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J Sekiguchi

Publications and source records attributed to J Sekiguchi.

At least 73 records · Page 4Linked to original sources
PubMed1996

Cloning and expression of the inorganic pyrophosphatase gene from thermophilic bacterium PS-3.

The thermophilic bacterium PS-3 ppa gene encoding inorganic pyrophosphatase (PPiase) has been cloned and sequenced. The deduced amino acid sequence was exactly the same as that determined on Edman degradation (Ichiba, T., Takenaka, O., Samejima, T. and Hachimori, A., J. Biochem. 108: 572-578, 1990) except that amino acid-70 is threonine instead of proline and two more amino acids, Asn-Lys, are present at the carboxyl terminus. The structural gene containing BamH I and Hind III restriction cleaving sites at the 5' and 3' ends, respectively, was amplified by the PCR method. Escherichia coli JM109 was transformed with a synthetic DNA, and we found that thermophilic ppa was expressed in E. coli, as judged on enzyme assaying, SDS-PAGE and immune assaying.

Amino Acid Sequence↗
PubMed1996

New method of endoscopic pterygomaxillary disjunction for a Le Fort Type I osteotomy.

Endoscopic pterygomaxillary (PM) disjunction in a Le Fort type I osteotomy is presented, and the versatility of this new technique is discussed on the basis of our own experience. We have reexamined the anatomy of the posterior part of the maxilla, focusing in particular on the neurovascular bundle, to attempt to perform the endoscopic PM disjunction through the maxillary sinus along a new osteotomy line. We found that the use of an endoscope provided a magnified field of vision and a view of the objects being manipulated on a television monitor, thereby reducing the necessity of working blind. We have applied this technique in 10 clinical cases, and in all cases the Le Fort type I osteotomy was accomplished safely and with minimal bleeding. The operative time using an endoscope was about 15 minutes to 20 minutes on each side, and the blood loss during this manipulation was less than 50 mL. The application of the endoscope should widen in craniofacial surgical field when its advantages are recognized and new operative approaches for using it are developed.

Adolescent↗
PubMed1996

Cloning and sequencing of a 40.6 kb segment in the 73 degrees-76 degrees region of the Bacillus subtilis chromosome containing genes for trehalose metabolism and acetoin utilization.

In the framework of the international project aimed at the sequencing of the Bacillus subtilis genome, a 40.6 kb chromosome segment, which contains the tre locus, has been cloned and sequenced. This region (40 601 bp; 73 degrees-76 degrees on the genetic map) contains 38 complete ORFs and one partial one. Three ORFs, the closest to the hsdC locus, correspond to the treP, treA and treR genes encoding enzyme IITre, trehalose-6-phosphate hydrolase and the repressor of the tre operon, respectively. A homology search for the products deduced from the 39 ORFs revealed that 23 exhibit significant similarity to known proteins, e.g. proteins involved in acetoin utilization, deoxyribonuclease, methyladenine glycosidase, hydroxyisobutyrate dehydrogenase, multidrug resistance proteins, protein phosphatase, cyclic-nucleotide phosphodiesterase, 5'-nucleotidase and NADP(H)-flavin oxidoreductase. Based on the gene organization and the results of the homology search, it is predicted that YfjG, YfjH, YfjI, YfjJ and YfjK form an acetoin dehydrogenase system (acetoin regulatory protein, and acetoin dehydrogenase components/subunits E3, E2, E1 beta and E1 alpha respectively). yfkN, an extremely large ORF comprising 4386 nucleotides, seems to correspond to the fusion of the genes for 2',3'-cyclic-nucleotide 2'-phosphodiesterase and 5'-nucleotidase precursor.

Acetoin↗
PubMed1996

Effects of mecA and mecB (clpC) mutations on expression of sigD, which encodes an alternative sigma factor, and autolysin operons and on flagellin synthesis in Bacillus subtilis.

