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Biomedical subjects

H Sakai

Publications and source records attributed to H Sakai.

At least 73 records · Page 4Linked to original sources
PubMed2001

Correlation between sequence conservation of the 5' untranslated region and codon usage bias in Mus musculus genes.

The codon adaptation index (CAI) values of all protein-coding sequences of the full-length cDNA libraries of Mus musculus were computed based on the RIKEN mouse full-length cDNA library. We have also computed the extent of consensus in flanking sequences of the initiator ATG codon based on the 'relative entropy' values of respective nucleotide positions (from -20 to +12 bp relative to the initiator ATG codon) for each group of genes classified by CAI values. With regard to the two nucleotides positions (-3 and +4) known to be highly conserved in Kozak's consensus sequence, a clear correlation between CAI values and relative entropy values was observed at position -3 but this was not significant at position +4, although a significant correlation was found at position -1 of the consensus sequence. Further, although no correlation was observed at any additional positions, relative entropy values were very high at positions -4, -6, and -8 in genes with high CAI values. These findings suggest that the extent of conservation in the flanking sequence of the initiator ATG codon including Kozak's consensus sequence was an important factor in modulation of the translation efficiency as well as synonymous codon usage bias particularly in highly expressed genes.

5' Untranslated Regions↗
PubMed2001

Crystal structure at 1.5-A resolution of Pyrus pyrifolia pistil ribonuclease responsible for gametophytic self-incompatibility.

The crystal structure of the Pyrus pyrifolia pistil ribonuclease (S(3)-RNase) responsible for gametophytic self-incompatibility was determined at 1.5-A resolution. It consists of eight helices and seven beta-strands, and its folding topology is typical of RNase T(2) family enzymes. Based on a structural comparison of S(3)-RNase with RNase Rh, a fungal RNase T(2) family enzyme, the active site residues of S(3)-RNase assigned were His(33) and His(88) as catalysts and Glu(84) and Lys(87) as stabilizers of an intermediate in the transition state. Moreover, amino acid residues that constitute substrate binding sites of the two RNases could be superimposed geometrically. A hypervariable (HV) region that has an S-allele-specific sequence comprises a long loop and short alpha-helix. This region is far from the active site cleft, exposed on the molecule's surface, and positively charged. Four positively selected (PS) regions, in which the number of nonsynonymous substitutions exceeds that of synonymous ones, are located on either side of the active site cleft, and accessible to solvent. These structural features suggest that the HV or PS regions may interact with a pollen S-gene product(s) to recognize self and non-self pollen.

Amino Acid Sequence↗
PubMed2001

Specific tissue distribution of megsin, a novel serpin, in the glomerulus and its up-regulation in IgA nephropathy.

Mesangial cells play critical roles in maintaining a structure and function of the glomerulus. We previously cloned a novel mesangium-predominant gene, megsin, a new serine protease inhibitor. To clarify localization and roles of megsin protein, we raised polyclonal antibodies to megsin. By immunohistochemistry, megsin protein was specifically identified in the mesangial area. The amount of megsin protein was increased in glomeruli of patients with IgA nephropathy than in those of normal individuals and of patients with minimal change nephrotic syndrome or membranous nephropathy, suggesting a pathophysiological role of megsin as a functional modulator of mesangial functions in situ.

Amino Acid Sequence↗
PubMed2001

Effects of Y-27632 on acetylcholine-induced contraction of intact and permeabilized intrapulmonary bronchial smooth muscles in rats.

In the present study, the effects of a selective Rho-associated coiled-coil forming protein kinase (ROCK) inhibitor, Y-27632 [(+)-(R)-trans-4-(1-aminoethyl)-(4-pyridyl)cyclohexanecarboxamide dihydrochloride] on acetylcholine-induced contraction and Ca(2+) sensitization of rat bronchial smooth muscle were examined. Intact and beta-escin-permeabilized muscles of the third branch of intrapulmonary bronchi were used. In intact muscles, Y-27632 (10(-6)-10(-4) M) concentration-dependently inhibited acetylcholine-induced contractile responses. In acetylcholine (10(-3) M)-precontracted intact muscles, the maximal relaxation (about 50% inhibition of contraction) was obtained by a concentration of 10(-4) M Y-27632, which had no effect on the resting tone. In beta-escin-permeabilized muscles, addition of acetylcholine (10(-5)-10(-3) M) plus GTP (100 microM) induced a further contraction, i.e., Ca(2+) sensitization at a constant Ca(2+) concentration of pCa=6.0. The acetylcholine-induced Ca(2+) sensitization was completely blocked in the presence of 10(-4) M Y-27632, whereas the Ca(2+)-induced contraction itself was not affected by Y-27632. Immunoblot study revealed the expression of ROCK-I and ROCK-II proteins in the intrapulmonary bronchi of rats. These findings suggest that Y-27632 dilates acetylcholine-mediated contraction of rat bronchial smooth muscle by inhibiting RhoA/ROCK-mediated Ca(2+) sensitization.

