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J Sekiguchi

Publications and source records attributed to J Sekiguchi.

At least 37 records · Page 2Linked to original sources
PubMed1999

Peptidoglycan hydrolase LytF plays a role in cell separation with CwlF during vegetative growth of Bacillus subtilis.

Peptidoglycan hydrolase, LytF (CwlE), was determined to be identical to YhdD (deduced cell wall binding protein) by zymography after insertional inactivation of the yhdD gene. YhdD exhibits high sequence similarity with CwlF (PapQ, LytE) and p60 of Listeria monocytogenes. The N-terminal region of YhdD has a signal sequence followed by five tandem repeated regions containing polyserine residues. The C-terminal region corresponds to the catalytic domain, because a truncated protein without the N-terminal region retained cell wall hydrolase activity. The histidine-tagged LytF protein produced in Escherichia coli cells hydrolyzed the linkage of D-gamma-glutamyl-meso-diaminopimelic acid in murein peptides, indicating that it is a D,L-endopeptidase. Northern hybridization and primer extension analyses indicated that the lytF gene was transcribed by EsigmaD RNA polymerase. Disruption of lytF led to slightly filamentous cells, and a lytF cwlF double mutant exhibited extraordinary microfiber formation, which is similar to the cell morphology of the cwlF sigD mutant.

Amino Acid Sequence↗
PubMed1999

Characterization of a new sigma-K-dependent peptidoglycan hydrolase gene that plays a role in Bacillus subtilis mother cell lysis.

Bacillus subtilis produces a 30-kDa peptidoglycan hydrolase, CwlH, during the late sporulation phase. Disruption of yqeE led to a complete loss of CwlH formation, indicating the identity of yqeE with cwlH. Northern blot analysis of cwlH revealed a 0.8-kb transcript after 6 to 7.5 h for the wild-type strain but not for the sigma(F), sigma(E), sigma(G), and sigma(K) mutants. Expression of the sigma(K)-dependent cwlH gene depended on gerE. Primer extension analysis also suggested that cwlH is transcribed by Esigma(K) RNA polymerase. CwlH produced in Escherichia coli harboring a cwlH plasmid is an N-acetylmuramoyl-L-alanine amidase (EC 3.5.1.28) and exhibited an optimum pH of 7.0 and high-level binding to the B. subtilis cell wall. A cwlC cwlH double mutation led to a lack of mother cell lysis even after 7 days of incubation in DSM medium, but the single mutations led to mother cell lysis after 24 h.

Amino Acid Sequence↗
PubMed1999

Characterization of Bacillus subtilis ExoA protein: a multifunctional DNA-repair enzyme similar to the Escherichia coli exonuclease III.

To discover the physiological role of the Bacillus subtilis ExoA protein, which is similar in amino acid sequence to Escherichia coli exonuclease III, an exoA::Cm disruption was constructed in the chromosomal DNA of B. subtilis. There was no clear difference in tolerance to hydrogen peroxide and alkylating agents between the disruptant and the wild type strain. An expression plasmid of the ExoA in E. coli was constructed by inserting the exoA gene into the expression vector pKP1500. The purified ExoA was used to clarify enzymatic characterizations using synthetic DNA oligomers as substrates. A DNA oligomer containing a 1', 2'-dideoxyribose residue as an AP site, a DNA-RNA chimera oligomer, and a 3' end 32P-labeled oligomer were synthesized. It has been shown that the ExoA has AP endonuclease, 3'-5' exonuclease, ribonuclease H, and 3'-phosphomonoesterase activities. Thus, it has been confirmed that ExoA is a multifunctional DNA-repair enzyme in B. subtilis that is very similar to E. coli exonuclease III except that ExoA has lower 3'-5' exonuclease activity than that of E. coli exonuclease III.

Bacillus subtilis↗
PubMed1998

The gene glvA of Bacillus subtilis 168 encodes a metal-requiring, NAD(H)-dependent 6-phospho-alpha-glucosidase. Assignment to family 4 of the glycosylhydrolase superfamily.

