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Biomedical subjects

N Okada

Publications and source records attributed to N Okada.

At least 145 records · Page 8Linked to original sources
PubMed1998

Existence of a bovine LINE repetitive insert that appears in the cDNA of bovine protein BCNT in ruminant, but not in human, genomes.

A novel protein, BCNT, originally isolated from bovine brain and named after Bucentaur, contains an internal portion that is translated from part of bovine LINE repetitive sequence (Bov-B LINE). Human cDNA highly homologous to the bovine bcnt (bbcnt) cDNA has been isolated but does not contain a sequence similar to the Bov-B LINE insert (Nobukuni, T., Kobayashi, M., Omori, A., Ichinose, S., Iwanaga, T., Takahashi, I., Hashimoto, K., Hattori, S., Kaibuchi, K., Miyata, Y., Masui, T., Iwashita, S., 1997. An Alu-linked repetitive sequence corresponding to 280 amino acids is expressed in a novel bovine protein, but not in its human homologue. J. Biol. Chem. 272, 2801-2807). In this study, we conducted a polymerase chain reaction analysis to investigate whether such a Bov-B LINE insert is present in bcnt orthologs in other animals and in the genomic sequence of the human BCNT (hBCNT) gene. The results indicate that the Bov-B LINE insert is present in the genomic sequences of bcnt orthologs from sheep, goats, axis deer, and mouse deer (chevrotain), that is in Ruminantia, but not in pigs or human. Analysis of the bbcnt genomic sequence around the Bov-B LINE insert revealed a large part of the inserted Bov-B LINE sequence to be included in an exon; this is followed by a 54-nucleotide sequence that is highly homologous to Bov-B LINE in the 3'-side intron. The hBCNT gene was isolated and found to consist of seven exons and six introns, among which the intron corresponding to the Bov-B LINE insertion site in the bbcnt genome is 16.5kb in length with no sequence similar to Bov-B LINE. Based on these results, it seems likely that the Bov-B LINE insert is derived from a long Bov-B LINE repetitive sequence transposed to an ancestral bcnt gene in Ruminantia and reformed as a new exon through new splicing sites in the transposed sequence.

Amino Acid Sequence↗
PubMed1998

Selection and analysis of a mutant cell line defective in the hypoxia-inducible factor-1 alpha-subunit (HIF-1alpha). Characterization of hif-1alpha-dependent and -independent hypoxia-inducible gene expression.

Hypoxia-inducible expression has been demonstrated for many groups of mammalian genes, and studies of transcriptional control have revealed the existence of hypoxia-responsive elements (HREs) in the cis-acting sequences of several of these genes. These sequences generally contain one or more binding sites for a heterodimeric DNA binding complex termed hypoxia-inducible factor-1 (HIF-1). To analyze this response further, Chinese hamster ovary cells were stably transfected with plasmids bearing HREs linked to genes encoding immunoselectable cell surface markers, and clones that showed reduced or absent hypoxia-inducible marker expression were selected from a mutagenized culture of cells. Analysis of these cells revealed several clones with transacting defects in HRE activation, and in one the defect was identified as a failure to express the alpha-subunit of HIF-1. Comparison of hypoxia-inducible gene expression in wild type, HIF-1alpha-defective, and HIF-1alpha-complemented cells revealed two types of response. For some genes (e.g. glucose transporter-1), hypoxia-inducible expression was critically dependent on HIF-1alpha, whereas for other genes (e.g. heme oxygenase-1) hypoxia-inducible expression appeared largely independent of the expression of HIF-1alpha. These experiments show the utility of mutagenesis and selection of mutant cells in the analysis of mammalian transcriptional responses to hypoxia and demonstrate the operation of HIF-1alpha-dependent and HIF-1alpha-independent pathways of hypoxia-inducible gene expression in Chinese hamster ovary cells.

Animals↗
PubMed1998

Functional differences among multiple isoforms of guinea pig decay-accelerating factor.