Acetylcholine↗
PubMed2001

Analysis of dominant-negative effects of mutant Env proteins of human immunodeficiency virus type 1.

The Env protein of human immunodeficiency virus type 1 is assembled into a stable trimer, and oligomerization is required for maintenance of viral infectivity. This property of Env suggests that Env mutants may have a dominant-negative effect on virus infectivity. To investigate this possibility, we established a packaging cell line in which both wild-type and mutant Env proteins could be expressed simultaneously in a single cell. We analyzed the effects of two types of Env mutants: cytoplasmic tail-truncated TM mutants and a mutant defective in gp120/gp41 cleavage. The cytoplasmic tail-truncated proteins were found to be incorporated into virions by forming an oligomer with wild-type TM, but could not inhibit the wild-type function. In contrast, phenotypic mixing of cleavage-defective Env with the wild-type protein caused dramatic inhibition of infectivity, indicating that this mutant has a strong dominant-negative phenotype.

Acquired Immunodeficiency Syndrome↗
PubMed2001

Possible involvement of G(i3) protein in augmented contraction of bronchial smooth muscle from antigen-induced airway hyperresponsive rats.

To investigate a possible involvement of pertussis toxin (PTX)-sensitive heterotrimeric G proteins in the pathogenesis of airway hyperresponsiveness, the effect of PTX treatment on the augmented contractile response to acetylcholine (ACh) in bronchial smooth muscle of antigen-induced airway hyperresponsive rats was determined. In bronchial smooth muscle of airway hyperresponsive rats that were actively sensitized and repeatedly challenged with 2,4-dinitrophenylated Ascaris suum antigen, ACh-induced contractions were markedly augmented. The augmented contractile responses in the airway hyperresponsive group were significantly inhibited after treatment with PTX (1 microg/mL for 6 hr, 37 degrees ), whereas only a slight attenuation was observed in the normal control group. The level of G(alpha)i3 (measured by immunoblotting), but not other alpha-subunits of G(i/o) family proteins, in bronchial smooth muscle of the airway hyperresponsive rats was significantly increased as compared with that of control animals. It is concluded that PTX-sensitive muscarinic contractile responses of bronchial smooth muscle might be augmented upon antigen-induced airway hyperresponsiveness in rats, probably due to an up-regulation of G(alpha)i3 protein of bronchial smooth muscle.

Acetylcholine↗
PubMed2001

Serum levels of HCV RNA and core protein before and after incubation at 37 degrees C for 24 h.

The kinetics of HCV during interferon (IFN) therapy have recently been described and the estimated virion half-life is an average of 2.7 h, suggesting that HCV infection is highly dynamic. The aim of this study was to evaluate serum levels of HCV-RNA and HCV core protein (HCV-Ag) before and after incubation at 37 degrees C for 24 h. We also evaluated the viral kinetics during IFN treatment by determining their serum levels at 0, 24 and 48 h, and day 8 after the start of treatment. The decay slope was calculated as the logarithm of the ratio of HCV-RNA levels at 0 and 24 h of incubation: log(virus load) 24 h-log(virus load) 0 h and the estimated half-life was also calculated. The decay slope was -1.66+/-0.75 (-4.12 to -0.18) (mean+/-S.D. (range)) and the estimated virion half-life was 6.2+/-6.9 h (1.8-39.3). The HCV-RNA level was rapidly decreased to 6.8+/-13.1% of the initial load after incubation independently of the serotype. In contrast, the HCV-Ag level after incubation for 24 h was 98.7+/-12.2% of the initial level. The synthesized naked HCV-RNA (equivalent to 10(7) copy/ml) was not detected after 1-min incubation. These data suggested that HCV virions are very unstable and collapsed rapidly and that HCV-RNA, existing outside of virions, is immediately degraded in serum, whereas HCV-Ag remains stable. IFN treatment caused a rapid decrease in the levels of both HCV-RNA and HCV-Ag. The HCV-RNA decay slope was -1.95+/-0.96 (range: -3.48 to -0.50) and was similar to that seen in the incubation study. Our result suggested the significance of measuring HCV-Ag during clinical management independently of HCV-RNA, especially because of its high stability.