The expression of the major vegetative phase-specific autolysin genes (cwlB [lytC] and cwlG [lytD]) was greatly reduced by mecA and mecB null mutations. In contrast to the negative effects on late competence genes (such as comG) and levansucrase gene (sacB) expression, this positive effect of mec genes on autolysin gene expression was not mediated through the ComK protein but apparently through the level of the SigD protein. The pleiotropic effects of the mec mutations, i.e., the reduction of sigD expression and the overexpression of the ComK protein, seem not to be interwoven since the SigD- and ComK-dependent functions are clearly separable in the mec mutants. We also show that the synthesis of the flagellin protein, which is encoded by the SigD-dependent hag gene, was similarly affected by the mec mutations. Complementation analysis with a SigD-overproducing plasmid, pHYSigD, in mec mutants revealed the reversion of almost all of the SigD-dependent phenotypes except motility. This finding suggested that Mec proteins act on motility genes at two levels, one of which is apparently SigD independent. Finally, we discuss the transcriptional regulation of the sigD gene by multiple regulators, i.e., MecA, MecB, SinR (FlaD), and DegS-DegU, and its implications for cells in a global context.

Adenosine Triphosphatases↗
PubMed1996

flaD (sinR) mutations affect SigD-dependent functions at multiple points in Bacillus subtilis.

A flaD (sinR) null mutation depressed sigD-lacZ expression only two- to fourfold, whereas a flaD1 point mutation depressed it almost completely. Introduction of pHYSigD, a sigmaD-overproducing plasmid, corrected the filamentous phenotype common to both sinR mutants; autolysin synthesis was restored partially and completely in the flaD1 and flaD (sinR) null strains, respectively. Flagellin synthesis and motility were not restored at all in either strain.

Bacillus subtilis↗
PubMed1995

Proteolytic footprinting of vaccinia topoisomerase bound to DNA.

Vaccinia DNA topoisomerase, a member of the eukaryotic type I enzyme family, binds duplex DNA and forms a covalent protein.DNA complex at sites containing a conserved sequence element 5'-CCCTT decreases. The structure of the enzyme in the free and DNA-bound states was probed by limited proteolysis. The free topoisomerase (a 314-amino acid polypeptide) consists of protease-resistant amino- and carboxyl-terminal structural domains flanking a protease-sensitive "hinge." The hinge region, located between residues 135 and 142, is defined by accessibility to three different proteases. The amino-terminal region is punctuated by a trypsin-sensitive "bridge" at Arg-80, suggesting at least a tripartite domain structure overall. A specific subset of residues accessible to proteases in the free enzyme becomes resistant to proteolysis in the DNA-bound state. The trypsin-sensitive site at Arg-80 is protected almost completely in the covalent complex. Within the hinge region, Lys-135, Tyr-136, and Glu-139 are protected from trypsin, chymotrypsin, and V8, respectively. Acquisition of altered protease sensitivity upon DNA binding occurs prior to covalent adduct formation. The 20-kDa carboxyl domain by itself binds noncovalently to duplex DNA, albeit without the sequence specificity characteristic of the full-sized topoisomerase.

Amino Acid Sequence↗
PubMed1995

Genome structure and nucleotide sequence of a lipolytic enzyme gene of Aspergillus oryzae.

Aspergillus oryzae, which is widely used for Japanese traditional fermentation, produced at least two lipolytic enzymes (L1 and L2). Southern hybridization analysis of restriction enzyme-digested genomic DNA fragments of Aspergillus oryzae with 23-mer oligonucleotides synthesized according to the amino acid sequence of the enzyme L1 as probes suggested that there is single copy of the L1 gene in the genome. DNA fragments containing the L1 gene were cloned in Escherichia coli. Nucleotide sequencing of the DNA fragments revealed an open reading frame consisting of 213 amino acid residues. It had three putative introns whose sizes were 52 bp, 48 bp and 53 bp, respectively. Putative CAAT and TATA boxes were found at positions -147 and -100 from A (+1) of the translational initiation codon, and a polyadenylation site at 158 bp downstream of the stop codon. The deduced amino acid sequence of the L1 gene was highly similar to those of cutinases from phytopathogenic fungi. Thus, it is interesting to note that the non-phytopathogenic fungus, A. oryzae, produces cutinase, which seems to play an important role in flavor formation.