Decay-accelerating factor (DAF, CD55) is a membrane inhibitor that protects host cells from the autologous C-mediated attack. The guinea pig homologue of DAF consists of multiple isoforms generated by alternative splicing from a single copy gene. These isoforms are mainly comprised of a glycosylphosphatidylinositol (GPI)-anchored form and a transmembrane form (TM) that is not present in human DAF. Both forms occur in at least three variations that differ in the length of the Ser/Thr-rich region (termed ST-a, ST-ab, and ST-abc). We have transfected cDNAs of the six major isoforms into Chinese hamster ovary cells, and their functional differences were evaluated in inhibition of C-mediated cytolysis and C3 deposition, using the transfectants expressing DAF at the same level on cell membranes. The degree of inhibition in both the classical and alternative pathways differed according to the length of the ST region in the order of abc > ab > a in both GPI and TM forms. When GPI and TM forms were compared, those with the ab or abc variation exhibited almost the same activity, whereas a-TM was less efficient than a-GPI. Although several isoforms are expressed constitutively in most of tissues, spermatozoa preferentially express the abc-GPI isoform, suggesting that this isoform offers effective protection to spermatozoa in the female genital tract.

Animals↗
PubMed1998

Conflict among individual mitochondrial proteins in resolving the phylogeny of eutherian orders.

The phylogenetic relationship among primates, ferungulates (artiodactyls + cetaceans + perissodactyls + carnivores), and rodents was examined using proteins encoded by the H strand of mtDNA, with marsupials and monotremes as the outgroup. Trees estimated from individual proteins were compared in detail with the tree estimated from all 12 proteins (either concatenated or summing up log-likelihood scores for each gene). Although the overall evidence strongly suggests ((primates, ferungulates), rodents), the ND1 data clearly support another tree, ((primates, rodents), ferungulates). To clarify whether this contradiction is due to (1) a stochastic (sampling) error; (2) minor model-based errors (e.g., ignoring site rate variability), or (3) convergent and parallel evolution (specifically between either primates and rodents or ferungulates and the outgroup), the ND1 genes from many additional species of primates, rodents, other eutherian orders, and the outgroup (marsupials + monotremes) were sequenced. The phylogenetic analyses were extensive and aimed to eliminate the following artifacts as possible causes of the aberrant result: base composition biases, unequal site substitution rates, or the cumulative effects of both. Neither more sophisticated evolutionary analyses nor the addition of species changed the previous conclusion. That is, the statistical support for grouping rodents and primates to the exclusion of all other taxa fluctuates upward or downward in quite a tight range centered near 95% confidence. These results and a site-by-site examination of the sequences clearly suggest that convergent or parallel evolution has occurred in ND1 between primates and rodents and/or between ferungulates and the outgroup. While the primate/rodent grouping is strange, ND1 also throws some interesting light on the relationships of some eutherian orders, marsupials, and montremes. In these parts of the tree, ND1 shows no apparent tendency for unexplained convergences.

Animals↗
PubMed1998

A new repetitive sequence uniquely present in the decay-accelerating factor genes.

The decay-accelerating factor (DAF, CD55) protects cells from autologous complement attack on self cell membranes. We have previously reported that the seventh exon encoding the serine/threonine-rich(S/T)-abc region of the guinea pig DAF gene is composed of five homologous repeats of about 51 base pairs, and that differential usage of these repeats produces the various lengths observed in the S/T region of guinea pig DAF. In this study, we found that the seventh intron of the guinea pig DAF gene was wholly composed of 18 tandem repeats homologous to the repeating unit of the S/T-abc exon. This type of repetitive structure, although the number of repeats was variable, was also found in the corresponding exons and introns of all DAF genes of other species so far tested including human and seven other primates and mouse, in which alternative splicing in this region has not been found. This suggested that generation of the repetitive sequences spanning the exon and intron regions had occurred before the diversification of these species. In addition, all the intron sequences of the tested DAF genes had no stop codon when they were presumably translated in the same reading frame as the seventh and eighth exons, except for that of one of two duplicated mouse DAF genes. These findings and significant interspecies identities of the intron sequence suggest that the intron sequence conceivably could be translated in some tissues and/or in some stages of development although to date we have not yet succeeded in detecting mRNA for this region.

