Biomedical subjects

S Karita

Publications and source records attributed to S Karita.

At least 19 recordsLinked to original sources

PubMed2006

[Surgical treatment for clinical stage II and III small cell lung cancer patients].

To evaluate a surgical resection in multidisciplinary treatment for clinical stage (c-stage) II and III small cell lung cancer (SCLC) patients, we examined the postoperative prognosis concerning in 18 patients who underwent complete surgical resection. Five-year survival rate of 8 c-stage II and 10 c-stage III patients showed 62.5% and 46.7%, respectively. Of these, 7 patients underwent preoperative induction chemotherapy resulting in down-staging to c-stage 0-I, and their survival rate at 5 years after sugery was as good as 68.6%. However, in 4 (57.1%) of 7 patients, tumor cells proved to be alive by pathological assessment of lymph nodes. As a consequence, it was suggested that surgery for c-stage II and III SCLC patients is a meaningful treatment considering their prognoses. In particular, even though the patients show a favorable response of preoperative chemotherapy, to completely eliminate remnant tumor cells, a surgical resection may be necessary.

Antineoplastic Combined Chemotherapy Protocols↗

PubMed2003

[Prevention of parenchymal air leakage after lung resection; comparison of effectiveness in drug formation of fibrin adhesive].

We investigated the comparative effectiveness of the seat and glue types of fibrin adhesive, to clarify which is more useful in preventing postoperative parenchymal air leaks after lung resection. One hundred sixteen patients received fibrin adhesive to prevent postoperative air leakage after lung resection carried out by the same surgeon. Ninety-two lobectomies and 24 partial resections were assessed. There were 29 patients with emphysematous lung. In the seat type group, an average of 2.6 postoperative days elapsed before chest drain removal. In the glue type, this average was 3.2 days, a significant difference. This difference was also evident in the lobectomy group. However, among emphysematous lung patients and the partial resection group, there was no apparent difference. Prolonged air leakage was seen in 2 patients with emphysematous lung, but no difference in terms of drug formation could be seen. The seated type of fibrin adhesive was more useful than the glue type in preventing postoperative air leaks, but in emphysematous lung patients, better surgical technique would seem to be the critical factor.

Aged↗

PubMed2001

Sequence of celQ and properties of celQ, a component of the Clostridium thermocellum cellulosome.

The nucleotide sequence of the Clostridium thermocellum F1 celQ gene, which codes for the endoglucanase CelQ, consists of 2,130 bp encoding 710 amino acids. The precursor form of CelQ has a molecular weight of 79,809 and is composed of a signal peptide, a family 9 cellulase domain, a family IIIc carbohydrate-binding module (CBM), and a dockerin domain. Truncated derivatives of CelQ were constructed: CelQdeltadoc consisted of the catalytic domain and the CBM; CelQcat consisted of the catalytic domain only. CelQdeltadoc showed strong activity toward carboxymethylcellulose (CMC) and barley beta-glucan and low activity toward Avicel, acid-swollen cellulose, lichenan, and xylan. The Vmax and Km values were 235 micromol/min/mg and 3.3 mg/ml, respectively, for CMC. By contrast, CelQcat, which was devoid of the CBM, showed negligible activity toward CMC, i.e., about 1/1,000 of the activity of CelQdeltadoc, supporting the previously proposed idea that family IIIc CBMs participate in the catalytic function of the enzyme. Immunological analysis using an antiserum raised against CelQdeltadoc confirmed that CelQ is a component of the C. thermocellum cellulosome.

Amino Acid Sequence↗

PubMed2001

Conversion of chitinous wastes to hydrogen gas by Clostridium paraputrificum M-21.

The chitinolytic bacterium Clostridium paraputrificum strain M-21 produced 2.2 and 1.5 mol hydrogen gas from 1 mol N-acetyl-D-glucosamine (GlcNAc) and ball-milled chitin equivalent to 1 mol of GlcNAc, respectively, at pH 6.0. In addition, strain M-21 efficiently degraded and fermented ball-milled raw shrimp and lobster shells to produce hydrogen gas: 11.4 mmol H2 from 2.6 g of the former and 7.8 mmol H2 from 1.5 g of the latter. Hydrogen evolution from these shell wastes were enhanced two fold by employing acid and alkali pretreatment. Waste from the starch industry was also converted to hydrogen. When C. paraputrificum M-21 was cultivated on ball-milled chitin and ball-milled shrimp shells for 14 and 12 h, respectively, chitinases ChiA and/or ChiB were detected as the major chitinase species in the supernatant of the cultures, suggesting that the play a critical role in the degradation of chitinous materials.

