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Biomedical subjects

R Sawada

Publications and source records attributed to R Sawada.

At least 19 recordsLinked to original sources
PubMed2005

Analysis of cellular functions by multipoint fluorescence correlation spectroscopy.

The biophysical investigation of living cells is currently possible by single molecular detection methods such as fluorescence correlation spectroscopy (FCS). FCS is applied for measuring the dynamic mobility of target molecules in living cells; however, the conventional FCS systems still lack quantitative analysis for many regions of interests (ROI) in real time. To improve this situation, we have developed a novel multipoint FCS system (M-FCS) that can measure multipoint correlation functions in the cell simultaneously. To evaluate its performance, we measured correlation functions for rhodamine 6G (Rh6G) in homogeneous conditions and for green fluorescence protein (GFP) in HeLa cells. We conclude that M-FCS possesses reliable performance. As a pharmacological application, glucocorticoid receptor protein fused GFP (GR-GFP) was transfected in HeLa cells and FCS measurements were carried out in the cytoplasm and the nucleus simultaneously. The translocation of GR-GFP from the cytoplasm to the nucleus by ligand stimulation was observed with laser scanning microscopy (LSM) and M-FCS. Particularly in the nucleus, the slower diffusion of GR-GFP suggested molecular interactions after the translocation. These data imply that M-FCS can be applied for quantitative analysis of kinetic processes in living cells.

Diffusion↗
PubMed2000

Purification and some properties of sulfur reductase from the iron-oxidizing bacterium Thiobacillus ferrooxidans NASF-1.

Thiobacillus ferrooxidans strain NASF-1 grown aerobically in an Fe2+ (3%)-medium produces hydrogen sulfide (H2S) from elemental sulfur under anaerobic conditions with argon gas at pH 7.5. Sulfur reductase, which catalyzes the reduction of elemental sulfur (S0) with NAD(P)H as an electron donor to produce hydrogen sulfide (H2S) under anaerobic conditions, was purified 69-fold after 35-65% ammonium sulfate precipitation and Q-Sepharose FF, Phenyl-Toyopearl 650 ML, and Blue Sepharose FF column chromatography, with a specific activity of 57.6 U (mg protein)(-1). The purified enzyme was quite labile under aerobic conditions, but comparatively stable in the presence of sodium hydrosulfite and under anaerobic conditions, especially under hydrogen gas conditions. The purified enzyme showed both sulfur reductase and hydrogenase activities. Both activities had an optimum pH of 9.0. Sulfur reductase has an apparent molecular weight of 120,000 Da, and is composed of three different subunits (M(r) 54,000 Da (alpha), 36,000 Da (beta), and 35,000 Da (gamma)), as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This is the first report on the purification of sulfur reductase from a mesophilic and obligate chemolithotrophic iron-oxidizing bacterium.

Journal Article↗
PubMed1998

Globotriaosyl ceramide and globoside as major glycolipid components of fibroblasts in scirrhous gastric carcinoma tissues.

Scirrhous gastric cancer is characteristic in that cancer cells proliferate and invade with prominent fibrosis. To search for the expression of specific carbohydrate chains in scirrhous gastric cancer, we have examined the glycosphingolipid composition of scirrhous cancer tissues (n=10) in comparison with that of non-scirrhous cancer tissues (n=10) by means of two-dimensional thin layer chromatography, followed by fast atom bombardment mass spectrometry of the individual glycolipids and immunostaining analysis. The major neutral glycosphingolipids from scirrhous gastric cancer tissues were identified as ceramide monohexoside, ceramide dihexoside, globotriaosyl ceramide (Gb3) and globoside (Gb4), while the major acidic glycosphingolipids were II3 NeuAcalpha-LacCer, II3 NeuAcalpha2-LacCer and sulfatide. Relative concentrations of Gb3 and Gb4 in scirrhous gastric cancer tissues (Gb3 + Gb4 = 58%) were two times higher than those in non-scirrhous gastric cancer tissues (29%). Orthotopic fibroblasts cloned from scirrhous gastric cancer tissues showed similar high concentrations of Gb3 and Gb4 to scirrhous gastric cancer tissues. Furthermore, immunohistochemical study revealed that Gb3 and Gb4 were expressed intensely on the fibroblasts. On the other hand, analysis of glycosphingolipids in four scirrhous gastric cancer cell lines yielded the following results. i) The contents of Gb3 and Gb4 were low (6%), compared with orthotopic fibroblasts (62%). ii) Significant amounts of Le(a) (pentaglycosylceramide) and Le(b) (hexa- and heptaglycosylceramides), which could not be detected in scirrhous cancer tissues, were observed. The results show that the major neutral glycosphingolipids such as Gb3 and Gb4 of scirrhous gastric cancer tissues were derived from orthotopic fibroblasts and not from the cancer cells.

