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Biomedical subjects

M Tokushige

Publications and source records attributed to M Tokushige.

At least 19 recordsLinked to original sources
PubMed2001

Clinical importance of preoperative carcinoembryonic antigen and carbohydrate antigen 19-9 levels in gastric cancer.

Although serum carcinoembryonic antigen (CEA) and carbohydrate antigen (CA) 19-9 are commonly measured before surgery for gastric carcinoma, this clinical significance is not fully understood. We evaluated a total of 549 patients with gastric cancer who underwent gastrectomy. Levels of CEA and CA19-9 were measured preoperatively in all patients. We retrospectively analyzed correlations between CEA or CA19-9 and clinicopathologic features, and estimated the prognostic utility of the tumor markers by analyzing clinicopathologic characteristics of the carcinoma as a function of seropositivity or negativity of the antigens in combination or by raising the levels. The positivity rates of CEA (> or =5 ng/mL) and CA19-9 (> or =37 U/mL) were 19.5% and 18%, respectively. Serum CEA and CA19-9 positivity significantly correlated with depth of invasion, hepatic metastasis, and curativity. Forty-nine patients positive for both CEA and CA19-9 had significantly higher frequencies of lymph node metastasis, deeper invasion by the tumor, lower rates of curative resection (p < 0.01), and higher rates of hepatic metastasis (p < 0.05) than 377 patients with normal levels of CEA and CA19-9. Surgical outcomes of patients who were CEA- and CA19-9-positive were poorer than those of patients with normal CEA and CA19-9 levels (p < 0.01). Significant correlation was found between serum CEA and CA19-9 level (p < 0.001, r = 0.24). Doubling the threshold level of serum positivity to 10 ng/mL (CEA) and 74 U/mL (CA19-9) improved the prognostic value of these factors. However, multivariate analysis using Cox's hazards model revealed that only CEA positivity using the doubled threshold value (10 ng/mL) (p = 0.04, hazard ratio = 1.7), nodal involvement (p = 0.01, hazard ratio = 1.9), and depth of invasion (p = 0.02 hazard ratio = 1.5) significantly predicted prognosis. Carcinoembryonic antigen positivity using the doubled threshold level (10 ng/mL) was an important prognostic factor in patients with gastric cancer.

Aged↗
PubMed2000

Cloning and over-expression of thermostable Bacillus sp. YM55-1 aspartase and site-directed mutagenesis for probing a catalytic residue.

A thermostable aspartase gene (aspB) from Bacillus sp. YM55-1 was cloned and the gene sequenced. The aspB gene (1407 bp ORF) encodes a protein with a molecular mass of 51 627 Da, consisting of 468 amino-acid residues. An amino-acid sequence comparison revealed that Bacillus YM55-1 aspartase shared 71% homology with Bacillus subtilis aspartase and 49% with Escherichia coli and Pseudomonas fluorescens aspartases. The E. coli TK237/pUCASPB strain, which was obtained by transforming E. coli TK237 (aspartase-null strain) with a vector plasmid (pUCASPB) containing the cloned aspB gene, produced a large amount of the enzyme corresponding to > 10% of the total soluble protein. The over-expressed recombinant enzyme (native molecular mass: 200 kDa) was purified effectively and rapidly using heat treatment and affinity chromatography. In order to probe the catalytic residues of this enzyme, two conserved amino-acid residues, Lys183 and His134, were individually mutated to alanine. Although the tertiary structure of each mutant was estimated to be the same as that of wild-type aspartase in CD and fluorescence measurements, the Lys183Ala mutant lost its activity completely, whereas His134Ala retained full activity. This finding suggests that Lys183 may be involved in the catalytic activity of this thermostable Bacillus YM55-1 aspartase.

Amino Acid Sequence↗
PubMed2000

Laparoscopic surgery for endometriosis: a long-term follow-up.

OBJECTIVE: To investigate if complete resolution of endometriosis by laparoscopic surgery is beneficial to postoperative fecundity, dysmenorrhea and dyspareunia. DESIGN: An observational comparative study on the outcome of laparoscopic surgery. PATIENTS: Laparoscopically-treated symptomatic women with endometriosis (total n = 236); complete (n = 185) and incomplete (n = 51) surgery groups. MEASUREMENTS: Postoperative fecundity and symptom reduction. RESULTS: With whole populations, no surgical completeness-related difference was observed in cumulative pregnancy rates during the postoperative days 0-400 (cycle fecundity rate = 0.0319). Further accumulation of pregnant cases was followed in the complete surgery group (final cumulative pregnancy rate = 80%), but not in the counterpart group (p = 0.003). The similar result was obtained when only r-AFS classification stages III and IV were compared (p = 0.007). No r-AFS stage-related difference was observed in cumulative pregnancy rates when only patients of complete surgery were selected for comparison. The surgery reduced dysmenorrhea (84.7%) and dyspareunia (80.0%). CONCLUSIONS: Laparoscopic conservative surgery for endometriosis, especially when it is complete, increases fecundity and reduces disease-related symptoms, such as dysmenorrhea and dyspareunia.