Journal Article↗

PubMed2001

Characterization of Clostridium paraputrificum chitinase A from a recombinant Escherichia coli.

Clostridium paraputrificum chitinase A (ChiA) was purified from a recombinant Escherichia coli. ChiA was active toward chitin from crab shells, colloidal chitin, glycol chitin, and 4-methylumbelliferyl beta-D-N,N'-diacetylchitobioside [4-MU-(GlcNAc)2]. ChiA showed maximum activity at pH 6.0 and its optimum temperature was 45 degrees C. ChiA was stable between pH 6.0 and 9.0 and at temperatures up to 40 degrees C. The K(m) and V(max) values of ChiA for 4-MU-(GlcNAc)2 were estimated to be 6.9 microM and 43 micromol/min/mg, respectively. Thin-layer chromatography indicated that ChiA hydrolyzes chitooligosaccharides to mainly chitobiose. ChiA was found to adsorb not only chitinous polymers but also cellulosic polymers.

Journal Article↗

PubMed2001

A cryptic plasmid, pAO1, from a compost bacterium, Bacillus sp.

The complete nucleotide sequence of a new cryptic plasmid, pAO1 isolated from a compost bacterium Bacillus sp., has been analyzed. Analysis of the PCR-based 16S rRNA sequence showed the bacterium harboring pAO1 was closely related to Bacillus pallidus. The plasmid pAO1 was 3,325 bp in size. Two open reading frames, ORF1 and ORF2, encoding putative polypeptides of 248 and 290 amino acids, respectively, were identified within the sequence. The ORF1 has a limited sequence similarity to an integrase/recombinase, while the ORF2 has high similarity with the replication protein of pBC1 from Bacillus coagulans. A putative origin sequence for a plus-strand was located between ORFs. Southern blot analysis indicates this plasmid replicates via a rolling circle-type mechanism.

Bacillus↗

PubMed2001

Importance of the carbohydrate-binding module of Clostridium stercorarium Xyn10B to xylan hydrolysis.

The Clostridium stercorarium xylanase Xyn10B is a modular enzyme comprising two thermostabilizing domains, a family 10 catalytic domain of glycosyl hydrolases, a family 9 carbohydrate-binding module (CBM), and two S-layer homologous (SLH) domains [Biosci. Biotechnol. Biochem., 63, 1596-1604 (1999)]. To investigate the role of this CBM, we constructed two derivatives of Xyn10B and compared their hydrolytic activity toward xylan and some preparations of plant cell walls; Xyn10BdeltaCBM consists of a catalytic domain only, and Xyn10B-CBM comprises a catalytic domain and a CBM. Xyn10B-CBM bound to various insoluble polysaccharides including Avicel, acid-swollen cellulose, ball-milled chitin, Sephadex G-25, and amylose-resin. A cellulose binding assay in the presence of soluble saccharides suggested that the CBM of Xyn10B had an affinity for even monosaccharides such as glucose, galactose, xylose, mannose and ribose. Removal of the CBM from the enzyme negated its cellulose- and xylan-binding abilities and severely reduced its enzyme activity toward insoluble xylan and plant cell walls but not soluble xylan. These findings clearly indicated that the CBM of Xyn10B is important in the hydrolysis of insoluble xylan. This is the first report of a family 9 CBM with an affinity for insoluble xylan in addition to crystalline cellulose and the ability to increase hydrolytic activity toward insoluble xylan.

Catalysis↗

PubMed2001

Sequence of the Clostridium thermocellum mannanase gene man26B and characterization of the translated product.