Journal Article↗
PubMed2001

Orbit-related long-term climate cycles revealed in a 12-Myr continental record from Lake Baikal.

Quaternary records of climate change from terrestrial sources, such as lake sediments and aeolian sediments, in general agree well with marine records. But continuous records that cover more than the past one million years were essentially unavailable until recently, when the high-sedimentation-rate site of Lake Baikal was exploited. Because of its location in the middle latitudes, Lake Baikal is highly sensitive to insolation changes and the entire lake remained uncovered by ice sheets throughout the Pleistocene epoch, making it a valuable archive for past climate. Here we examine long sediment cores from Lake Baikal that cover the past 12 million years. Our record reveals a gradual cooling of the Asian continental interior, with some fluctuations. Spectral analyses reveal periods of about 400 kyr, 600 kyr and 1,000 kyr, which may correspond to Milankovitch periods (reflecting orbital cycles). Our results indicate that changes in insolation were closely related to long-term environmental variations in the deep continental interior, over the past 12 million years.

Journal Article↗
PubMed2001

Effect of adsorption of bovine serum albumin on liposomal membrane characteristics.

The effect of adsorption of bovine serum albumin (BSA) on the membrane characteristics of liposomes at pH 7.4 was examined in terms of zeta potential, micropolarity, microfluidity and permeability of liposomal bilayer membranes, where negatively charged L-alpha-dipalmitoylphosphatidylglycerol (DPPG)/L-alpha-dipalmitoylphosphatidylcholine (DPPC), negatively charged dicetylphosphate (DCP)/DPPC and positively charged stearylamine (SA)/DPPC mixed liposomes were used. BSA with negative charges adsorbed on negatively charged DPPG/DPPC mixed liposomes but did not adsorb on negatively charged DCP/DPPC and positively charged SA/DPPC mixed liposomes. Furthermore, the adsorption amount of BSA on the mixed DPPG/DPPC liposomes increased with increasing the mole fraction of DPPG in spite of a possible electrostatic repulsion between BSA and DPPG. Thus, the adsorption of BSA on liposomes was likely to be related to the hydrophobic interaction between BSA and liposomes. The microfluidity of liposomal bilayer membranes near the bilayer center decreased by the adsorption of BSA, while the permeability of liposomal bilayer membranes increased by the adsorption of BSA on liposomes. These results are considered to be due to that the adsorption of BSA brings about a phase separation in liposomes and that a temporary gap is consequently formed in the liposomal bilayer membranes, thereby the permeability of liposomal bilayer membranes increases by the adsorption of BSA.

Journal Article↗
PubMed2001

Effects of lysozyme and bovine serum albumin on membrane characteristics of dipalmitoylphosphatidylglycerol liposomes.

The effects of adsorption of two kinds of proteins on the membrane characteristics of liposomes were examined at pH 7.4 in terms of adsorption amounts of proteins on liposomes, penetrations of proteins into liposomal bilayer membranes, phase transition temperature, microviscosity and permeability of liposomal bilayer membranes, using positively charged lysozyme (LSZ) and negatively charged bovine serum albumin (BSA) as proteins and negatively charged L-alpha-dipalmitoylphosphatidylglycerol (DPPG) liposomes. The saturated adsorption amount of LSZ was 720 g per mol of liposomal DPPG, while that of BSA was 44 g per mol of liposomal DPPG. The penetration of LSZ into DPPG lipid membranes was greater than that of BSA. The microviscosity in the hydrophobic region of liposomal bilayer membranes increased due to adsorption (penetration) of LSZ or BSA, while the permeability of liposomal bilayer membranes increased. The gel-liquid crystalline phase transition temperature of liposomal bilayer membranes was not affected by adsorption of LSZ or BSA, while the DSC peak area (heat of phase transition) decreased with increasing adsorption amount of LSZ or BSA. It is suggested that boundary DPPG makes no contribution to the phase transition and that boundary DPPG and bulk DPPG are in the phase-separated state, thereby increasing the permeability of liposomal bilayer membranes through adsorption of LSZ or BSA. A possible schematic model for the adsorption of LSZ or BSA on DPPG liposomes was proposed.