Adenocarcinoma, Scirrhous↗
PubMed1997

Sequence analysis of SRSV in fecal specimens from an epidemic of infantile gastroenteritis, October to December 1995, Japan.

From October to December in 1995, an epidemic of infantile gastroenteritis occurred all over Japan except in Hokkaido and Okinawa prefectures. The number of infected infants and young children was estimated to be over 5 million cases [Editorial, IASR 1996]. The stool specimens from patients were examined for the presence of small round structured viruses (SRSVs) by reverse transcription-polymerase chain reaction (RT-PCR) and nucleotide sequencing of parts of the RNA-dependent RNA polymerase region. Thirty-five of 87 stool specimens examined gave positive results. Genomic variation was investigated by sequence analysis of a 327 bp cDNA region. The nucleotide and deduced amino acid sequences of the ten strains segregated into two distinct groups; one showed 96.0-100% nucleotide and 99.1-100% amino acid identity, the others showed 91.4-99.7% nucleotide and 93.5-100% identity. The main mechanism of transmission remains unknown. However, these data suggest the possibility of person-to-person spread by two or more kinds of SRSV.

Base Sequence↗
PubMed1995

Characterization of chitin synthase 2 of Saccharomyces cerevisiae. Implication of two highly conserved domains as possible catalytic sites.

Chitin synthase 2 of Saccharomyces cerevisiae was characterized by means of site-directed mutagenesis and subsequent expression of the mutant enzymes in yeast cells. Chitin synthase 2 shares a region whose sequence is highly conserved in all chitin synthases. Substitutions of conserved amino acids in this region with alanine (alanine scanning) identified two domains in which any conserved amino acid could not be replaced by alanine to retain enzyme activity. These two domains contained unique sequences, Glu561-Asp562-Arg563 and Gln601-Arg602-Arg603-Arg604-Trp605, that were conserved in all types of chitin synthases. Glu561 or arginine at 563, 602, and 603 could be substituted by glutamic acid and lysine, respectively, without significant loss of enzyme activity. However, even conservative substitutions of Asp562 with glutamic acid, Gln601 with asparagine, Arg604 with lysine, or Trp605 with tyrosine drastically decreased the activity, but did not affect apparent Km values for the substrate significantly. In addition to these amino acids, Asp441 was also found in all chitin synthase. The mutant harboring a glutamic acid substitution for Asp441 severely lost activity, but it showed a similar apparent Km value for the substrate. Amounts of the mutant enzymes in total membranes were more or less the same as found in the wild type. Furthermore, Asp441, Asp562, Gln601, Arg604, and Trp605 are completely conserved in other proteins possessing N-acetylglucosaminyltransferase activity such as NodC proteins of Rhizobium bacterias. These results suggest that Asp441, Asp562, Gln601, Arg604, and Trp605 are located in the active pocket and that they function as the catalytic residues of the enzyme.

Amino Acid Sequence↗
PubMed1995

The dithiane Ro 44-5912 enhances vinblastine sensitivity of drug resistant and parental KB lines in vivo.