The man26B gene of Clostridium thermocellum strain F1 was found in pKS305, which had been selected as a recombinant plasmid conferring endoglucanase activity on Escherichia coli. The open reading frame of man26B consists of 1,773 nucleotides encoding a protein of 591 amino acids with a predicted molecular weight of 67,047. Man26B is a modular enzyme composed of an N-terminal signal peptide and three domains in the following order: a mannan-binding domain, a family 26 mannanase domain, and a dockerin domain responsible for cellulosome assembly. We found that this gene was a homologue of the man26A gene of C. thermocellum strain YS but that there were insertion or deletion mutations that caused a frame-shift mutation affecting a stretch of 26 amino acids in the catalytic domain. Man26B devoid of the dockerin domain was constructed and purified from a recombinant E. coli, and its enzyme properties were examined. Immunological analysis indicated that Man26B was a catalytic component of the C. thermocellum F1 cellulosome.

Amino Acid Sequence↗

PubMed2000

Identification and characterization of Clostridium paraputrificum M-21, a chitinolytic, mesophilic and hydrogen-producing bacterium.

A strictly anaerobic, mesophilic and chitinolytic bacterial strain, M-21, was isolated from a soil sample collected from Mie University campus and identified as Clostridium paraputrificum based on morphological and physiological characteristics, and 16S rRNA sequence analysis. C. paraputrificum M-21 utilized chitin and N-acetyl-D-glucosamine (GlcNAc), a constituent monosaccharide of chitin, to produce a large amount of gas along with acetic acid and propionic acid as major fermentation products. Hydrogen and carbon dioxide accounted for 65% and 35% of the gas evolved, respectively. The conditions for 1 l batch culture of C. paraputrificum, including pH of the medium, incubation temperature and agitation speed, were optimized for hydrogen production with GlcNAc as the carbon source. The bacterium grew rapidly on GlcNAc with a doubling time of around 30 min, and produced hydrogen gas with a yield of 1.9 mol H2/mol GlcNAc under the following cultivation conditions: initial medium pH of 6.5, incubation temperature of 45 degrees C, agitation speed of 250 rpm, and working volume of 50% of the fermentor. The dry cell weight harvested from this culture was 2.0 g/l.

Journal Article↗

PubMed2000

Characterization of the binding of spike H protein of bacteriophage phiX174 with receptor lipopolysaccharides.

The spike H protein of bacteriophage phiX174 was prepared as a hexa histidine-tagged fusion (HisH). On enzyme-linked plate assaying, HisH was found to bind specifically to the lipopolysaccharides (LPSs) of phiX174-sensitive strains, Escherichia coli C and Salmonella typhimurium Ra chemotype, having the complete oligosaccharide sequence of the R-core on the LPSs. In sharp contrast, HisH bound weakly to the LPSs of phiX174-insensitive strains, i.e. E. coli F583 (Rd(2)) lacking some terminal saccharides and E. coli O111: B4 (smooth strain) having additional O-repeats on the R-core. The fluorescence spectra of HisH changed dose-dependently in the case of the LPS of E. coli C, the intensity increasing and the emission peak shifting to the shorter wavelength side, which was attributable to the hydrophobic interaction of HisH with the LPS. The binding equilibrium was analyzed by fluorometric titration to determine the dissociation constant K(d), 7.02 +/- 0.37 microM, and the Gibbs free energy change DeltaG(0), -29.1 kJ mol(-1) (at 22 degrees C, pH 7.4). Based on the temperature dependence of (K)d in a van't Hoff plot, the standard enthalpy change DeltaH(0) and the entropy change DeltaS(0) were calculated to be +23.7 kJ mol(-1) and 179 J mol(-1) K(-1) at 22 degrees C, respectively, and this binding was thereby concluded to be an entropy-driven reaction.

Bacteriophage phi X 174↗

PubMed2000

A large gene cluster for the Clostridium cellulovorans cellulosome.

A large gene cluster for the Clostridium cellulovorans cellulosome has been cloned and sequenced upstream and downstream of the cbpA and exgS genes (C.-C. Liu and R. H. Doi, Gene 211:39-47, 1998). Gene walking revealed that the engL gene cluster (Y. Tamaru and R. H. Doi, J. Bacteriol. 182:244-247, 2000) was located downstream of the cbpA-exgS genes. Further DNA sequencing revealed that this cluster contains the genes for the scaffolding protein CbpA, the exoglucanase ExgS, several endoglucanases of family 9, the mannanase ManA, and the hydrophobic protein HbpA containing a surface layer homology domain and a hydrophobic (or cohesin) domain. The sequence of the clustered genes is cbpA-exgS-engH-engK-hbpA-engL-man A-engM-engN and is about 22 kb in length. The engN gene did not have a complete catalytic domain, indicating that engN is a truncated gene. This large gene cluster is flanked at the 5' end by a putative noncellulosomal operon consisting of nifV-orf1-sigX-regA and at the 3' end by noncellulosomal genes with homology to transposase (trp) and malate permease (mle). Since gene clusters for the cellulosome are also found in C. cellulolyticum and C. josui, they seem to be typical of mesophilic clostridia, indicating that the large gene clusters may arise from a common ancestor with some evolutionary modifications.

