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Biomedical subjects

M Seki

Publications and source records attributed to M Seki.

At least 73 records · Page 4Linked to original sources
PubMed2000

Glucocorticoid-induced expression of a foreign gene by the GVG system in transformed tobacco BY-2 cells.

A glucocorticoid-induced target gene expression system was used to control the expression of the uidA gene, whose product was beta-glucuronidase (GUS), in tobacco BY-2 cell suspension culture. This targeting system showed quick, sensitive, and reversible response to dexamethazone (DEX), an artificial glucocorticoid hormone. Addition of DEX greatly and quickly enhanced uidA gene expression, whose level was as high as that under the control of the CaMV 35S promoter whereas in the absence of DEX, the GUS specific activity was suppressed to be as low as that of nontransformed BY-2 cells. The dilution of DEX decreased GUS specific activity showing that the concentration of DEX plays a major role in controlling the expression level of the target. The use of the glucocorticoid-induced system in plant cell suspension culture was demonstrated to precisely control target gene expression.

Journal Article↗
PubMed2000

Optimization of perfusion decalcification for bones and joints in rats.

Decalcification of osseous tissues by perfusion of decalcifying solution into the vascular system has never been applied to the study of peripheral joints. To optimize perfusion methods, rats were decalcified by direct immersion or by one of two perfusion techniques: 1) systemic perfusion circulating the decalcifying solution from the ascending aorta; and 2) regional perfusion circulating the solution to the lower extremities from the abdominal aorta. The process of decalcification was monitored by serial radiographic examinations. After decalcification, bone and joint samples were stained for histochemistry and immunohistochemistry. With systemic perfusion, the decalcification time, dependent on weight, was markedly reduced compared to immersion. Regional perfusion decalcification was faster than all other methods studied. Microstructural preservation was comparable and immunostaining quality often improved. Applications of this work will improve the study of basic skeletal and articular problems.

Animals↗
PubMed2000

Differential regulation of human RecQ family helicases in cell transformation and cell cycle.

Three human RecQ DNA helicases, WRN, BLM and RTS, are involved in the genetic disorders associated with genomic instability and a high incidence of cancer. RecQL1 and RecQL5 also belong to the human RecQ helicase family, but their correlation with genetic disorders, if any, is unknown. We report here that in human B cells transformed by Epstein-Barr virus (EBV), human fibroblasts and umbilical endothelial cells transformed by simian virus 40, the expression of WRN, BLM, RTS and RecQL1 was sharply up-regulated. In B cells this expression was stimulated within 5-40 h by the tumor promoting agent phorbol myristic acetate (PMA). Interestingly, RecQL5beta, an alternative splicing product of RecQL5 with a nuclear localization signal, is expressed in resting B cells without significant modulation of its synthesis by EBV or PMA, suggesting it has a role in resting cells. We also roughly determined the number of copies per cell for the five RecQ helicase in B cells. In addition, levels of the different RecQ helicases are modulated in different ways during the cell cycle of actively proliferating fibroblasts and umbilical endothelial cells. Our results support the view that the levels of WRN, BLM, RTS and RecQL1 are differentially up-regulated to guarantee genomic stability in cells that are transformed or actively proliferating.

Adenosine Triphosphatases↗
PubMed2000

Covalent modification of the Werner's syndrome gene product with the ubiquitin-related protein, SUMO-1.