The multidrug resistance modifying activity of a dithiane analogue of tiapamil, Ro 44-5912, was examined in vivo. Results of acute toxicity studies in mice indicated that lethal toxicity occurred with doses greater than 1 mmol/kg of body weight. In a preliminary pharmacokinetic investigation, Ro 44-5912 appeared to have a longer half-life in mice than did its (R) enantiomer Ro 44-5911 (3.15 +/- 0.02 h versus 2.15 +/- 0.14 h) as measured by total radiolabel in plasma. In non-tumour bearing mice, Ro 44-5912 enhanced the toxicity of vinblastine in a manner that was dependent on the dose of both drugs. Vinblastine did not have a significant effect on tumour growth when given to nude mice bearing the parental cell line KB-3-1 at a dose of 1.5 mg/kg once per week for 3 weeks. Combination treatment with Ro 44-5912 markedly enhanced the antitumour activity of vinblastine. Similar results were seen when KB-3-1 tumours were treated with the combination of vinblastine plus cyclosporin A. Another tiapamil analogue, Ro 11-2933, had no enhancing activity with this tumour when used at an equitoxic combination dose. Ro 44-5912 also significantly enhanced vinblastine activity with P-glycoprotein-expressing KB-8-5 tumours. In three independent experiments, Ro 44-5912 enhanced the growth inhibiting activity of vinblastine by a mean of approximately 40%. Neither Ro-11-2933 nor cyclosporin A, at the maximal tolerated doses in combination with vinblastine, led to significant inhibition of KB-8-5 tumour growth compared to treatment with the two vehicles alone. These results show that Ro 44-5912 is an active modulator of drug resistance in vivo.

Animals↗
PubMed1994

Differential E-selectin-dependent adhesion efficiency in sublines of a human colon cancer exhibiting distinct metastatic potentials.

Previously we have shown that high metastatic colonic carcinoma cells express relatively more lamp molecules and sialyl Le(x) structures on the cell surface than their corresponding low metastatic counterparts (Saitoh, O., Wang, W.-L., Lotan, R., and Fukuda, M. (1992) J. Biol. Chem. 267, 5700-5711). In the present study, we extended these findings by testing whether these high and low metastatic colonic carcinoma cells differ in their adhesion efficiency to E-selectin-expressing cells. First, it was found that the high metastatic cells, as compared to their low metastatic counterparts, bind more efficiently to activated human endothelial cells that express E-selectin. This was also true when the adhesion was tested for Chinese hamster ovary cells stably expressing E-selectin. In addition, it was found that the high metastatic cells also adhere more efficiently to mouse endothelioma cells after activation with interleukin-1 beta. It was also shown that the adhesion can be inhibited by soluble lamp-1 or soluble leukosialin that contain sialy Le(x) termini. The inhibition was not, however, observed when these soluble glycoproteins lack sialyl Le(x) structures. The results indicate that the efficiency of the E-selectin-mediated binding of colonic carcinoma cells to human and mouse endothelial cells correlates with the metastatic potential of the cells and suggest that this adhesive event may be one of the critical factors for the metastatic spread of tumor cells. Soluble forms of leukosialin or lamp-1 may be useful as therapeutic agents for the inhibition of E-selectin-mediated binding to tumor cells.

Animals↗
PubMed1994

Carbohydrate structures of recombinant soluble lamp-1 and leukosialin containing sialyl Le(x) terminus.

Recombinant soluble lamp-1 and soluble leukosialin can be produced from CHO cells which express sialyl Le(x) structures after stable transfection of fucosyltransferase-III. It was shown previously that those soluble lamp-1 and leukosialin are potent inhibitors for E-selectin-mediated adhesion of human colonic tumor cells (Sawada, R.; Tsuboi, S.; Fukuda, M. J. Biol. Chem., 1994, 269, 1425). In the present study, we have determined the amount of the sialyl Le(x) structure present in recombinant, soluble lamp-1 and soluble leukosialin. CHO cells were metabolically labeled with [3H]-galactose and recombinant soluble lamp-1 and leukosialin were purified from the spent medium. Glycopeptides containing N-glycans derived from lamp-1 were fractionated by sequential lectin affinity chromatography. Similarly, O-glycans released from leukosialin were fractionated by Bio-Gel P-4 gel filtration. The terminal structures of carbohydrate chains were determined by sequential digestion with specific glycosidases. The results clearly indicate that soluble lamp-1 contains much more sialyl Le(x) structure than soluble leukosialin. Considering that soluble leukosialin and lamp-1 are almost equally effective as inhibitors for E-selectin-mediated adhesion, the results strongly suggest that densely clustered O-glycans are better presenters for E-selectin ligands than N-glycans.