Amino Acid Sequence↗
PubMed1995

Functioning free muscle transplantation to the lower leg.

Many authors have reported on transplantation of functioning free muscle to the upper extremities, but few reports are available on functioning free muscle transfers to the lower extremities, where injuries have hitherto been treated mainly with tendon transfers. However, under suitable conditions, better results can be obtained using a functioning free musculocutaneous flap transfer to treat extensor muscle loss in the lower leg. The authors reconstructed the anterior extensors in two patients suffering from lower-leg necrosis. Ankle-joint active dorsiflexion was restored, and dorsiflexion in the ankle joint of about 0 degree was achieved; however, recovery of ankle-joint range of motion was limited by contracture of the ankle joint, due to associated injury, even when the strength and excursion of the grafted muscle were sufficient.

Adult↗
PubMed1995

Nucleotide sequence and characterization of the large mitochondrial rRNA gene of Penicillium urticae, and its comparison with those of other filamentous fungi.

The nucleotide sequence of a large rRNA gene and its flanking regions in cloned fragments of mitochondrial DNA from a patulin producer, Penicillium urticae NRRL2159A, was determined by dideoxy sequencing, and the 5' end and intron-exon border of the 1-rRNA gene were determined by primer extension analysis and RNA sequencing, respectively. In addition to the extensive sequence homology of the 3' end of the P. urticae mt 1-rRNA gene with those of Aspergillus nidulans and Neurospora crassa, the P. urticae gene had a 1,685 bp intron which separates a 3,307 bp 5' exon and a 583 bp 3' exon. In spite of being closely related Penicillium species, the size of the 5' exon of the P. urticae mt 1-rRNA is 472 bp larger than that of P. chrysogenum, whereas the sizes of the 3' exon and intron of P. urticae are very similar to those of P. chrysogenum (581 bp for the 3' exon and 1,678 bp for the intron). The intron of P. urticae contains a structure similar to the consensus one of the self splicing group IA intron and a large open reading frame suggested to be a gene for ribosomal protein S5. A sequence similar to the I-SceI recognition sequence was found at the exon-intron border. Extensive sequence homology was observed between P. urticae and P. chrysogenum, exceptions being in four regions in the 5' exon. These non-homologous regions were located in the hairpin and variable regions outside of the core structures. Comparison of the mt 1-rRNA sequences of several filamentous fungi revealed that the above four non-homologous regions are greatly expanded, and two other non-homologous regions appear at the 3' ends of the 5' exon and 3' exon.

Aspergillus nidulans↗
PubMed1995

Johanson-Blizzard syndrome facial anomaly and its correction using a microsurgical bone graft and tripartite osteotomy.

The facial anomaly of Johanson-Blizzard syndrome and its correction are described. The facial anomaly was characterized by cleft-like bony defects located in the inferomedial portion of the orbit apart from the hypoplastic maxilla and the absence of nasal alae. Correction of the facial skeleton was performed using a free vascularized iliac bone graft and tripartite osteotomy to correct the shape of the orbit and elongate the severely hypoplastic maxilla. Those procedures were effective to a certain degree in correcting the facial anomaly of Johanson-Blizzard syndrome.

Bone Lengthening↗
PubMed1995

Retroauricular hairline flap transfer to the face.

