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Biomedical subjects

T Sekiguchi

Publications and source records attributed to T Sekiguchi.

At least 55 records · Page 3Linked to original sources
PubMed1998

Induction of growth arrest and cell death by overexpression of the cyclin-Cdk inhibitor p21 in hamster BHK21 cells.

p21cip1/waf1/sdi1 is a universal cyclin-Cdk kinase inhibitor that has two functional domains; one binds and inhibits cyclin-Cdk activity and the other binds PCNA and thereby inhibits elongation by DNA polymerase. When transiently expressed in hamster BHK21 cells we found that human p21 was able to cause cell cycle arrest in G1 phase; this arrest was counteracted by coexpression of E2F-1 or SV40 large T antigen. To study the effect of p21 overexpression in vivo, BHK21 cell clones inducibly expressing human p21 (Tet-p21) driven by the tetracycline (Tet)-repressible promoter were established. The maximum induced p21 levels in the absence of Tet were estimated to be ten times that of endogenous hamster p21. As p21 levels rose following removal of Tet, p21-associated histone H1 kinase activity was increased and concomitantly cell growth and DNA synthesis were reduced. Tet-p21 BHK21 cells became arrested in G1 phase and lost colony forming ability irreversibly 2-4 days after removal of Tet. The induction of cyclin E- and cyclin A-associated kinase activities was diminished when G0-synchronized Tet-p21 BHK21 cells were serum stimulated in the absence of Tet. Increased binding of p21 to PCNA and cyclin D1-Cdk4 was detected in induced cells. Overexpression of p21 led to cell death in BHK21 cells at 39.5 degrees C within 4 days.

Animals↗
PubMed1998

Soft X-ray beamline specialized for actinides and radioactive materials equipped with a variably polarizing undulator.

This report presents the design of an undulator beamline at SPring-8 to be used for soft X-ray spectroscopy focused on radioactive materials. Photoemission spectroscopy experiments are carried out in a radioisotope (RI)-controlled area where actinide compounds as well as unsealed radioactive materials are usable. Intrusion of the radioactive materials into the electron storage ring or to the outside of the evacuated beamline components can be avoided by a specially devised RI protection/inspection mechanism. The combination of a variably polarizing undulator and a varied-line-spacing plane-grating monochromator provides linearly or circularly polarized soft X-rays with a high resolving power in the energy range 0.28-1.5 keV. The beamline will become operational in December 1997.

Journal Article↗
PubMed1998

Crystal structure of eucaryotic E3, lipoamide dehydrogenase from yeast.

The crystal structure of eucaryotic lipoamide dehydrogenase from yeast has been determined by an X-ray analysis at 2.7 (partially at 2.4) A resolution. The enzyme has two identical subunits related by a pseudo twofold symmetry. The tertiary structure is similar to those of other procaryotic enzymes. The active site, consisting of FAD, Cys44, and Cys49 from one subunit and His457' from the other subunit, is highly conserved. This enzyme is directly bound to the core protein E2 of the 2-oxoglutarate dehydrogenase complex, whereas it is bound to the pyruvate dehydrogenase complex through a protein X. The calculated electrostatic potential suggests two characteristic regions for binding with these two proteins.

Amino Acid Sequence↗
PubMed1998

RagA is a functional homologue of S. cerevisiae Gtr1p involved in the Ran/Gsp1-GTPase pathway.

Human RagA and RagB is reported to be 52% identical to a putative GTPase of Saccharomyces cerevisiae, Gtr1p. According to the reported nucleotide sequence, we amplified human RagA and RagBs cDNAs from the human B cell cDNA library with PCR. Both cDNAs rescued a cold sensitivity of S. cerevisiae, gtr1-11. Furthermore, we introduced into the cloned human RagA cDNA, the mutation 'T21L' corresponding to the gtr1-11 mutation which has been reported to suppress not only all of rcc1-, temperature-sensitive mutants of Ran/Gsp1p GTPase GDP/GTP-exchanging factor, but also rna1-1, a temperature-sensitive mutant of Ran/Gsp1p GTPase-activating protein. The resulting RagAgtr1-11 cDNA partially, but significantly, suppressed both rcc1- and rna1-1 mutations. These results indicated that RagA and RagBs are functional homologues of S. cervisiae Gtr1p. Interestingly, while wild-type human RagA and RagBs were localized within the cytoplasm, similar to S. cerevisiae Gtr1p, the mutated human RagAgtr1-11 corresponding to a dominant negative form of RagA was distributed in discrete speckles in the nucleus, being localized side by side with SC-35, a non-snRNP of the splicing complex. In contrast, a dominant positive form of RagA, Q66L was localized in the cytoplasm. Thus, RagA was suggested to shuttle between the cytoplasm and the nucleus, depending on the bound nucleotide state.

Amino Acid Sequence↗
PubMed1998

Production of superoxide and nitric oxide by alveolar macrophages in the bleomycin-induced interstitial pneumonia mice model.