Journal Article↗
PubMed2001

Preparation of liposomes containing Ceramide 3 and their membrane characteristics.

Liposomes composed of Ceramide 3, [2S,3S,4R-2-stearoylamide-1,3,4-octadecanetriol], and L-alpha-dipalmitoylphosphatidylcholine (DPPC) were prepared by varying the amount of Ceramide 3, and the effects of Ceramide 3 on the liposome formation, particle size, dispersibility, microviscosity and phase transition temperature were examined by means of a microscopy, a dynamic light scattering method, a fluorescence polarization method, a differential scanning calorimetry (DSC) and so on. All the DPPC was able to contribute to the formation of liposomes up to 0.130 mol fraction of Ceramide 3. The particle size of liposomes was almost unaffected by the addition of Ceramide 3. The dispersibility of liposomes containing Ceramide 3 was maintained for at least 15 days. The microviscosity of liposomal bilayer membranes in the liquid crystalline state was increased with increasing the mole fraction of Ceramide 3, while that in the gel state was independent of the mole fraction of Ceramide 3. The phase transition temperature from gel to liquid crystalline states of DPPC bilayer membranes was shifted upwards with the addition of Ceramide 3, indicating a cooperative interaction between DPPC and Ceramide 3 molecules. However, a sharp DSC peak became broad and split at higher mole fractions of Ceramide 3, suggesting a phase separation in the mixed DPPC/Ceramide 3 liposomal bilayer membranes. These phenomena were suggested to be related to the previously observed fact for the mixed DPPC/Ceramide 3 monolayers that Ceramide 3 interacts with DPPC in the liquid-expanded phase with consequent phase separation accompanied with domain formation.

Journal Article↗
PubMed2001

Solution properties of amino acid-type new surfactant.

The solution properties of an amino acid-type new surfactant, N-[3-lauryloxy-2-hydroxypropyl]-L-arginine L-glutamate (C12HEA-Glu), were examined by means of pK(a), surface tension, light scattering and fluorescence measurements. The dissociation state of C12HEA-Glu was changed by varying pH of the aqueous solution. The critical micelle concentration (cmc) of aqueous solution of C12HEA-Glu was lowest at pH 5.6 and the cmc increased with decreasing pH. The aggregation number of C12HEA-Glu micelles was largest at pH 5.6 and decreased with decreasing pH. The surface tension-concentration curve for C12HEA-Glu in water (non-buffered solution) showed a minimum. This is considered to be due to the coexistence of a trace species and a main species of C12HEA-Glu with various dissociation states since the pH of aqueous solution of C12HEA-Glu changes with the concentration of C12HEA-Glu. The micropolarity in the micelles of C12HEA-Glu was almost independent of pH, while the microfluidity in the micelles increased with increasing pH.

Journal Article↗
PubMed2001

Telomerase activity is repressed during differentiation along the hepatocytic and biliary epithelial lineages: verification on immortal cell lines from the same origin.

Recent investigations indicate that telomerase activity regulates the life span of cells by compensating for telomere shortening during DNA replication. In addition, as differentiation progresses, telomerase activity is reduced in several different cell lineages. These findings lend support to the theory that more immature cells have greater remaining proliferative capacity and longer life span. However, it has not been directly demonstrated that the differentiation along a hepatocytic or a bile ductal lineage is accompanied by reduction of telomerase activity. In this study, we present direct evidence that telomerase activity is reduced during hepatocytic and biliary epithelial differentiation by using our unique cell lines including a stem-like cell line, ETK-1. When hepatocytic differentiation was induced in ETK-1 by 5-azacytidine, telomerase activity decreased significantly. Similarly, when we compared the telomerase activity on SSP-25 and RBE cell lines from the same origin but representing different maturation stages of cholangiocarcinoma, more mature cells were found to possess significantly lower activity. These results indicate that the generally accepted relationship between telomerase activity and differentiation stage also applies in the hepatocytic and biliary epithelial lineages.

Azacitidine↗
PubMed2001

Limitations of successive transradial approach in the same arm: the Japanese experience.