The gene glvA (formerly glv-1) from Bacillus subtilis has been cloned and expressed in Escherichia coli. The purified protein GlvA (449 residues, Mr = 50,513) is a unique 6-phosphoryl-O-alpha-D-glucopyranosyl:phosphoglucohydrolase (6-phospho-alpha-glucosidase) that requires both NAD(H) and divalent metal (Mn2+, Fe2+, Co2+, or Ni2+) for activity. 6-Phospho-alpha-glucosidase (EC 3.2.1.122) from B. subtilis cross-reacts with polyclonal antibody to maltose 6-phosphate hydrolase from Fusobacterium mortiferum, and the two proteins exhibit amino acid sequence identity of 73%. Estimates for the Mr of GlvA determined by SDS-polyacrylamide gel electrophoresis (51,000) and electrospray-mass spectroscopy (50,510) were in excellent agreement with the molecular weight of 50,513 deduced from the amino acid sequence. The sequence of the first 37 residues from the N terminus determined by automated analysis agreed precisely with that predicted by translation of glvA. The chromogenic and fluorogenic substrates, p-nitrophenyl-alpha-D-glucopyranoside 6-phosphate and 4-methylumbelliferyl-alpha-D-glucopyranoside 6-phosphate were used for the discontinuous assay and in situ detection of enzyme activity, respectively. Site-directed mutagenesis shows that three acidic residues, Asp41, Glu111, and Glu359, are required for GlvA activity. Asp41 is located at the C terminus of a betaalphabeta fold that may constitute the dinucleotide binding domain of the protein. Glu111 and Glu359 may function as the catalytic acid (proton donor) and nucleophile (base), respectively, during hydrolysis of 6-phospho-alpha-glucoside substrates including maltose 6-phosphate and trehalose 6-phosphate. In metal-free buffer, GlvA exists as an inactive dimer, but in the presence of Mn2+ ion, these species associate to form the NAD(H)-dependent catalytically active tetramer. By comparative sequence alignment with its homologs, the novel 6-phospho-alpha-glucosidase from B. subtilis can be assigned to the nine-member family 4 of the glycosylhydrolase superfamily.

Amino Acid Sequence↗
PubMed1998

Free flap transfers for the treatment of osteomyelitis of the lower leg.

Since 1974, 31 cases of soft tissue defects associated with osteomyelitis of the lower leg have been treated by free flap transfer and followed up for more than three years. Four patients developed recurrences. Two of them were reoperated on and healed well. One patient was reoperated on three times, but remained infected. One patient developed a fever, but improved after intravenous infusion of antibiotics. Of the eight patients who underwent vascularised muscle transfer to the dead space created by saucerisation of the osteomyelitis, none developed signs of recurrence. By contrast, the four patients who developed recurrences were those in whom the dead space could not be completely filled with the free flap. It therefore seems important that after saucerisation, well-vascularised soft tissue should be used to pack the dead space completely to prevent recurrence of the osteomyelitis.

Adolescent↗
PubMed1998

Blood coagulation activity during microsurgery.

The authors investigated blood coagulation activity in patients who underwent microsurgery. Hemostatic parameters were measured in 9 patients (10 operations) who were undergoing free tissue transfers. These parameters included prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinopeptide A (FPA), prothrombin fragment 1 + 2 (F1 + 2), and thrombin-antithrombin complex (TAT). The flap totally necrosed owing to vasospasm in 1 patient with osteomyelitis of the heel, and the FPA, F1 + 2, and TAT values significantly increased. Reexploration was required because of flap cyanosis in 1 patient with a hemangioma on the wrist, and the F1 + 2 and TAT values increased during the salvage procedure. These molecular markers could be important in indicating hypercoagulable state sensitivity, and they serve as a warning of possible vascular compromise to a surgeon.

Adult↗
PubMed1998

Endoscopic management of subcutaneous gunshot wound with irrigation technique.