Adult↗
PubMed1999

Purification and characterization of thermostable aspartase from Bacillus sp. YM55-1.

A thermostable aspartase was purified from a thermophile Bacillus sp. YM55-1 and characterized in terms of activity and stability. The enzyme was isolated by a 5-min heat treatment at 75 degrees C in the presence of 11% (w/v) ammonium sulfate and 100 mM aspartate, followed by Q-Sepharose anion-exchange and AF-Red Toyopearl chromatographies. The native molecular weight of aspartase determined by gel filtration was about 200,000, and this enzyme was composed of four identical monomers with molecular weights of 51,000 determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Unlike Escherichia coli aspartase, the enzyme was not activated by the presence of magnesium ion at alkaline pH. At the optimum pH, the Km and Vmax were 28.5 mM and 700 units/mg at 30 degrees C and 32.0 mM and 2200 units/mg at 55 degrees C, respectively. The specific activity was four and three times higher than those of E. coli and Pseudomonas fluorescens enzymes at 30 degrees C, respectively. Eighty percent of the activity was retained after a 60-min incubation at 55 degrees C, and the enzyme was also resistant to chemical denaturants; 80% of the initial specific activity was detected in assay mixtures containing 1.0 M guanidine hydrochloride. The purified enzyme shared a high sequence homology in the N-terminal region with aspartases from other organisms.

Amino Acid Sequence↗
PubMed1999

Kinetics of the bioleaching of chalcopyrite concentrate by acidophilic thermophile acidianus brierleyi

The kinetics of leaching of high-grade chalcopyrite (CuFeS(2)) concentrate (38-53 &mgr;m) by the acidophilic thermophilic bacterium, Acidianus brierleyi, was studied at 65 degrees C and pH 1.2 in a batch stirred reactor. The adsorption of A. brierleyi cells between the sulfide surface and solution was attained within the first 20 min of exposure to the mineral, and the equilibrium adsorption data were correlated with the Langmuir isotherm. The bioleaching was markedly accelerated in the presence of A. brierleyi, and greater than 90% leaching of copper in the concentrate was achieved within 10 days. The chalcopyrite leaching with A. brierleyi was found to take place with a direct attack by adsorbed cells on the mineral surface, the chemical leaching of chalcopyrite by ferric iron being insignificant. Rate data collected under a wide variety of operating variables were analyzed to determine kinetic and stoichiometric parameters for the microbial chalcopyrite leaching.

Journal Article↗
PubMed1998

Carcinomatous infiltration into the submucosa as a predictor of lymph node involvement in early gastric cancer.

The clinicopathologic features of 114 patients with resectable early gastric cancer (EGC) invading the submucosa were examined retrospectively with respect to lymph node involvement and the possibility of performing a minimally invasive operation. Patients were divided into node-positive (n = 25) and node-negative (n = 81) groups. Among several pathologic factors, the diameter of the tumor and lymphatic involvement were significantly correlated with nodal involvement. Within the submucosal layer the depth of invasion and the horizontal cancerous expansion also correlated with lymph node disease (p < 0.05). The size of the tumor did not correlate with the length of submucosal infiltration (r = 0.12, p = 0.1). Patients with both slight invasion into the submucosa and less than 5 mm of horizontal expansion were often negative for lymph node involvement and thus may benefit from local surgery as an alternative to gastrectomy.

Adenocarcinoma↗
PubMed1995

Minimal deviation adenocarcinoma of endometrioid type may arise in the isthmus: clinicopathological and immunohistochemical study of two cases.

Clinicopathological and immunohistochemical features of minimal deviation adenocarcinoma (MDA) of endometrioid type which may have arisen in the isthmus are presented. Hysterectomy specimens from two women, one 39 years of age and the other 64 years of age, showed the presence of endometrioid-type adenocarcinoma with minimal atypia and without back-to-back arrangement of the glands, which was consistent with a histological diagnosis of endometrioid MDA. However, the tumorous glands were most abundant in the isthmus, and extensively infiltrated into the cervical stroma, vagina, parametrium, and corpus in one case, and the tumor was confined to the isthmus in the other. Immunohistochemically, the tumorous glands were CEA-positive and vimentin-negative in both cases. p53 protein expression was found in one case. Histogenetic origin of MDA of endometrioid type is discussed.