Amino Acid Sequence↗

PubMed2000

Purification, characterization, and molecular cloning of acidophilic xylanase from penicillium sp.40.

Penicillum sp. 40, which can grow in an extremely acidic medium at pH 2.0 was screened from an acidic soil. This fungus produces xylanases when grown in a medium containing xylan as a sole carbon source. A major xylanase was purified from the culture supernatant of Penicillium sp. 40 and designated XynA. The molecular mass of XynA was estimated to be 25,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. XynA has an optimum pH at 2.0 and is stable in pH 2.0-5.0. Western blot analysis using anit-XynA antibody showed that XynA was induced by xylan and repressed by glucose. Also, its production was increased by an acidic medium. The gene encoding XynA (xynA) was isolated from the genomic library of Penicillium sp. 40. The structural part of xynA was found to be 721 bp. The nucleotide sequence of cDNA amplified by RT-PCR showed that the open reading frame of xynA was interrupted by a single intron which was 58 bp in size and encoded 221 amino acids. Direct N-terminal amino acid sequencing showed that the precursor of XynA had a signal peptide composed of 31 amino acids. The molecular mass caliculated from the deduced amino acid sequence of XynA is 20,713. This is lower than that estimated by gel electrophoresis, suggesting that XynA is a glycoprotein. The predicted amino acid sequence of XynA has strong similarity to other family xylanases from fungi.

Amino Acid Sequence↗

PubMed2000

Recognition of receptor lipopolysaccharides by spike G protein of bacteriophage phiX174.

The spike G protein of bacteriophage phiX174 was prepared as a hexa histidine-tagged G protein (HisG). In the enzyme-linked plate assay, HisG bound specifically to lipopolysaccharides (LPSs) of the phiX174-sensitive strains, and did not bind to LPSs of the phiX174-insensitive strains. The truncated G protein obtained after trypsin digestion of HisG had the similar affinity to the LPSs to HisG, indicating that eight amino acid residues from the N-terminus are not essential to the binding with the LPSs.

Bacteriophage phi X 174↗

PubMed2000

Characterization of the cellulolytic complex (cellulosome) from Ruminococcus albus.

The cellulolytic complex was isolated from the culture supernatant of Ruminococcus albus strain F-40 grown on cellulose by a Sephacryl S-300HR column chromatography. The molecular mass of the cellulolytic complex was found to be larger than 1.5 x 10(6) Da. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis indicated that the cellulolytic complex contained at least 15 proteins with molecular weights from 40kDa to 250 kDa. Among them, 11 proteins showed endoglucanase and/or xylanase activities on the zymograms. Immunological analysis using an antiserum raised against the dockerin domain of endoglucanase VII of R. albus (DocVII) suggested that at least 7 proteins in the cellulolytic complex contained a dockerin domain immunoreactive with the anti-Doc-VII antiserum. Furthermore, DocVII was shown to specifically interact with a 40-kDa protein of the cellulolytic complex by Far-Western blot analysis. These results strongly suggest that the cellulolytic complex produced by R. albus resembles the cellulosome specified for the cellulolytic complex of several clostridia such as Clostridium thermocellum and respective components are assembled into the cellulosome by the mechanism common in all of the cellulolytic clostridia, i.e., the cellulosome is formed by the interaction between a dockerin domain of catalytic components and a cohesin domain of a scaffolding protein.

Amino Acid Sequence↗

PubMed2000

Cloning, sequencing, and expression of the gene encoding a cell-bound multi-domain xylanase from Clostridium josui, and characterization of the translated product.