The application of the endoscope to the treatment of gunshot wounds has never been reported to our knowledge, with the exception of the laparoscope and the sigmoidscope in abdominal gunshot wounds. We report a patient with a subcutaneous gunshot wound who was treated endoscopically using the irrigation technique. The patient was a 25-year-old man who had been shot in the upper arm. The bullet was found near the thoracic spine via radiograph. At surgery the endoscope was inserted into the wound and irrigation was started. Excellent views could be obtained by irrigation, and the endoscope was advanced along the path of the bullet. The bullet and several fragments were removed with two additional small incisions. The method described herein seems beneficial in that (1) it is less invasive than conventional surgical debridement with a long incision, (2) it may provide information helpful in diagnosing the condition and the location of the bullet and its path, and (3) saline irrigation may have a cleansing effect.

Adult↗
PubMed1998

Regulation and characterization of a newly deduced cell wall hydrolase gene (cwlJ) which affects germination of Bacillus subtilis spores.

The predicted amino acid sequence of Bacillus subtilis ycbQ (renamed cwlJ) exhibits high similarity to those of the deduced C-terminal catalytic domain of SleBs, the specific cortex-hydrolyzing enzyme of B. cereus and the deduced one of B. subtilis. We constructed a cwlJ::lacZ fusion in the B. subtilis chromosome. The beta-galactosidase activity and results of Northern hybridization and primer extension analyses of the cwlJ gene indicated that it is transcribed by EsigmaE RNA polymerase. cwlJ-deficient spores responded to both L-alanine and AGFK, the A580 values of spore suspensions decreased more slowly than in the case of the wild-type strain, and the mutant spores released less dipicolinic acid than did those of the wild-type strain during germination. However, the mutant spores released only slightly less hexosamine than did the wild-type spores. In contrast, B. subtilis sleB spores did not release hexosamine at a significant level. While cwlJ and sleB spores were able to germinate, CJSB (cwlJ sleB) spores could not germinate but exhibited initial germination reactions, e.g., partial decrease in A580 and slow release of dipicolinic acid. CJSB spores became slightly gray after 6 h in the germinant, but their refractility was much greater than that of sleB mutant spores. The roles of the sleB and cwlJ mutations in germination and spore maturation are also discussed.

Alanine↗
PubMed1998

Regulation of a new cell wall hydrolase gene, cwlF, which affects cell separation in Bacillus subtilis.

Bacillus subtilis produces a 35-kDa cell wall hydrolase, CwlF, during vegetative growth. The CwlF protein was extracted from B. subtilis cwlB sigD mutant cells and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. N-terminal amino acid sequencing revealed that its sequence is completely identical to that of the internal region of the papQ gene product. Disruption of the papQ gene in the B. subtilis chromosome led to the complete loss of CwlF, indicating that papQ is identical to cwlF. CwlF exhibits high sequence similarity to the p60 proteins of Listeria species, NlpC proteins of Escherichia coli and Haemophilus influenzae, and Enp2 protein of Bacillus sphaericus. The beta-galactosidase activity of the cwlF-lacZ transcriptional fusion and Northern blot analysis of the cwlF gene indicated that the gene is expressed as a monocistronic operon during the exponential growth phase, and primer extension analysis suggested that the cwlF gene is transcribed mainly by EsigmaA RNA polymerase and weakly by EsigmaH RNA polymerase. While the cells of the cwlF-deficient mutant were about twice as long as those of the wild-type strain, the cwlF sigD double mutant cells exhibited extraordinary microfiber formation, in contrast to the filamentation of the sigD mutant. The CwlF production was not affected by the pleiotropic mutations flaD1 and degU32(Hy), which endow cells with the ability of extensive filamentation.

Amino Acid Sequence↗
PubMed1998

Purification and characterization of triacylglycerol lipase from Aspergillus oryzae.

Triacylglycerol lipase (L3) was purified from Aspergillus oryzae RIB128 by ammonium sulfate fractionation, acetone precipitation, anion-exchange chromatography, and gel filtration. The purified enzyme was formed from a glycoprotein and a monomeric protein with molecular masses of 25 and 29 kDa, by SDS-PAGE and gel filtration, respectively. The optimum pH at 40 degrees C was 5.5 and the optimum temperature at pH 5.5 was 40 degrees C. The enzyme was stable between a pH range of 4.0-7.5 at 30 degrees C for 24 h, and at up to 30 degrees C at pH 5.5 for 1 h. Heavy metal ions, detergents, DFP, and DEP strongly inhibited the enzyme activity. The lipase hydrolyzed not only triacylglycerols but also monoacylglycerols and diacylglycerols. The enzyme had higher specificity toward triacylglycerols of middle-chain saturated fatty acids than short-chain or long-chain fatty acids. The enzyme had 1,3-positional specificity. The N-terminal amino acid sequence of the enzyme was not significantly similar to that of other lipases with published sequences.