Amino Acid Sequence↗
PubMed1998

Expression of membrane-type matrix metalloproteinase-1 in human pancreatic adenocarcinomas.

The expression of a new type of matrix metalloproteinase, membrane-type matrix metalloproteinase-1 (MT-MMP-1), was examined in 24 cases of primary pancreatic adenocarcinomas and 9 cases of secondary liver tumors derived from pancreatic adenocarcinomas, using a non-radioactive in situ hybridization and immunohistochemical methods. Out of 24 cases of primary pancreatic adenocarcinomas, 18 showed positive expression of MT-MMP-1 transcripts in cancer cells and 20 of 24 showed positive expression in the tumor stromal cells. The immunoreactivity of the gene products for MT-MMP-1 was demonstrated to be almost the same, as shown by in situ hybridization in these 24 cases. In particular, both the staining intensity for MT-MMP-1 transcripts and the immunoreactivity of the gene products in the tumor stromal cells of mucinous cystadenocarcinomas were significantly weaker than those of common-type ductal adenocarcinomas among the 24 cases. All of the 9 cases of secondary liver tumors derived from pancreatic adenocarcinomas showed positive expression for MT-MMP- transcripts but less immunoreactivity for the gene products. These results suggest that MT-MMP-1 is transcribed and translated in both cancer cells and the tumor stromal cells in human pancreatic adenocarcinomas. Furthermore, considering that common-type ductal adenocarcinoma of the pancreas usually shows a strong desmoplastic reaction, while mucinous cystadenocarcinoma typically does not, MT-MMP-1 expressed in the tumor stromal cells of common-type adenocarcinomas may be involved in processes leading to the desmoplastic reaction.

Adenocarcinoma↗
PubMed1998

Post-transplant diabetes with anti-glutamic acid decarboxylase antibody during tacrolimus therapy.

A 54-year-old man undergoing hemodialysis because of end-stage renal failure was transplanted with a cadaver kidney in November 1997. He had no history of diabetes. Tacrolimus was used as the primary immunosuppressant. Three weeks after transplantation, he developed insulin-requiring diabetes mellitus. Anti-glutamic acid decarboxylase antibody was not detected on the third post-operative day, but appeared 4 weeks after transplantation. The recipient had DNA haplotypes that indicated susceptibility to Type 1 diabetes in Japanese subjects. Immunosuppressive therapy was changed from tacrolimus to cyclosporin. Thereafter, titer of anti-glutamic acid decarboxylase antibody disappeared and the patient's insulin requirement was notably reduced. The mechanism underlying the development of diabetes in this case appears to be, in part, direct beta-cell toxicity due to tacrolimus therapy, resulting in secondary beta-cell autoimmunity. This case suggests that tacrolimus therapy after transplantation should be used with caution in patients with genetic susceptibility to Type 1 diabetes.

Antibodies↗
PubMed1998

Relative contributions of decay accelerating factor (DAF), membrane cofactor protein (MCP) and CD59 in the protection of melanocytes from homologous complement.

Complement regulatory molecules, membrane cofactor protein (MCP), decay accelerating factor (DAF) and CD59, protect body cells from autologous complement. They have wide tissue distribution but nothing is known about the expression of these molecules on human melanocytes. Since melanocytes are lysed in the lesional skin of patients with a depigmentary disorder vitiligo, it is important to compare the protection offered by complement regulatory molecules to melanocytes present in normal and vitiligo epidermis, against autologous complement. From this point of view, we investigated the differential expression of MCP, DAF and CD59 on normal cultured human melanocytes and assessed their individual contribution in the protection of these cells against complement-mediated damage. Flow cytometric analysis showed that MCP and DAF but not CD59 were expressed on cultured melanocytes. When heat inactivated sera of patients with vitiligo were used as a source of anti-melanocyte antibody to sensitize melanocytes, and guinea pig serum (GpS) or normal human serum (NHS) as a source of complement, GpS was found to be more effective in causing the lysis of melanocytes than NHS. When melanocytes were sensitized with autoantibody as well as F(ab')2 fragment of either anti-MCP or anti-DAF and subsequently incubated with NHS or GpS, both antibody fragments increased the killing of melanocytes by NHS as well as by GpS. F(ab')2 fragment of anti-DAF was much more effective in causing enhancement of lysis than that of anti-MCP. Thus, cultured normal human melanocytes express functionally active MCP and DAF but not CD59. Contribution of DAF in protecting melanocytes against complement attack was much more than that of MCP.