For facial skin defects including the hair-bearing area of preauricle-sideburn, lateral forehead-temporal hairline, and upper eyelid-eyebrow region, the transfer of a hairline flap that incorporates a skin flap is one option available for creating a natural hairline. An island or free retroauricular hairline flap (retroauricular skin flap including retroauricular hairline) based on the superficial temporal vessels or posterior auricular vessels was used to reconstruct facial skin defects including hair-bearing areas in seven patients. All flaps "took" perfectly, and aesthetically satisfactory results were achieved with minimal donor-site morbidity. However, flap vessels must be selected with great care during the surgical procedure because of the anatomic variation found among patients in the positions of the superficial temporal and posterior auricular vessels.

Adult↗
PubMed1995

Glucosaminidase of Bacillus subtilis: cloning, regulation, primary structure and biochemical characterization.

The 90 kDa glucosaminidase protein was purified to apparent homogeneity from vegetative cells of Bacillus subtilis AC327, and then the corresponding gene was cloned into Escherichia coli in two inactive forms by standard procedures. Nucleotide sequencing of the glucosaminidase region revealed a monocistronic operon, (designated lytD = cwIG) encoding a 95.6 kDa protein, comprising 880 amino acid residues, which has a typical signal peptide. Moreover, another monocistronic operon (designated pmi = orfX), encoding a 35.4 kDa protein, was found upstream of the glucosaminidase gene. Expression of a lytD-lacZ fusion gene, driven by lytD regulatory sequences, was observed during the exponential growth phase. The introduction of a sigD null mutation greatly reduced (by about 95%) the expression of the fusion. Amino acid sequence analysis of the glucosaminidase showed two types of direct repeats, each type being present twice, in the N-terminal-to-central region of the glucosaminidase: these repeats probably represent the cell-wall-binding domain. Zymographic analysis revealed that the 90 kDa glucosaminidase is partly processed to several smaller proteins (35-39 kDa), retaining lytic activity. Processing of these proteins occurred between the N-terminal cell-wall-binding and C-terminal catalytic domains of the glucosaminidase, the site being located between the 569th and 606th codons of the glucosaminidase. Serial deletions from the N-terminus of the glucosaminidase revealed that the loss of more than one repeating unit drastically reduces its lytic activity toward cell walls. The lytD gene product, in either an intact or a truncated form, was found to be lethal for E. coli, and the N-terminally truncated glucosaminidase proteins, produced in E. coli, were very unstable. The partially purified glucosaminidase from B. subtilis was found to be very unstable at low ionic strength at 37 degrees C, but this instability was overcome by the addition of either SDS-purified cell wall or protease inhibitor (PMSF) to the enzyme or after purification of the glucosaminidase to apparent homogeneity.

Amino Acid Sequence↗
PubMed1995

Nucleotide sequence and regulation of a new putative cell wall hydrolase gene, cwlD, which affects germination in Bacillus subtilis. .

DNA sequencing of a region upstream of the mms223 gene of Bacillus subtilis showed the presence of two open reading frames, orf1 and orf2, which may encode 18- and 27-kDa polypeptides, respectively. The predicted amino acid sequence of the latter shows high similarity to a major autolysin of B. subtilis, CwlB, with 35% identity over 191 residues, as well as to other autolysins (CwlC, CwlM, and AmiB). The gene was tentatively named cwlD. Bright spores produced by a B. subtilis mutant with an insertionally inactivated cwlD gene were committed to germination by the addition of L-alanine, and spore darkening, a slow and partial decrease in A580, and 72% dipicolinic acid release compared with that of the wild-type strain were observed. However, degradation of the cortex was completely blocked. Spore germination of the cwlD mutant measured by colony formation after heat treatment was less than 3.7 x 10(-8). The germination deficiency of the cwlD mutant was only partially removed when the spores were treated with lysozyme. Analysis of the chromosomal transcription of cwlD demonstrated that a transcript (RNA2) appearing 3 h after initiation of sporulation may have originated from an internal sigma E-dependent promoter of the cwlD operon, and a longer transcript (RNA1) appearing 4.5 h after sporulation may have originated from a sigma G-dependent promoter upstream of the orf1 gene. The cwlD mutant harboring a B. subtilis vector plasmid containing the intact cwlD gene recovered germination at a frequency 26% of the wild-type level.