Amino Acid Sequence↗
PubMed1997

Intramolecular synapsis of duplex DNA by vaccinia topoisomerase.

Complexes formed by vaccinia topoisomerase I on plasmid DNA were visualized by electron microscopy. The enzyme formed intramolecular loop structures in which non-contiguous DNA segments were synapsed within filamentous protein stems. At high enzyme concentrations the DNA appeared to be zipped up within the protein filaments such that the duplex was folded back on itself. Formation of loops and filaments was also observed with an active site mutant, Topo-Phe274. Binding of Topo-Phe274 to relaxed DNA circles in solution introduced torsional strain, which, after relaxation by catalytic amounts of wild-type topo-isomerase, resulted in acquisition of negative supercoils. We surmise that the topoisomerase-DNA complex is a plectonemic supercoil in which the two duplexes encompassed by the protein filaments are interwound in a right handed helix. We suggest that topoisomerase-mediated DNA synapsis plays a role in viral recombination and in packaging of the 200 kbp vaccinia genome during virus assembly.

Binding Sites↗
PubMed1997

Mutational analysis of vaccinia virus topoisomerase identifies residues involved in DNA binding.

Vaccinia DNA topoisomerase catalyzes the cleavage and re-joining of DNA strands through a DNA-(3'-phosphotyrosyl)-enzyme intermediate formed at a specific target sequence, 5'-(C/T)CCTT downward arrow. The 314 aa protein consists of three protease-resistant structural domains demarcated by protease-sensitive interdomain segments referred to as the bridge and the hinge. The bridge is defined by trypsin-accessible sites at Arg80, Lys83 and Arg84. Photocrosslinking and proteolytic footprinting experiments suggest that residues near the interdomain bridge interact with DNA. To assess the contributions of specific amino acids to DNA binding and transesterification chemistry, we introduced alanine substitutions at 16 positions within a 24 aa segment from residues 63 to 86(DSKGRRQYFYGKMHVQNRNAKRDR). Assays of the rates of DNA relaxation under conditions optimal for the wild-type topoisomerase revealed significant mutational effects at six positions; Arg67, Tyr70, Tyr72, Arg80, Arg84 and Asp85. The mutated proteins displayed normal or near-normal rates of single-turnover transesterification to DNA. The effects of amino acid substitutions on DNA binding were evinced by inhibition of covalent adduct formation in the presence of salt and magnesium. The mutant enzymes also displayed diminished affinity for a subset of cleavage sites in pUC19 DNA. Tyr70 and Tyr72 were subjected to further analysis by replacement with Phe, His, Gln and Arg. At both positions, the aromatic moiety was important for DNA binding.

Amino Acid Sequence↗
PubMed1997

Novobiocin inhibits vaccinia virus replication by blocking virus assembly.

Novobiocin inhibits the replication of vaccinia virus in cultured BSC40 cells. All classes of viral proteins were synthesized during synchronous infection in the presence of drug. The onset of DNA replication was delayed slightly, yet the extent of DNA replication in the presence of novobiocin was comparable to that of a control infection. A delay in the temporal transition to late viral protein synthesis was in keeping with the effects on DNA replication. Although the precursor forms of the major viral structural proteins were synthesized normally at late times, the proteolytic processing of these polypeptides was inhibited, which suggested an impediment to virus assembly. Electron microscopy revealed that novobiocin blocked virus morphogenesis at an early stage. Conversion of the concatemeric DNA replication intermediates into hairpin telomeres occurred in the presence of novobiocin, confirming that telomere resolution was not coupled to virus assembly. Novobiocin is the latest addition to a class of antipoxviral agents, which includes rifampin and IMCBH, that arrest morphogenesis.