Animals↗
PubMed1994

Two isolectins from leaves of winged bean, Psophocarpus tetragonolobus (L.) DC.

Two isolectins were isolated from leaves of winged bean and characterized. They differed from each other in terms of their immunological properties, hemagglutinating activities, sugar inhibition patterns, and amino acid compositions. Both lectins were acidic and one of them (L-I), which was inactive toward trypsinized human type O erythrocytes, was similar to one of green shell lectins (WGS-1); which resembled basic seed lectin in its immunological properties. The amino-terminal sequence of L-I was homologous to that of WGS-1. The amino acid composition of L-I was similar to that of basic seed lectin, but the extent of the homology between amino-terminal sequences was low when L-I and basic seed lectin were compared. Examination by ELISA revealed that L-I and WGS-1 were distinct from the basic lectins of seeds and tuberous roots. L-I had a disulfide bridge between two subunits and it exhibited high hemagglutinating activity toward human type A erythrocytes, as compared to its activity toward other erythrocytes. By contrast, the properties of a second acidic lectin from winged bean leaves (L-II) were very similar to those of acidic lectins from seeds and tuberous roots, and the similarities extended as far as the immunological properties.

Amino Acid Sequence↗
PubMed1993

E-selectin-dependent adhesion efficiency of colonic carcinoma cells is increased by genetic manipulation of their cell surface lysosomal membrane glycoprotein-1 expression levels.

Lysosomal membrane glycoprotein (lamp)-1 and lamp-2 are the most abundant glycoproteins within the lysosomal membrane. A small amount of lamp-1 and lamp-2 molecules, however, can be present on the cell surface. We have shown previously that highly metastatic colonic carcinoma L4 cells express more lamp-1 and lamp-2 on the cell surface than low metastatic SP cells (Saitoh, O., Wang, W.-L., Lotan, R., and Fukuda, M. (1992) J. Biol. Chem. 267, 5700-5711). Since lamp-1 and lamp-2 are the major carriers for poly-N-acetyllactosamines that are able to display sialyl-Le(x) termini, we sought to determine if an increased amount of lamp-1 on the cell surface would lead to increased expression of cell surface sialyl-Le(x) determinants and to the increased adhesion of those cells to E-selectin. Expression of increased amounts of lamp-1 on the cell surface was achieved either by overexpression of lamp-1 or by expressing a mutant lamp-1 molecule preferentially at the plasma membrane, rather than in lysosomes. Cells that express variable amounts of cell surface lamp-1 were tested for their adhesion to activated endothelial cells or E-selectin expressing Chinese hamster ovary cells. The results clearly show that the extent of adhesion to E-selectin and cell surface sialyl-Le(x) determinants is proportional to the amount of cell surface lamp-1. Moreover, it was demonstrated that such adhesion can be inhibited by soluble lamp-1 generated from Chinese hamster ovary cells expressing sialyl-Le(x) structures. These results indicate that lamp-1 can efficiently present ligands for E-selectin and at the same time can be a useful reagent for inhibition of E-selectin (and possibly P-selectin)-mediated interaction.

Animals↗
PubMed1993

The genes of major lysosomal membrane glycoproteins, lamp-1 and lamp-2. 5'-flanking sequence of lamp-2 gene and comparison of exon organization in two genes.