Adult↗
PubMed1995

A case of laparoscopic complication: injury of the left common iliac vessels and subsequent acute compartment syndrome of the left leg.

A 45-year-old Japanese woman underwent a laparoscopy-assisted vaginal hysterectomy. Insertion of a trocar injured the left common iliac artery and vein, which were repaired within 2.5 hours. Postoperatively the patient presented an acute compartment syndrome of the left leg. Fasciotomy and rehabilitation rescued her from functional disturbances.

Acute Disease↗
PubMed1992

Determination of the subunit contact region of aspartase.

The subunits of tetrameric enzyme aspartase from Escherichia coli and Pseudomonas fluorescens were incapable of forming hybrid tetramers, suggesting that the subunit contact regions of these two enzymes are not conserved in spite of significant homology between the total sequences of the enzymes. To locate the subunit contact region, we modified cysteine residues of the intermediate species formed during the assembly of the subunits of aspartase from E. coli. Subunits modified with N-ethylmaleimide were unable to assemble into tetramers. Further experiments showed that Cys-88 was the primary residue that was modified. The sequence flanking Cys-88 is quite different from that in the the enzyme from P. fluorescens, suggesting that this region participates in the mutual recognition of subunits.

Amino Acid Sequence↗
PubMed1992

Cloning and overproduction of biodegradative threonine deaminase from Escherichia coli W strain.

We have cloned the structural gene (tdcB) of biodegradative threonine deaminase from Escherichia coli W strain by utilizing the polymerase chain reaction. The JM109/pUCTDA strain, which was obtained by transforming E. coli JM109 with a vector plasmid (pUCTDA) containing the cloned tdcB gene, produced a large amount of the enzyme corresponding to more than 5% of the total soluble protein. Amino acid sequence analysis of this recombinant enzyme showed that the amino acid sequence is identical to the nucleotide-deduced sequence of biodegradative threonine deaminase from E. coli K-12.

Amino Acid Sequence↗
PubMed1991

Preliminary X-ray crystallographic analysis of tryptophanase from Escherichia coli.

Tryptophanase (L-tryptophan indole-lyase) from Escherichia coli has been crystallized from ammonium sulfate solution using a vapor diffusion method. The crystals are tetragonal and belong to space group P4(1)2(1)2 or its enantiomorph. The cell dimensions of the crystals are a = b = 113.4 A, and c = 232.2 A, with two subunits per asymmetric unit. The crystals diffract to at least 3 A resolution, and are suitable for X-ray structural analysis.

Ammonium Sulfate↗
PubMed1991

Activation of aspartase by site-directed mutagenesis.

To elucidate the role of sulfhydryl groups in the enzymatic reaction of the aspartase from Escherichia coli, we used site-directed mutagenesis which showed that the enzyme was activated by replacement of Cys-430 with a tryptophan. This mutation produced functional alterations without appreciable structural change: The kcat values became 3-fold at pH 6.0; the Hill coefficient values became higher under both pH conditions; the dependence of enzyme activity on divalent metal ions increased; and hydroxylamine, a good substrate for the wild-type enzyme, proved a poor substrate for the mutant.

Amino Acid Sequence↗
PubMed1991

Rapid purification and characterization of homoserine dehydrogenase from Saccharomyces cerevisiae.

Homoserine dehydrogenase of Saccharomyces cerevisiae has been rapidly purified to homogeneity by heat and acid treatments, ammonium sulfate fractionation, and chromatography on Matrex Gel Red A and Q-Sepharose columns. The final preparation migrated as a single entity upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a Mr of 40,000. The Mr of the native enzyme was 81,000 as determined by gel filtration, suggesting that the enzyme is composed of two identical subunits. This feature was also confirmed by cross-linking analysis using the bifunctional reagent dimethyl suberimidate. Feedback inhibition by L-methionine and L-threonine was observed using the purified enzyme. The enzyme was markedly stabilized against heat treatment at high salt concentrations. Additions of feedback inhibitors or high concentrations of salts failed to cause any dissociation or aggregation of the enzyme subunits unlike enzymes from other sources such as Rhodospirillum rubrum. The enzyme denatured in 3 M guanidine-HCl was refolded by simple dilution with a concomitant restoration of the activity. Cross-linking analysis of the renaturation process suggested that the formation of the dimer is required for activity expression. Amino acid sequence analysis of peptides obtained by digestion of the enzyme protein with Achromobacter lyticus protease I revealed that several amino acid residues are strictly conserved among homoserine dehydrogenases from S. cerevisiae, Escherichia coli, and Bacillus subtilis.

Amino Acid Sequence↗
PubMed1991

Purification and characterization of two types of fumarase from Escherichia coli.