The transradial approach (TRA) has been used for diagnostic and interventional cardiology. It has not previously been determined how many times the same radial artery can be cannulated without complications. A total of 812 patients (502 men and 310 women) underwent angiography or angioplasty via the TRA between 1997 and 1999 at our institution with a total of 1,438 procedures. Sheaths were 5 (55%) or 6 Fr (45%). Dropout rates of 3.5% and 7.9% were found at the second TRA attempt in the men and the women, respectively. Of the 62 TRA failures, 56 (90%) were due to narrowing or occlusion of the radial artery after the previous TRA procedure. A third TRA procedure was possible in 90% of the men and 80% of the women. A fifth TRA procedure was possible in 70% of the men and 50% of the women. The dropout rates for TRA increased as successive punctures were performed. This was primarily due to vessel narrowing and occlusion occurring as a function of multiple punctures.

Aged↗
PubMed2001

Site difference in RhoA expression between rat bronchial and tracheal smooth muscles after antigen challenge--relation to development of hyperresponsiveness.

OBJECTIVE: The present study compared the effects of repeated antigen exposure on the development of hyperresponsiveness and the expression of RhoA in the main bronchial and lower tracheal smooth muscles of sensitized METHODS: Actively sensitized rats were repeatedly challenged by antigen inhalation. Twenty-four hours after the final antigen challenge the isometrical contractions of the bronchial and tracheal smooth muscles were measured. Immunoblottings were also performed using bronchial and tracheal homogenates and the density ratios of RhoA/beta-actin were calculated to quantify the levels of RhoA. RESULTS: Acetylcholine-induced contraction of bronchial, but not tracheal, smooth muscle of antigen-treated rats was significantly augmented as compared with that of control rats, indicating that airway hyperresponsiveness appeared by antigen challenge in bronchial smooth muscle. RhoA expression in bronchial, but not tracheal, smooth muscle was significantly increased in the antigen-treated animals. CONCLUSION: The increased expression of RhoA is suggested to have an important role in developing hyperresponsiveness of bronchial smooth muscle.

Acetylcholine↗
PubMed2001

Correlation between Shine--Dalgarno sequence conservation and codon usage of bacterial genes.

In this study, we analyzed the correlation between codon usage bias and Shine--Dalgarno (SD) sequence conservation, using complete genome sequences of nine prokaryotes. For codon usage bias, we adopted the codon adaptation index (CAI), which is based on the codon usage preference of genes encoding ribosomal proteins, elongation factors, heat shock proteins, outer membrane proteins, and RNA polymerase subunit proteins. To compute SD sequence conservation, we used SD motif sequences predicted by Tompa and systematically aligned them with 5'UTR sequences. We found that there exists a clear correlation between the CAI values and SD sequence conservation in the genomes of Escherichia coli, Bacillus subtilis, Haemophilus influenzae, Archaeoglobus fulgidus, Methanobacterium thermoautotrophicum, and Methanococcus jannaschii, and no relationship is found in M. genitalium, M. pneumoniae, and Synechocystis. That is, genes with higher CAI values tend to have more conserved SD sequences than do genes with lower CAI values in these organisms. Some organisms, such as M. thermoautotrophicum, do not clearly show the correlation. The biological significance of these results is discussed in the context of the translation initiation process and translation efficiency.

Base Sequence↗
PubMed2001

Novel glucoamylase-type enzymes from Thermoactinomyces vulgaris and Methanococcus jannaschii whose genes are found in the flanking region of the alpha-amylase genes.

A region downstream of the gene for pullulan-hydrolyzing alpha-amylase, TVA II, of Thermoactinomyces vulgaris R-47 was sequenced, and an open reading frame encoding an enzyme homologous to glucoamylase was found. The nucleotide sequence of this enzyme, designated TGA, consists of 1,953 base pairs corresponding to a protein of 651 amino acid residues. The TGA gene was subcloned and expressed in Escherichia coli. Enzymatic analyses showed that, like other glucoamylases, TGA produced beta-D-glucose from its substrate. However, TGA hydrolyzed maltooligosaccharides such as maltotetraose and maltose more efficiently than starch, while fungal glucoamylases preferred starch to maltooligosaccharides. The primary structure of TGA resembled a putative glucoamylase from the hyperthermophilic archaeon Methanococcus jannaschii (MGA), while homologies between TGA and the fungal glucoamylases were low. The enzymatic properties of recombinant MGA produced in E. coli cells were similar to those of TGA. These findings indicate that TGA and MGA are novel glucoamy-lase-type enzymes with oligosaccaharide-metabolizing activity.

Amino Acid Sequence↗