CD55 Antigens↗
PubMed1998

Response of human insulinoma cells to extracellular calcium is different from normal B cells.

The preoperative determination of the localization of a small insulinoma is sometimes difficult using routine imaging techniques. We have used the selective arterial calcium injection (SACI) test to determine the location of the tumor preoperatively. The pathophysiologic basis of the SACI test is based on the responsiveness of insulinomas to calcium injected into the feeding artery. In this study, we demonstrated the in vitro response of the insulinoma cells to the extracellular calcium challenge by using primary-cultured insulinoma cells. Human insulinoma cells were obtained from three patients. MIN6 cells (normal pancreatic B cells) were used as a control; their insulin response to various stimuli resembles that of normal B cells. The insulin secretory dynamics in response to extracellular calcium were observed using a perfusion system. Second, the change of the concentration of cytosolic free calcium ([Ca2+]i) was monitored by fluorometry using fura-2/AM. When the concentration of extracellular calcium ([Ca2+]o) was changed from 2.54 mM to 10 mM, insulin secretion from the insulinoma cells was markedly increased within 6 min (10- to 18-fold at maximum), and rapidly returned to the basal level; at the same time, [Ca2+]i was immediately elevated and reached a peak within 1 min. In contrast, in the MIN6 cells, the insulin secretion and [Ca2+]i were not significantly changed when [Ca2+]o was switched to 10 mM. The results of these in vitro experiments agreed with the clinical results of the SACI test. The positive response of the insulinoma to the SACI test is probably due to the different response of insulinoma cells to the extracellular calcium challenge compared with normal B cells. The role of [Ca2+]i may be important in the mechanism underlying the SACI test.

Animals↗
PubMed1998

Thrombosed varix in the submandibular space.

Thrombosed varix in the oral region often appears as a pigmented mass in the mucosa simulating a hemangiomatous lesion. It rarely occurs in the deeper orofacial region. We report a case of thrombosed varix in the submandibular space with symptoms of a tumor. The clinical symptoms and MRI and CT findings are discussed. The clinical symptoms were not characteristics. High signal intensity on CT (80 HU) and a concentric signal distribution on CT and MRI may suggest a thrombosed varix.

Chin↗
PubMed1998

Altered expression of membrane inhibitors of complement in human gastric epithelium during Helicobacter-associated gastritis.

AIMS: Membrane inhibitors of complement are thought to protect bystander cells from complement mediated damage. Expression of these proteins is enhanced in the colonic mucosa of patients with ulcerative colitis. Our aim was to investigate the regulation of complement activity in Helicobacter-associated chronic gastritis. METHODS AND RESULTS: We immunohistochemically assayed expression of three membrane inhibitors of complement, decay accelerating factor (DAF; CD55), 20 kDa homologous restriction factor (HRF20; CD59) and membrane cofactor protein (MCP; CD46), in 55 biopsy specimens of the human gastric mucosa. DAF, expressed in 33 (60%) of biopsy specimens, and HRF20, expressed in 45 (82%) of the samples, were located mainly on the apical surface of the epithelial cells, whereas MCP, expressed in 48 (87%) of the biopsies, was found on the basolateral surface. We found strong correlation between expression of DAF on gastric mucosal epithelium and the severity of mucosal infiltration of neutrophils (rs = 0.875, P < 0.0001) and mononuclear cells (rs = 0.773, P < 0.0001). No significant correlation was observed between HRF20 expression and neutrophil or mononuclear cell infiltration, while there was a weak negative correlation between MCP expression and these cellular infiltrations. When we assayed immunostaining of Helicobacter pylori (H. pylori) in these biopsy specimens, strong correlation with the degree of neutrophil (P < 0.0001) and mononuclear cell (P < 0.0001) infiltration were observed. The expression of DAF and H. pylori infection in these biopsies were also significantly (P < 0.0001) correlated. No correlation between HRF20 expression and H. pylori infection was observed, but we did find a significant negative correlation (P < 0.005) between the expression of MCP and H. pylori infection. CONCLUSIONS: Our results, demonstrating altered expression of membrane inhibitors of complement in gastric mucosa during inflammation and/or H. pylori infection, suggest that complement may significantly participate in the pathology of gastric inflammation. Moreover, DAF and MCP may play an important role in the regulation of complement activation in the alimentary tract.