Werner's syndrome is a potential model of accelerated human aging. The gene responsible for Werner's syndrome encodes a protein that has a helicase domain homologous to Escherichia coli RecQ. To identify binding partners that regulate the function in concert with Wrn, we screened for proteins using the yeast two-hybrid system with mouse Wrn as bait and found three. One was a novel protein, and the other two were mouse Ubc9 and SUMO-1. Ubc9 also interacted with the mouse homologue of the Bloom's syndrome gene product, another eukaryotic RecQ-type helicase, but not mouse DNA helicase Q1/RecQL (RecQL1). Deletion experiments indicated that both proteins interacted with the N-terminal segment of Wrn (amino acid 272-514). The interaction between Wrn and SUMO-1 was weaker than that between Wrn and Ubc9. Positive interaction was observed in the heterogeneous combination of Wrn and yeast Ubc9 (yUbc9), as well as yUbc9 and SUMO-1, in the two-hybrid system. The interaction between yUbc9 and SUMO-1 was abolished by deleting the C-terminal Gly residue of SUMO-1, which is reportedly required for the formation of Ubc9-SUMO-1 thioester linkage. The interaction of Wrn and SUMO-1 was also abolished by deleting the Gly residue, indicating that the interaction of Wrn and SUMO-1 is mediated by yUbc9 in the two-hybrid system. Finally, we confirmed by immunoblotting with an anti-SUMO-1 antibody that Wrn was covalently attached with SUMO-1.

Adenosine Triphosphatases↗
PubMed2000

Possible association of BLM in decreasing DNA double strand breaks during DNA replication.

Bloom's syndrome (BS) is a rare genetic disorder and the cells from BS patients show genomic instability and an increased level of sister chromatid exchange (SCE). We generated BLM(-/-) and BLM(-/-)/RAD54(-/-) DT40 cells from the chicken B-lymphocyte line DT40. The BLM(-/-) DT40 cells showed higher sensitivity to methyl methanesulfonate and elevated levels of SCE as expected. The targeted integration frequency was also increased remarkably in BLM(-/-) cells. The SCE frequency increase in BLM(-/-) cells was considerably reduced and the enhanced targeted integration observed in BLM(-/-) cells was almost completely abolished in BLM(-/-)/RAD54(-/-) cells, indicating that a large portion of the SCE in BLM(-/-) cells occurs via homologous recombination, and homologous recombination events increase with the defect of BLM function. The BLM(-/-)/RAD54(-/-) cells showed a slow growth phenotype and an increased incidence of chromosome-type breaks/gaps while each single mutant showed relatively small numbers of chromosome-type breaks/gaps.

Adenosine Triphosphatases↗
PubMed2000

Preparation of Monodispersed Solid Lipid Microspheres Using a Microchannel Emulsification Technique.

Monodispersed solid lipid microspheres consisting of high melting point edible oil were prepared as a novel sophisticated material. To prepare the monodispersed solid lipid microspheres a temperature-controlled microchannel (MC) emulsification process was devised. The prepared microspheres had diameters of approximately 20 µm or more and the standard deviation of the diameters was less than 1 µm. The effects of different surfactants, the pressure and the shape of the MC on the MC emulsification, and the diameter of the prepared droplet were studied. The experimental results are discussed using the new mechanism of droplets formation which is caused by the interfacial tension. Copyright 2000 Academic Press.

Journal Article↗
PubMed2000

Quantitative determination of cultured strawberry-cell heterogeneity by image analysis: effects of medium modification on anthocyanin accumulation.

Cultured plant cells are often highly heterogeneous in terms of secondary metabolite production. We have developed a quantitative determination method that uses an image processing system to estimate such individual cell characteristics as content of the secondary metabolite, anthocyanin. In this study, strawberry cells producing anthocyanins were grown in modified Linsmaier-Skoog medium. Anthocyanin accumulation profiles of individual cells depended on medium compositions and were quantitatively determined using the new method. The modified medium supplemented with riboflavin and high sugar concentration showed a markedly higher anthocyanin accumulation profile and pigmented cell ratio than the other modified media. The maximum content was about 11mg (g-fresh cell weight)(-1), which was three times higher than that in the control medium. Moreover, the anthocyanin accumulation profiles in the individual cells cultured in all modified media could be approximated to the parts of the normal distribution curves with the constant variance.