Animals↗
PubMed1997

Cloning and sequencing of a 35.7 kb in the 70 degree-73 degree region of the Bacillus subtilis genome reveal genes for a new two-component system, three spore germination proteins, an iron uptake system and a general stress response protein.

In the framework of the international project aimed at sequencing the Bacillus subtilis (Bs) genome, a 35.7-kb chromosome segment around the pel locus has been cloned and sequenced. This region (35,745 bp; 70 degrees-73 degrees of the genetic map) contains two partial and 38 complete orfs. A homology search for the products deduced from the 39 orfs revealed that 26 of them exhibit significant similarity to known proteins, e.g. germination proteins, sodium-alanine symporter, PTS system, methionine amino peptidase, 2-oxoglutarate/malate translocater, pectate lyase, general stress response protein, RNA helicase, iron uptake and two-component systems.

Bacillus subtilis↗
PubMed1997

Ligation of RNA-containing duplexes by vaccinia DNA ligase.

Vaccinia virus DNA ligase repairs nicked duplex DNA substrates consisting of a 5'-phosphate-terminated strand and a 3'-hydroxyl-terminated strand annealed to a bridging template strand. This study addresses the ability of vaccinia DNA ligase to seal nicked substrates containing one or more RNA strands. We found that the viral enzyme rapidly and efficiently joined a 3'-OH RNA to 5'-phosphate DNA when the reacting polynucleotides were annealed to a bridging DNA strand. In contrast, ligation of 3'-OH DNA to 5'-phosphate RNA was slow (0.2% of the rate of RNA-to-DNA ligation) and entailed the accumulation of high levels of RNA-adenylate intermediate. A native gel mobility shift assay showed that vaccinia DNA ligase discriminates at the substrate binding step between ligands containing 5'-phosphate DNA versus 5'-phosphate RNA at the nick. The enzyme displayed weak activity in RNA-to-RNA ligation on a bridging DNA template (0.01% of RNA-to-DNA activity). Vaccinia DNA ligase was incapable of joining two DNAs annealed on an RNA template. These results can be explained by a requirement for B-form helical conformation on the 5'-phosphate side of the nick. The robust RNA-to-DNA strand joining activity underscores the potential for vaccinia DNA ligase to catalyze RNA-based integration of host cell genetic information into the genome of cytoplasmic poxviruses.

Base Sequence↗
PubMed1997

Kinetic analysis of DNA and RNA strand transfer reactions catalyzed by vaccinia topoisomerase.

Vaccinia topoisomerase binds duplex DNA and forms a covalent DNA-(3'-phosphotyrosyl) protein adduct at the sequence 5'-CCCTT downward arrow. The enzyme reacts readily with a 36-mer CCCTT strand (DNA-p-RNA) composed of DNA 5' and RNA 3' of the scissile bond. However, a 36-mer composed of RNA 5' and DNA 3' of the scissile phosphate (RNA-p-DNA) is a poor substrate for covalent adduct formation. Vaccinia topoisomerase efficiently transfers covalently held CCCTT-containing DNA to 5'-OH-terminated RNA acceptors; the topoisomerase can therefore be used to tag the 5' end of RNA in vitro. Religation of the covalently bound CCCTT-containing DNA strand to a 5'-OH-terminated DNA acceptor is efficient and rapid (krel > 0.5 s-1), provided that the acceptor DNA is capable of base pairing to the noncleaved DNA strand of the topoisomerase-DNA donor complex. The rate of strand transfer to DNA is not detectably affected by base mismatches at the 5' nucleotide of the acceptor strand. Nucleotide deletions and insertions at the 5' end of the acceptor slow the rate of religation; the observed hierarchy of reaction rates is as follows: +1 insertion > -1 deletion > +2 insertion >> -2 deletion. These findings underscore the importance of a properly positioned 5'-OH terminus in transesterification reaction chemistry, but they also raise the possibility that topoisomerase may generate mutations by sealing DNA molecules with mispaired or unpaired ends.

Catalysis↗