Alanine↗
PubMed1995

Purification and characterization of a lipase from Aspergillus oryzae.

A lipase from Aspergillus oryzae was purified by ammonium sulfate fractionation, anion exchange chromatography, hydrophobic interaction chromatography, and anion exchange chromatography. The purified enzyme was a monomeric protein with a molecular mass of 41 kDa estimated by SDS-PAGE and 39 kDa by gel filtration. The optimum pH at 30 degrees C and optimum temperature at pH 7.0 were 7.0 and 30 degrees C, respectively. The enzyme was stable over a pH range of 6-9 at 25 degrees C for 18 h, and up to 30 degrees C at pH 7.0 for 3 h. Ag+, Fe3+, Hg2+, Cu2+, and Zn2+ inhibited the enzyme activity severely. The enzyme was a lipase that hydrolyzed monoacylglycerols and diacylglycerols, but did not hydrolyze triacylglycerols. The N-terminal amino acid sequence of the enzyme was highly homologous with that of the mono- and diacylglycerol lipase from Penicillium camembertii U-150.

Amino Acid Sequence↗
PubMed1995

Radiological services in rural mission hospitals in Ghana.

The provision of basic radiological services in rural, first-referral hospitals is an essential component of any country's attempt to achieve health for all. We report the results of a review of examination frequency trends, operator training background, and machine operational and safety status in the X-ray facilities in rural mission hospitals in Ghana in 1991-92. The radiological workload at the reporting hospitals was low and declined by more than 50% over the study period. Although most of the X-ray operators had little or no formal training, they produced adequate imaging results. Most of the X-ray machines seen were over 20 years old, yet remained functional, but less than 25% had standard radiation safety (beam limitation) devices. These results suggest that many rural, first-referral hospitals in developing countries could benefit from a careful review of their services and adoption of the WHO Basic Radiological System (WHO-BRS).

Ghana↗
PubMed1995

The recognition of DNA containing an AP site by E.coli endonuclease VI (exonuclease III).

The major apurinic/apyrimidinic (AP) DNA-repair endonuclease of Escherichia coli is the endonuclease VI (exonuclease III) protein. To elucidate the substrate specificity of the AP endonuclease, we used the double- and single-stranded oligo deoxyribonucleotides containing an AP residue such as 2-deoxyribosylformamide (1), 2-deoxyribose (2), 1,2-dideoxyribofuranose (3), and propanediol (4) as the substrate. The endonuclease VI cleaved the phosphodiester bond 5' at these AP sites of the duplexes. The endonucleolytic activity was not influenced by the kind of nucleotide residue on the opposite side of the AP site. Further, it was observed that the AP endonuclease cleaved single-stranded oligomers containing an AP site.

Base Sequence↗
PubMed1994

Vaccinia topoisomerase binds circumferentially to DNA.

Vaccinia DNA topoisomerase, a member of the eukaryotic type I enzyme family, binds duplex DNA and forms a covalent adduct at sites containing a conserved sequence element 5'-CCCTT decreases in the scissile strand. The protein-DNA interface entails essential contacts with four phosphate moieties within the CCCTT motif, including the scissile phosphate, and three phosphates within the GGGAA sequence on the noncleaved strand. Critical protein-phosphate contacts are arrayed across the minor groove of the DNA helix. Base-specific contacts with the pentamer element are within the major groove and are situated on the opposite face of the helix. Thus, vaccinia topoisomerase binds circumferentially to its target site in duplex DNA. This binding mode suggests that the eukaryotic enzyme adopts a toroidal shape in the DNA-bound state. Conformational isomerization of the bound protein provides a plausible mechanism for DNA relaxation.

Base Sequence↗