The nucleotide sequence of the Clostridium josui FERM P-9684 xyn10A gene, encoding a xylanase Xyn10A, consists of 3,150 bp and encodes 1,050 amino acids with a molecular weight of 115,564. Xyn10A is a multidomain enzyme composed of an N-terminal signal peptide and six domains in the following order: two thermostabilizing domains, a family 10 xylanase domain, a family 9 carbohydrate-binding module (CBM), and two S-layer homologous (SLH) domains. Immunological analysis indicated the presence of Xyn10A in the culture supernatant of C. josui FERM P-9684 and on the cell surface. The full-length Xyn10A expressed in a recombinant Escherichia coli strain bound to ball-milled cellulose (BMC) and the cell wall fragments of C. josui, indicating that both the CBM and the SLH domains are fully functional in the recombinant enzyme. An 85-kDa xylanase species derived from Xyn10A by partial proteolysis at the C-terminal side, most likely at the internal region of the CBM, retained the ability to bind to BMC. This observation suggests that the catalytic domain or the thermostabilizing domains are responsible for binding of the enzyme to BMC. Xyn10A-II, the 100-kDa derivative of Xyn10A, was purified from the recombinant E. coli strain and characterized. The enzyme was highly active toward xylan but not toward p-nitrophenyl-beta-D-xylopyranoside, p-nitrophenyl-beta-D-cellobioside, or carboxymethylcellulose.

Amino Acid Sequence↗

PubMed2000

Molecular cloning, overexpression, and purification of a major xylanase from Aspergillus oryzae.

The gene encoding xylanase G2 (xynG2) was isolated from a genomic library of Aspergillus oryzae KBN616, used for making shoyu koji. The structural part of xynG2 was found to be 767 bp. The nucleotide sequence of cDNA amplified by RT-PCR showed that the open reading frame of xynG2 was interrupted by a single intron which was 71 bp in size and encoded 232 amino acids. Direct N-terminal amino acid sequencing showed that the precursor of XynG2 had a signal peptide of 44 amino acids. The predicted amino acid sequence of XynG2 has strong similarity to other family 11 xylanases from fungi. The xynG2 gene was successfully overexpressed in A. oryzae and the overpexpressed XynG2 was purified. The molecular weight of XynG2 estimated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 21,000. This was almost the same as the molecular weight of 20,047 calculated from the deduced amino acid sequence. The purified XynG2 showed an optimum activity at pH 6.0 and 58 degrees C. It had a Km of 5.1 mg/ml and a Vmax of 123 micromol/min/mg when birch wood xylan was used as a substrate.

Amino Acid Sequence↗

PubMed2000

Sequence of egV and properties of EgV, a Ruminococcus albus endoglucanase containing a dockerin domain.

The Ruminococcus albus F-40 egV gene, encoding endoglucanase V (EGV), consists of an open reading frame of 1,833 nucleotides and encodes 611 amino acids with a deduced molecular weight of 67,103. The deduced EGV is a modular enzyme composed of a catalytic domain of family 5 of glycosyl hydrolases, a domain of unknown function, and a dockerin domain responsible for cellulosome assembly, suggesting that R. albus F-40 produces a cellulosome, and EGV is a component of the cellulosome. A truncated form of EGV with an apparent molecular weight of 42,000 was purified from a recombinant Escherichia coli and characterized since EGV suffered from partial proteolysis by E. coli protease(s). The truncated EGV was active toward carboxylmethyl cellulose, xylan, lichenan, and acid-swollen cellulose. The pH and temperature optima of the enzyme were 7.0 and 40 degrees C, respectively. By Western blot analysis using the antiserum raised against the truncated enzyme, EGV was detected in the whole cells but not in the culture supernatant of R. alubus F-40, suggesting that EGV was located on the cell surface.

Amino Acid Sequence↗

PubMed2000

Molecular breeding of cellulolytic microbes, plants, and animals for biomass utilization.

Cellulosic materials are the major components of fibrous biomass produced as a result of photosynthesis and are considered as a reservoir of solar energy and organic materials. In order to cope with the problems of food and energy shortages expected in the near future, biotechnologists are encouraged to develop new technologies for the more effective utilization of the world's sustainable resources, i.e., biomass. One way is to engineer microorganisms and animals with the capability of digesting and utilizing cellulosic materials, and plants which can be easily degraded by cellulolytic enzymes. In this article, we summarize recent studies on the molecular breeding of cellulolytic organisms for biomass utilization along with some considerations regarding cellulolytic enzymes.

Journal Article↗