To elucidate the potential role of superoxide (O2-) and nitric oxide (NO) in the pathogenesis of interstitial pneumonia, the quantity of O2- and NO produced by the alveolar macrophages (AM) were determined in the bleomycin (BLM)-induced interstitial pneumonia mouse model. The production of O2- and NO increased on days 7, 14 and 21 after BLM injection. Strong expression of peroxynitrite (ONOO-) was seen in AM by using immunostaining for nitrotyrosine. The hydroxyproline contents increased on day 21 after BLM injection. O2- and NO are thought to play an important role in the pathology of fibrosis.

Animals↗
PubMed1998

Effects of O-glycosylation inhibitors on the differentiation of HL-60 cells.

The effects of O-glycosylation inhibitors on the growth and differentiation of the human acute promyeloblastic leukemia cell line HL-60 were studied to examine whether the O-glycosylation is needed for HL-60 cells to differentiate into granulocyte-like cells or monocyte-macrophage-like cells. N-Acetyl-alpha-D-galactosaminides, inhibitors of mucin-type oligosaccharide synthesis, and N-acetyl-beta-D-galactosaminides did not affect either growth or differentiation. beta-D-Xylosides, the artificial initiators of glycosaminoglycan synthesis, also were tested. Only 4-methylumbelliferyl-beta-D-xyloside induced HL-60 cells, to differentiate, and they differentiated into granulocyte-like cells, assessed by reduction of nitroblue tetrazolium, Giemsa staining, and esterase double-staining. The aglycon portion of 4-methylum-belliferyl-beta-D-xyloside, 4-methylumbelliferone, caused the differentiation. Thus we could find a new drug that induces the differentiation of HL-60 cells.

Antimetabolites, Antineoplastic↗
PubMed1997

Determination of the amount of native structural bacteriorhodopsin in purple membrane Langmuir-Blodgett films by a spectroscopic surface denaturation quantifying technique.

Purple membrane (PM) shows denaturation when spread over an air/water interface. We established a technique, which we call the spectroscopic surface denaturation quantifying (SSDQ) technique, that uses infrared linear dichroism to determine the amount of native structural bacteriorhodopsin (BR) in PM Langmuir-Blodgett (LB) films. Using the SSDQ technique we found that the conformational change after surface denaturation of BR was the same as that caused by ethanol treatment. By extrapolating the data of the amount of non-denatured BR molecules in PM LB films vs. the area of a single BR molecule on an air/water interface, we also found that the surface area of a single non-denatured BR molecule was 11.5 nm2, which is consistent with that determined by high-resolution electron cryo-microscopy and electron diffraction (EMD). These results demonstrate that the SSDQ technique is effective in quantifying the amount of native structural BR in PM LB films. The SSDQ technique is also applicable to other types of protein consisting of alpha-helical conformation.

Bacteriorhodopsins↗
PubMed1997

FMRF amide-like-immunoreactive primary sensory neurons in the olfactory system of the terrestrial mollusc, Limax marginatus.

The distribution of FMRF amide-like immunoreactivity was investigated in the olfactory organs in the tentacle tip of the terrestrial slug, Limax marginatus. Approximately 0.7% of the neurons in the lobules of the tentacle ganglia demonstrated FMRF amide-like immunoreactivity. Most of the FMRF amide-like-immunoreactive somata lay at superficial positions within the lobules, and dendritic processes extended to the outer surface of the sensory epithelium, whereas the axons traveled toward the cerebral ganglion through the ventral part of the tentacle nerve. From their morphological features, FMRF amide-like-immunoreactive cells were considered to be primary sensory neurons.

Animals↗
PubMed1997

Prediction of effect of interferon on chronic hepatitis C.

Clinical, pathological, and virological analysis including hypervariable region-1 of hepatitis C virus (HCV) was performed to predict the effect of interferon (IFN) on 41 patients with chronic hepatitis type C. The low virus load, low frequency of the mutation in the hypervariable region-1 as the change of amino acid and high level of serum aminotransferase make one estimate the good effect of IFN on patients with HCV. Mutation in the hypervariable region-1 of HCV measured by fast assay fluorescence single-stranded conformational polymorphism was more frequent in nonresponders to IFN than responders. The most frequently mutated position was amino acid number 406. This indicates that the specific mutation site might affect the response of IFN.

Amino Acid Sequence↗
PubMed1997

Crystallization of eukaryotic E3, lipoamide dehydrogenase, from yeast, for exhibiting X-ray diffraction beyond 2.5 A resolution, and preliminary structure analysis.