Journal Article↗
PubMed2000

Intermittent light irradiation with second- or hour-scale periods controls anthocyanin production by strawberry cells*

Anthocyanin production by strawberry cells depends not only on light intensity but also on the light/dark cycle operation with hour- or second-scale periods. These findings are useful for designing and operating photobioreactors for enhanced anthocyanin production. Intermittent illumination with a second-scale period produces the same amount of anthocyanin as continuous light, suggesting that the light intensity distribution within a photobioreactor does not cause suppressed production. In the hour-scale cycle, continuous light operation enhanced anthocyanin production more than the light/dark cycle process.

Journal Article↗
PubMed2000

Elevation of sister chromatid exchange in Saccharomyces cerevisiae sgs1 disruptants and the relevance of the disruptants as a system to evaluate mutations in Bloom's syndrome gene.

The SGS1 of Saccharomyces cerevisiae is a homologue of the Bloom's syndrome and Werner's syndrome genes. The sgs1 disruptants show hyperrecombination, higher sensitivity to methyl methanesulfonate and hydroxyurea, and poor sporulation. In this study, we found that sister chromatid exchange was increased in sgs1 disruptants. We made mutated SGS1 genes coding a protein proved to lack DNA helicase activity (sgs1-hd), having equivalent missense mutations found in Bloom's syndrome patients (sgs1-BS1, sgs1-BS2). None of the mutated genes could suppress the higher sensitivity to methyl methanesulfonate and hydroxyurea and the increased frequency of interchromosomal recombination and sister chromatid exchange of sgs1 disruptants. On the other hand, all of the mutant genes were able to complement the poor sporulation phenotype of sgs1 disruptants, although the values were not as high as that of wild-type SGS1.

Alleles↗
PubMed2000

Requirement of divalent galactoside-binding activity of ecalectin/galectin-9 for eosinophil chemoattraction.

We have previously isolated and cloned a novel eosinophil chemoattractant (ECA) from a human T-cell-derived expression library. This ECA, termed ecalectin, is a variant of human galectin-9, a member of a beta-galactoside binding animal lectin family, which contains two conserved carbohydrate recognition domains (CRDs). In the present study, we addressed whether carbohydrate binding activity is required for the ECA activity of ecalectin and whether both CRDs are essential for this activity. Recombinant full-length wild-type ecalectin (ecalectin-WT) and N-terminal and C-terminal CRD (ecalectin-NT and -CT, respectively) were generated. All of these recombinant proteins exhibited affinity for lactose, a property shared by galectins, but ecalectin-WT exhibited substantially higher hemagglutination activities than ecalectin-NT and -CT. Furthermore, ecalectin-WT showed over 100-fold higher ECA activity than ecalectin-NT and -CT; combination of recombinant domain fragments did not reconstitute the ECA and hemagglutination activities of the full-length protein. ECA activity of ecalectin-WT was inhibited by lactose in a dose-dependent manner. Site-directed mutation of positions Arg(65) of ecalectin-NT and Arg(239) of ecalectin-CT to an aspartic acid residue resulted in the loss of both lactose-binding and ECA activities. We conclude that divalent galactoside-binding activity is required for eosinophil chemoattraction by ecalectin.

Antigens, Differentiation↗
PubMed2000

Stereoselective synthesis of beta-benzyl-alpha-alkyl-beta-amino acids from L-aspartic acid.

A stereoselective synthesis of beta-benzyl-alpha-alkyl-beta-amino acids 1 and 2 from L-aspartic acid 3 has been developed. Methyl 5-phenyloxazolidin-2-one-4-acetate 4 was prepared from L-aspartic acid 3 through the acylation of benzene or phenyllithium with alpha-amino carboxyl group of L-aspartic acid skeleton. Alkylation of a dianion of 4 with alkyl halides and subsequent hydrogenation afforded anti-disubstituted beta-amino acids 1b and 1c in high stereoselectivities. Complete reversal of the stereoselection was realized by the alkylation of 4-phenyl-3-tert-butoxycarbonylamino-4-butanolide 6 which was obtained in a single step from 4. The 2,3,4-trisubstituted amino lactone 7 thus obtained was hydrogenated to give a syn-disubstituted beta-amino acid 2a. The syn-products 2b, 2c, and 2dwere alternatively prepared via aldol condensation of 6 with aromatic or aliphatic aldehydes followed by stereoselective reduction of the double bond with nickel chloride-sodium borohydride.