Two distinct types of fumarase were purified to homogeneity from aerobically grown Escherichia coli W cells. The amino acid sequences of their NH2-terminals suggest that the two enzymes are the products of the fumA gene (FUMA) and fumC gene (FUMC), respectively. FUMA was separated from FUMC by chromatography on a Q-Sepharose column, and was further purified to homogeneity on Alkyl-Superose, Mono Q, and Superose 12 columns. FUMA is a dimer composed of identical subunits (Mr = 60,000). Although the activity of FUMA rapidly decreased during storage, reactivation was attained by anaerobic incubation with Fe2+ and thiols. Studies on the inactivation and reactivation of FUMA suggested that oxidation and the concomitant release of iron inactivated the enzyme in a reversible manner. While the inactivated FUMA was EPR-detectable, through a signal with g perpendicular = 2.02 and g = 2.00, the active enzyme was EPR-silent. These results suggested FUMA is a member of the 4Fe-4S hydratases represented by aconitase. After the separation of FUMC from FUMA, purification of the former enzyme was accomplished by chromatography on Phenyl-Superose and Matrex Gel Red A columns. FUMC was stable, Fe-independent and quite similar to mammalian fumarases in enzymatic properties.

Amino Acid Sequence↗
PubMed1991

Identification of lysyl residues at the AMP-binding site of biodegradative threonine deaminase from Escherichia coli.

The biodegradative threonine deaminase from Escherichia coli is activated allosterically by AMP. To identify the residues interacting with the phosphate group of AMP at the binding site, we used the affinity labeling reagent, adenosine diphosphopyridoxal (AP2-PL). In the absence of AMP, the enzyme formed the Schiff base with AP2-PL and Scatchard plot analysis showed a biphasic pattern, the respective Kd values for the high- and low-affinity binding phases being 20 and 110 microM. The former value is comparable to the Kd value of the enzyme for AMP. In the presence of AMP, the Schiff base formation was greatly reduced. Although the maximal activating effect of adenosine diphosphopyridoxine, a non-reactive derivative of AP2-PL, was about 13% of that of AMP, the half-saturation concentration was almost the same. These findings suggest that AP2-PL specifically labeled the lysyl residue(s) at the AMP-binding site of the enzyme. To identify the labeled residue(s), we reduced the modified enzyme with sodium borohydride, then cleaved it with cyanogen bromide and Achromobacter lyticus protease I. Reverse-phase HPLC was used to isolate two labeled peptides from the digest. Their amino acid compositions and sequences showed that Lys-111 and Lys-113 were labeled. We conclude that these two lysyl residues are located around the phosphate group of AMP at the allosteric regulation site of the enzyme.

Adenosine Diphosphate↗
PubMed1990

Role of tryptophan 248 in the active site of tryptophanase from Escherichia coli.

Tryptophan 248, located in the active site of tryptophanase from Escherichia coli, has been replaced with phenylalanine by site-directed mutagenesis. Judging from CD and fluorescence spectra, the global structure of the mutant enzyme was found to be the same as that of the wild-type enzyme. The binding affinity of the mutant enzyme for the coenzyme pyridoxal 5'-phosphate (PLP) was reduced tenfold compared to the wild-type enzyme. Kinetic analyses under PLP-saturated conditions indicated that the Km values of the mutant enzyme for substrates are the same as those of wild-type enzyme but the kcat values are decreased to about 85%, which accounts for the overall activity decrease. These findings suggest that tryptophan 248 interacts closely with PLP and plays an important role in the catalytic reaction.

Animals↗
PubMed1990

Studies on T-lineage cells in human decidua of first trimester pregnancies.

T-lineage cells in human decidua of early pregnancies were tested for surface markers, proliferative response, interleukin-2 (IL-2) production, and natural killer (NK) activity. T-lineage (CD2+) cells that were obtained from decidua by the use of E-rosette formation contained fewer CD3+ mature T cells and CD4+ cells than those from the peripheral blood of the same donors, while no differences were seen in the frequencies of CD8+ cells. P55 molecules of IL-2 receptor (IL-2R/p55, Tac antigen) were hardly detected on fresh decidual T-lineage cells, though approximately 20% were positive for HLA-DR. More than a half of decidual T-lineage cells expressed CD56 molecules on their surface and killed K562 cells, the prototype target of NK cells, while most of them were negative for CD16 and CD57. Upon stimulation with IL-2, decidual T-lineage cells demonstrated dose-dependent proliferative response. In addition, they were induced to produce high amounts of IL-2 by stimulation with mitogens but not with alloantigens. These results suggest that human decidua contains high numbers of CD2+3-CD16 +/- 56+ lymphocytes and that this population responds to IL-2, produces IL-2 and mediates NK activity.

Decidua↗