Antigens, CD↗
PubMed1998

Tissue distribution of the guinea-pig decay-accelerating factor.

MCA44 is a monoclonal antibody (mAb) to guinea-pig decay-accelerating factor (DAF) and, using this mAb, tissue distribution of guinea-pig DAF was studied by immunofluorescence. Guinea-pig DAF was found to be expressed not only on the vascular endothelium but also on different types of cells, such as the tubular epithelium of the kidney, epidermal cells of the skin and synovial lining cells. As there was no significant reduction in staining intensity with MCA44 following treatment with phosphatidylinositol-specific phospholipase C, many guinea-pig DAF molecules expressed in these tissues may be of the transmembrane form.

Animals↗
PubMed1998

Transcutaneous electrical stimulation of thigh muscles in the treatment of detrusor overactivity.

OBJECTIVE: To investigate the clinical effects on detrusor overactivity of a new method of transcutaneous reciprocal electrical stimulation of the thigh muscles. PATIENTS AND METHODS: Nineteen patients with detrusor overactivity, comprising 14 with detrusor hyperreflexia (DH) and five with idiopathic detrusor instability (IDI), were studied. Electrical stimulation was applied alternately to the quadriceps and hamstring muscles of one or both legs through surface electrodes for 20 min. The treatment was given once a day for 14 days and then the patients were evaluated urodynamically. RESULTS: All 19 patients tolerated the therapy well and none reported any adverse effects. The mean maximum cystometric capacity increased significantly (P < 0.05) after treatment. In 11 of the 19 patients, the maximum cystometric capacity was increased by > 50% of the pretreatment value; this occurred in eight of 14 of those with DH and in three of five of those with IDI. In six of the 11 who responded in this way, there was a clinical improvement in their urinary incontinence and frequency for several weeks to 3 months after the period of therapy. A second 14-day treatment was also effective in all four patients who underwent a repeat trial. CONCLUSION: This method of transcutaneous electrical stimulation can inhibit DH as well as IDI with no adverse effects. The suppressive effect on detrusor overactivity may persist for several months and repeat trials appear to be effective. Thus, we believe that this new stimulation technique should be tried as an alternative to other types of electrical stimulation and augmentation cystoplasty.

Adult↗
PubMed1998

Effects of UVB on the synthesis of complement proteins by keratinocytes.

UVB exposure of the skin results in increased production of several cytokines by keratinocytes and infiltration of inflammatory cells. We hypothesized that UVB may increase the expression of complement (C) components and C-regulatory proteins by keratinocytes. In vivo, UVB may upregulate these proteins by direct effects or via cytokines released by keratinocytes or infiltrating inflammatory cells. In vitro, UVB may upregulate these proteins only directly, because of dilution of released cytokines in the medium. To test this, we exposed cultured human keratinocytes to UVB (0-64 J per m2) and monitored C3 and Factor B release in the medium by enzyme-linked immunosorbent assay, and surface expression of decay accelerating factor, membrane cofactor protein, and CD59 by flow cytometry. Keratinocytes produced small amounts of C3 and Factor B, which remained unaffected by UVB. UVB (32 J per m2) caused a transient upregulation of all three C-regulatory proteins. Decay accelerating factor expression was maximal at 48 h (1.81 +/- 0.06-fold increase in mean fluorescence intensity over nonexposed cells), membrane cofactor protein at 72 h (2.13 +/- 0.09-fold increase in mean fluorescence intensity), and CD59 at 120 h (1.96 +/- 0.09-fold increase in mean fluorescence intensity), returning to baseline values within 96, 192, and 192 h, respectively. Exposure to 64 J per m2 resulted in significant cell death; cells surviving this dose up to 48 h expressed a higher level of all the three proteins than those surviving 32 J per m2. In conclusion, UVB upregulated membrane cofactor protein, decay accelerating factor, and CD59 on keratinocytes without affecting the constitutive release of C3 and Factor B. Thus, UVB can increase the resistance of keratinocytes against their own C known to be produced excessively in response to cytokines of inflammatory cells that infiltrate the skin following UVB exposure.