Lipoamide dehydrogenase, which is a common component of alpha-keto acid dehydrogenase complexes, has been highly purified from yeast (Saccharomyces cerevisiae) to reveal its structure at higher resolution. New crystals obtained by a desalting method exhibited diffraction beyond 2.5 A resolution. The cell dimensions are a = 97.1, b = 158.7, and c = 67.9 A, and the space group is P2(1)2(1)2(1). There is a dimeric enzyme in the asymmetric unit. The crystal structure was solved by means of the molecular-replacement technique and refined in a preliminary manner.

Crystallization↗
PubMed1997

Crystal structure of 3-isopropylmalate dehydrogenase from the moderate facultative thermophile, Bacillus coagulans: two strategies for thermostabilization of protein structures.

The crystal structure of 3-isopropylmalate dehydrogenase from the moderate facultative thermophile Bacillus coagulans (BcIPMDH) has been determined by the X-ray method. BcIPMDH is a dimeric enzyme composed of two identical subunits, each of which takes an open alpha/beta structure with 11 alpha-helices and 14 beta-strands. The polypeptide is folded into two domains. The first domain is composed of residues 1-101 and 257-356, and the second domain, of residues 102-256. The latter domains of the two subunits are associated with one another by a dyad axis to make the dimer, locally forming a beta-sheet and a four-helix bundle. As compared with the structure of the enzyme from the extreme thermophile Thermus thermophilus (TtIPMDH), a new short beta-sheet (residues 329-330 and 340-341) absent in TtIPMDH is formed by the insertion of 5 residues in BcIPMDH. In terms of determinants for thermostabilization, both consistent and inconsistent changes were found between the two enzymes. The regions including inconsistent changes are formed by different usages of the determinants for stabilizing the loops at different levels. Those in BcIPMDH contain some structural redundancies in length of amino acid sequence and flexibility of residues, which seem to be unnecessary for the enzymatic reaction. Such redundancies are also found in the primary structure of the enzyme of the mesophile Bacillus subtilis, but these parts are more stabilized in BcIPMDH by hydrogen bonds and salt bridges. On the other hand, TtIPMDH is stabilized by reducing such redundant parts. This contrast suggests that different strategies may be preferred for thermostabilization, depending on temperature.

3-Isopropylmalate Dehydrogenase↗
PubMed1997

Effect of lecithinized-superoxide dismutase on the rat colitis model induced by dextran sulfate sodium.

Lecithinized-superoxide dismutase (PC-SOD), which is synthesized with a lecithin derivative bound covalently to recombinant human Cu,Zn-SOD, has a longer half-life in blood and higher cell affinity than unmodified SOD. The effects of PC-SOD were evaluated using the rat ulcerative colitis model induced by 3% dextran sulfate sodium. Intravenous injection of rats with 0.5 or 1 mg/kg of PC-SOD suppressed the progression of bloody stools, the formation of erosion, and the infiltration of the colon with inflammatory cells. Furthermore, it also reduced the increase of leukocytes in blood. Thus, PC-SOD may have therapeutic potential in the treatment of ulcerative colitis.

Animals↗
PubMed1997

Effect of lecithinized-superoxide dismutase on the interstitial pneumonia model induced by bleomycin in mice.

Superoxide anion (O2-) acts as an exacerbation factor in interstitial pneumonia. Lecithinized-superoxide dismutase (PC-SOD), which is synthesized with a lecithin derivative bound covalently to recombinant human Cu,Zn-SOD, has a longer half-life in plasma and higher affinity to cell membranes than unmodified SOD. The effect of PC-SOD was evaluated using the bleomycin-induced interstitial pneumonia mouse model. Treatment with PC-SOD at 10 mg/kg significantly reduced the hydroxyproline content and fibrosis score. Namely, PC-SOD suppressed the progression of pulmonary fibrosis on the bleomycin-induced interstitial pneumonia mouse model. PC-SOD may be a potential drug for interstitial pneumonia therapy.

Animals↗
PubMed1997

Further classification of dysmotility-like dyspepsia by interdigestive gastroduodenal manometry and plasma motilin level.

OBJECTIVE: To investigate interdigestive gastroduodenal motility with an infused catheter and measure plasma motilin levels in eight normal individuals and 18 patients with dysmotility-like dyspepsia. METHODS AND RESULTS: All normal individuals had normal gastroduodenal interdigestive migrating complexes. Patients with dysmotility-like dyspepsia were classified into three subgroups on the basis of gastric antral motility: 1) seven patients with normal interdigestive migrating complexes, 2) five patients without interdigestive migrating complexes and with gastric phase II predominant over phase I, and 3) six patients without interdigestive migrating complexes and with phase I predominance. The maximum and mean plasma motilin levels were significantly different in normal individuals from those in subgroup 3 (Kruskal-Wallis test, p < 0.05). CONCLUSIONS: Dysmotility-like dyspepsia appears to be a heterogeneous condition. Abnormal motilin secretion may cause dysmotility in subgroup 3, but dyspepsia in subgroup 1 and the absence of interdigestive migrating complexes in subgroup 2 could not be explained only on this basis.

Adult↗