Human lysosomal membrane glycoproteins lamp-1 and lamp-2 are the major sialoglycoproteins present in lysosomal membranes. The expression of lamp-2 molecules is uniquely regulated, whereas lamp-1 is constitutively synthesized. In order to investigate the unique expression of lamp-2, and the gene evolution of lamp-1 and lamp-2, we isolated genomic phage clones encoding these glycoproteins. Comparison of the genomic and cDNA sequences revealed that the lamp-2 gene consists of nine exons. The transcriptional start site of the lamp-2 gene was determined by primer extension analysis. In order to locate the transcriptional regulatory region of this gene, various regions of 5'-sequences were tested for promoter activity using chloramphenicol acetyltransferase as a reporter molecule. The results revealed that the 5'-flanking sequence from -172 to -20 base pairs has strong promoter activity. In this sequence, potential SP1 and AP-1 binding sites and CAAT boxes are found. Most notably, the promoter activity is suppressed if the 5' farther upstream KpnI repeat sequence is included in the tested 5'-flanking sequence, thus suggesting that the KpnI repeat sequence may have some regulatory function in the lamp-2 gene expression. Comparison of the exon organization of human lamp-2 and lamp-1 genes, or chicken lamp-1 gene, reveals that these two proteins utilize the same exon phase in corresponding introns. Furthermore, each exon encodes almost identical portions of the proteins. On the other hand, the amino acid sequence of human lamp-1 is more homologous to lamp-1 of other species than it is to human lamp-2. These results indicate that lamp-1 and lamp-2 genes were most likely produced by duplication of a primordial gene, which took place early in evolution.

Amino Acid Sequence↗
PubMed1991

[Superoxide anion (O2-) production by polymorphonuclear leukocytes in insulin dependent diabetes mellitus (IDDM)].

The superoxide anion (O2-) production in polymorphonuclear leukocytes stimulated by phorbol myristate acetate in IDDM and non-insulin dependent diabetes mellitus (NIDDM) was determined by the method of Johnston et al, compared with that of each age matched controls. And the correlation between O2- production and hemoglobin (Hb) A1 and A1c value was investigated. The O2- production in IDDM was 24.4 +/- 7.4 (mean +/- SD, n mol per 4 X 10(5) cells) at 10 min. and 51.4 +/- 8.7 at 30 min., in NIDDM each 31.6 +/- 9.3, 60.2 +/- 14.4, and in controls each 40.5 +/- 4.2, 72.4 +/- 3.1. O2- production in IDDM was significantly lower than that in NIDDM (p less than 0.001 at 10 min. and p less than 0.01 30 min.) and controls (p less than 0.001 at 10 and 30 min.). O2- production at 10 and 30 min. possessed a negative correlation with Hba1 and A1c value (HbA1: p less than 0.01 at 10 min. p less than 0.05 at 30 min., HbA1c: p less than 0.01 at 10 and 30 min.). These findings suggest that impaired O2- production might be one of the factors accounting for depressed bactericidal activity of polymorphonuclear leukocytes in IDDM, and that a protracted hyperglycemia might shed some effect on O2- production.

Adult↗
PubMed1990

Isolation and expression of the full-length cDNA encoding CD59 antigen of human lymphocytes.

To identify the primary structure of CD59 antigen and to elucidate its function, a full-length cDNA clone of CD59 was isolated. The cDNA sequence contained an open reading frame that encodes an 128-amino-acid peptide. The amino-terminal 25 amino acids represented a typical signal peptide sequence and the carboxy-terminal hydrophobic amino acids were characteristic for phosphatidylinositol-anchored proteins. The predicted mature protein sequence showed 35% homology with murine Ly-6C.1 and 31% with Ly-6A.2. The number and the distribution of cysteine residues were conserved, implying that the CD59 represented a human homologue of murine Ly-6. RNA blot hybridization analysis revealed the expression of CD59 mRNA in placental, lung, and pancreatic tissues. The mRNA was not only expressed in T-cell lines but in some of monocytic, myeloid, and B-cell lines. In all of these tissues and cell lines, at least four mRNA species were detected. DNA blot hybridization analysis revealed a rather simple genomic structure, which suggested a single gene as compared with the complex multigene family of murine Ly-6.

Amino Acid Sequence↗