Antigens, CD↗
PubMed1998

Detection of the ongoing sorting of ancestrally polymorphic SINEs toward fixation or loss in populations of two species of charr during speciation.

The FokI family of short interspersed repetitive elements (SINEs) has been found only in the genomes of charr fishes (genus Salvelinus). In an analysis of the insertion of FokI SINEs using PCR, we characterized six loci at which FokI SINEs have been inserted into the genomes of Salvelinus alpinus (Arctic charr) and/or S. malma (Dolly Varden). An analysis of one locus (Fok-223) suggested that a sister relationship exists between S. alpinus and S. malma and the SINE at this locus might have been inserted in a common ancestor of these two species, being fixed in all extant populations examined. By contrast, SINEs at two other loci (Fok-211 and Fok-206) were present specifically in the genome of S. alpinus, with polymorphism among populations of this species. Moreover, the presence or absence of the SINEs of the other three loci (Fok-214, Fok-217, and Fok-600) varied among populations of these two species. The most plausible interpretation of this result is that SINEs, which were ancestrally polymorphic in the genome of a common ancestor of these two species, are involved in an ongoing process of differential sorting and subsequent fixation in the various populations of each species.

Animals↗
PubMed1998

Complement-mediated cytolysis and azidothymidine are synergistic in HIV-1 suppression.

Some normal human sera contain a natural IgM antibody against gangliotetraose and GM2 which is capable of initiating complement-mediated cytolysis of HIV-1-infected cells. A potent cytolytic serum (lytic serum) harboring this antibody also caused lysis of HIV-1 virions. When lytic serum was added to a mixed culture of HIV-infected cells and naive cells at a ratio of 1:100, expansion of HIV-1 infection was postponed by 1 week. Furthermore, the co-presence of both the lytic serum and azidothymidine continued to significantly suppress infection, even after 4 weeks of cultivation.

Anti-HIV Agents↗
PubMed1998

SINE cousins: the 3'-end tails of the two oldest and distantly related families of SINEs are descended from the 3' ends of LINEs with the same genealogical origin.

AFC short interspersed elements (SINEs) were isolated from cichlids from Madagascar, the New World, and Africa and characterized. A new family of long interspersed elements (LINEs), designated the CiLINE2 family, was also isolated from African cichlids, and its consensus sequence was deduced. Upon aligning all of the consensus sequences, we found that the 3'-tail regions of the AFC SINEs and the CiLINE2 family were very similar, providing another example in which a reverse transcriptase responsible for retroposition of SINEs might be contributed in trans by a LINE. Sequence comparisons showed that CiLINE2 in cichlids was closely related to LINE2 in mammals. Furthermore, we found that the 3'-tail sequence shared by the AFC SINEs and CiLINE2 in cichlids was very similar to the 3'-tail sequence shared by the MIR SINEs and LINE2 in mammals, even though the remaining parts of the AFC SINEs and the MIR SINEs were totally different from each other. Thus, the present report not only describes a new pair of SINEs and LINEs with the same 3' tail in cichlids, but also provides a new example of the phenomenon whereby the 3' ends of LINEs with the same genealogical origin can be incorporated into the 3'-end tails of different families of SINEs that have been generated independently in two different lineages during evolution.

3' Flanking Region↗