Acylation↗
PubMed2000

Analysis of pigment accumulation heterogeneity in plant cell population by image-processing system.

Plant-cultured cells are often highly heterogeneous in secondary metabolite productivity. The industrial application for large-scale metabolite production requires establishment of a stable high-producing cell line. In this study, image analysis of the individual cell is investigated as a method for evaluation of a heterogeneous cell population, and compared with the conventional method of estimation, which is based on average-cell productivity. Among strawberry cells producing anthocyanins, cells with a wide-range of pigment concentration were observed and maximum anthocyanin content was 10 times higher than the average value. In addition, a change of the frequency distribution was revealed in batch cultivation.

Anthocyanins↗
PubMed2000

An experimental study of the effects of nerve root retraction on the posterior ramus.

STUDY DESIGN: The histologic and ultrastructural changes in the posterior ramus after posterior lumbar surgery were studied in rabbits. OBJECTIVE: To investigate the structural changes in the posterior ramus after posterior lumbar surgery that may cause injury to the posterior ramus after the procedure. SUMMARY OF BACKGROUND DATA: Investigators in previous studies have pointed out that low back discomfort after lumbar discectomy relates to neurogenic changes and/or myogenic changes of paravertebral muscle. However, no previous study has demonstrated the effects of excessive nerve root retraction on spinal posterior rami. METHODS: Eighteen male Japanese White rabbits were used. The posterior ramus arising from the S1 nerve root was examined after exposure of the lamina only, fenestration, or retraction of the S1 nerve root, with light microscopy and transmission electron microscopy at 2, 4, and 6 weeks after the procedure. Results were compared with a those in control specimens that did not undergo the procedure. RESULTS: In the exposed group, no distinct difference was found compared with the control specimen. In the fenestration group, especially at 6 weeks, some attenuation and splitting of myelin sheaths was observed. In the retraction group, the structural alteration was most severe. Even at 2 weeks, fragmentation of many myelin sheaths was detected. Examination of specimens by electron microscopy indicated phagocytosis of myelinated fibers at 4 and 6 weeks. CONCLUSIONS: Findings showed that posterior lumbar procedures, including retraction of paravertebral muscle, fenestration of the lamina, and retraction of the nerve root affect the posterior ramus. Excessive retraction of the nerve root has an especially disastrous effect on the posterior ramus. Such a violent maneuver within the spinal canal must be avoided.

Animals↗
PubMed2000

Requirement of B7 costimulation for Th1-mediated inflammatory bone resorption in experimental periodontal disease.

The CD28 costimulation at TCR signaling plays a pivotal role in the regulation of the T cell response. To elucidate the role of T cells in periodontal disease, a system of cell transfer with TCR/CD28-dependent Th1 or Th2 clones was developed in rats. Gingival injection of specific Ag, Actinobacillus actinomycetemcomitans 29-kDa outer membrane protein, and LPS could induce local bone resorption 10 days after the transfer of Ag-specific Th1 clone cells, but not after transfer of Th2 clone cells. Interestingly, the presence of LPS was required not only for the induction of bone resorption but also for Ag-specific IgG2a production. LPS injection elicited the induction of expression of both B7-1 and B7-2 expression on gingival macrophages, which otherwise expressed only MHC class II when animals were injected with Ag alone. The expression of B7 molecules was observed for up to 3 days, which corresponded to the duration of retention of T clone cells in gingival tissues. Either local or systemic administration of CTLA4Ig, a functional antagonist of CD28 binding to B7, could abrogate the bone resorption induced by Th1 clone cells combined with gingival challenge with both Ag and LPS. These results suggest that local Ag-specific activation of Th1-type T cells by B7 costimulation appeared to trigger inflammatory bone resorption, whereas inhibition of B7 expression by CTLA4Ig might be a therapeutic approach for intervention with inflammatory bone resorption.

