma in situ) are composed of a clonal neoplastic population of cells and (ii) most cases of HSIL are unifocal in origin.

Carcinoma in Situ↗
PubMed1997

Alteration of p16 and p15 genes in common epithelial ovarian tumors.

We have examined the roles of 2 putative tumor-suppressor genes, the p16 and p15 inhibitor-of-cyclin-dependent-kinase genes, in the most commonly occurring epithelial tumors of the human ovary. Expression of p16 mRNA, examined by RT-PCR, was significantly reduced in 15 of the 48 tumors. Aberrant expression of p16 protein, detected by immunohistochemistry, occurred in 22 of 60 tumors, more frequently in low-grade tumors, and had significant correlation with low p16 mRNA expression. Hypermethylation of a site within the 5'-CpG island of the p16 gene was significantly associated with loss of p16 mRNA and protein expression. Homozygous gene deletion, evaluated by differential PCR analysis, was found in 2 tumors for the p16 gene and in 1 tumor for the p15 gene among 70 ovarian tumors examined. PCR-SSCP analysis detected point mutations in p16 in 4 tumors and in p15 in 1 tumor. One was a 38-bp deletion, from codons 48 to 60, in a mucinous tumor of low malignant potential; another was a non-sense mutation in codon 60 in a mucinous adenocarcinoma. The remaining 2 mutations were mis-sense mutations, one in codon 58 and the other in codon 60, in 2 endometrioid adenocarcinomas. We conclude that inactivation of p16, by loss of p16 mRNA and protein expression as a consequence of hypermethylation of the 5'-CpG island, rather than by gene deletion or point mutation, may play an important role in the genesis of human ovarian epithelial tumors.

Actins↗
PubMed1996

Sensitivity of opioid receptor-like receptor ORL1 for chemical modification on nociceptin, a naturally occurring nociceptive peptide.

Nociceptin or orphanin FQ is a novel neuropeptide that activates an opioid-like G protein-coupled receptor ORL1. This heptadecapeptide FGGFTGARKSARKLANQ resembles kappa-opioid peptide dynorphin A but exhibits an opposite effect to make animals hyperreactive to nociceptive stimulations (Meunier, J.-C., Mollereau, C., Toll, L., Suaudeau, C., Moisand, C., Alvinerie, P., Butour, J.-L., Guillemot, J.-C., Ferrara, P., Monsarrat, B., Mazarguil, H., Vassart, G., Parmentier, M., and Costentin, J. (1995) Nature 377, 532-535; Reinscheid, R. K., Nothacker, H.-P., Bourson, A., Ardati, A., Henningsen, R. A., Bunzow, J. R., Grandy, D. K., Langen, H., Monsma, F. J., Jr., and Civelli, O. (1995) Science 270, 792-794). In the present study, it was found that guinea pig brain contains receptors to which nociceptin binds much more strongly than to ORL1 receptors expressed in human 293 cells. Although the Tyr1 --> Phe substitution for dynorphin A eliminates almost completely an ability to bind to opioid receptors, the Phe1 --> Tyr substitution in nociceptin was found to retain almost fully both receptor binding affinity and in vivo hyperalgesic activity in tail-flick assay. Nociceptin was extremely weak to bind to opioid receptors, while Tyr1-nociceptin exhibited 10-40 times increased affinity, especially for mu receptors, due to its N-terminal sequential identity to opioid peptides. Shortened analogs of dynorphin A are known to retain receptor binding ability and analgesic activity, whereas the removal of C-terminal hexa- or decapeptide from nociceptin totally abolished the affinity for the ORL1 receptor. These results indicated that the mode of interaction between nociceptin and ORL1 receptor is quite different from that between dynorphin and opioid receptor and that the C-terminal portion of nociceptin is crucial for receptor recognition.

Amino Acid Sequence↗
PubMed1996

The whole structure of the 13-subunit oxidized cytochrome c oxidase at 2.8 A.

The crystal structure of bovine heart cytochrome c oxidase at 2.8 A resolution with an R value of 19.9 percent reveals 13 subunits, each different from the other, five phosphatidyl ethanolamines, three phosphatidyl glycerols and two cholates, two hemes A, and three copper, one magnesium, and one zinc. Of 3606 amino acid residues in the dimer, 3560 have been converged to a reasonable structure by refinement. A hydrogen-bonded system, including a propionate of a heme A (heme a), part of peptide backbone, and an imidazole ligand of CuA, could provide an electron transfer pathway between CuA and heme a. Two possible proton pathways for pumping, each spanning from the matrix to the cytosolic surfaces, were identified, including hydrogen bonds, internal cavities likely to contain water molecules, and structures that could form hydrogen bonds with small possible conformational change of amino acid side chains. Possible channels for chemical protons to produce H2O, for removing the produced water, and for O2, respectively, were identified.

Amino Acid Sequence↗