Abatacept↗
PubMed2000

High frequency of K-ras mutations in biliary duct carcinomas of cases with a long common channel in the papilla of Vater.

The frequency of K-ras mutation in biliary duct carcinomas in different locations and the relationship to the form of the junction of the pancreaticobiliary duct (JPBD) are not understood clearly. These points were investigated in the present study. Thirty-seven biliary duct carcinomas in patients without anomalous JPBD were investigated for K-ras mutations. Regarding location, 12 were hilar, 4 in the upper, 11 in the middle, and 10 in the lower portion of the duct. Furthermore, with 14 cases for which the form of the JPBD could be confirmed by endoscopic retrograde cholangiopancreatography or postoperative cholangiopancreatography, division was made into two types: those with a long common channel (>5 mm) in the papilla of Vater (type 1, n = 4), and the other with a shorter or nonapparent common channel (type 2, n = 10). The overall frequency of K-ras mutation was 30%, the incidence gradually increasing from upper to lower regions. K-ras mutations were significantly more frequent in biliary duct carcinomas associated with long common channels (P < 0.05). These results suggest that a long common channel may bear a relation to K-ras mutations in biliary duct carcinogenesis, presumably through its influence on pancreatic juice regurgitation.

Bile Duct Neoplasms↗
PubMed2000

Practical synthesis of (R)-4-mercaptopyrrolidine-2-thione from L-aspartic acid. Preparation of a novel orally active 1-beta-methylcarbapenem, TA-949.

A facile and economical synthesis of a novel orally active 1-beta-methylcarbapenem, TA-949 (1), is described. The key process involves an efficient synthesis of the C-2 side chain (R)-4-mercaptopyrrolidine-2-thione 2 from L-aspartic acid and the construction of the 1-beta-methylcarbapenem skeleton. The mercapto group of 2 with an R-configuration was formed via deaminative bromination of the amino group of L-aspartic acid beta-methyl ester hydrochloride 12 followed by a complete S(N)2-type substitution with potassium benzenemethanethiolate. High-yield amination and cyclization of the chloride 15 to the pyrrolidin-2-one 16 was accomplished by a simple treatment with ammonia. Thiation of 16 and the Birch reduction of the resultant thiolactam 18 provided the C-2 side chain 2 in high yield with the asymmetric center retained as such. The side chain 2 was installed into the 1-beta-methylcarbapenem skeleton either by coupling with the vinyl phosphate 5 or by the use of the counterattack strategy involving the Dieckmann-type cyclization of the thioester 8. Removal of the protective groups of the coupling product 6 followed by esterification provided TA-949 (1) in high yield.

Administration, Oral↗
PubMed2000

Induction of IgA against Haemophilus parainfluenzae antigens in tonsillar mononuclear cells from patients with IgA nephropathy.

Much evidence suggests that IgA production in vivo and in vitro is enhanced in patients with IgA nephropathy (IgAN). We have demonstrated glomerular deposition of the outer membranes of Haemophilus parainfluenzae (HP) antigens (OMHP) and the presence of HP-specific IgA in the serum of patients with IgAN. In this study, we investigated the production of IgA and several cytokines by tonsillar mononuclear cells (TMC) from IgAN patients induced by stimulation with OMHP. The spontaneous production of total IgA and TGF-beta by TMC from IgAN patients was higher than that by TMC from patients with chronic tonsillitis (CT) (P < 0.05). Stimulation with OMHP in vitro enhanced the production of HP-specific IgA by TMC from IgAN patients (P < 0.01), but not by TMC from CT patients. OMHP stimulation also enhanced the production of TGF-beta and IL-10 by TMC from IgAN patients (P < 0.001). These results suggest that the infection of HP in the tonsil may be involved in the etiology of IgAN.

Adolescent↗