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Biomedical subjects

N Tsukagoshi

Publications and source records attributed to N Tsukagoshi.

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

Sequence analysis, overexpression, and antisense inhibition of a beta-xylosidase gene, xylA, from Aspergillus oryzae KBN616.

beta-Xylosidase secreted by the shoyu koji mold, Aspergillus oryzae, is the key enzyme responsible for browning of soy sauce. To investigate the role of beta-xylosidase in the brown color formation, a major beta-xylosidase, XylA, and its encoding gene were characterized. beta-Xylosidase XylA was purified to homogeneity from culture filtrates of A. oryzae KBN616. The optimum pH and temperature of the enzyme were found to be 4.0 and 60 degrees C, respectively, and the molecular mass was estimated to be 110 kDa based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The xylA gene comprises 2,397 bp with no introns and encodes a protein consisting of 798 amino acids (86,475 Da) with 14 potential N-glycosylation sites. The deduced amino acid sequence shows high similarity to Aspergillus nidulans XlnD (70%), Aspergillus niger XlnD (64%), and Trichoderma reesei BxII (63%). The xylA gene was overexpressed under control of the strong and constitutive A. oryzae TEF1 promoter. One of the A. oryzae transformants produced approximately 13 times more of the enzyme than did the host strain. The partial-length antisense xylA gene expressed under control of the A. oryzae TEF1 promoter decreased the beta-xylosidase level in A. oryzae to about 20% of that of the host strain.

Amino Acid Sequence↗
PubMed1999

Pectin methylesterase gene (pmeA) from Aspergillus oryzae KBN616: its sequence analysis and overexpression, and characterization of the gene product.

A gene (pmeA) encoding pectin methylesterase was isolated from a shoyu koji mold, Aspergillus oryzae KBN616, and characterized. The structural gene comprised 1,370 bp with six introns. The PMEA protein consisted of 331 amino acids with a putative signal peptide of 17 amino acids. The deduced amino acid sequence was very similar to those of Aspergillus niger PMEA and Aspergillus aculeatus PME1. The pmeA gene was efficiently expressed under control of the A. oryzae TEF1 gene promoter for purification and characterization of the ezymatic properties. PMEA had a molecular mass of 38.5 kDa, a pH optimum of 5.0, and a temperature optimum of 55 degrees C.

Amino Acid Sequence↗
PubMed1999

Purification and characterization of the overexpressed Aspergillus oryzae xylanase, XynF1.

The Aspergillus oryzae xynF1 gene coding for a xylanase, XynF1, was successfully overexpressed under the strong A. oryzae TEF1 gene promoter. The high-XynF1-producing transformant secreted about 180 mg/l of XynF1 in the glucose-containing medium. The overexpressed XynF1 was purified by only one chromatographic step. The purified XynF1 had a molecular mass of 35.0 kDa, a pH optimum of 5.0, and a temperature optimum of 60 degrees C.

Aspergillus oryzae↗
PubMed1999

Molecular role of ascorbate in enhancement of NO production in activated macrophage-like cell line, J774.1.

Ascorbate-enhanced nitric oxide (NO) production in lipopolysaccharide (LPS)- and interferon-gamma (IFN-gamma)-activated macrophage J774.1 cells through the inducible nitric oxide synthase (iNOS) pathway. The iNOS gene was synergistically induced by LPS and IFN-gamma. The inductive mechanism of ascorbate on the iNOS gene was studied by examining the degradation of I kappa B alpha by Western blotting, activation of the nuclear factor kappa B (NF-kappa B) by gel shift assays, and protein levels of interferon regulatory factor 1 (IRF-1) in LPS- and IFN-gamma-activated cells. Ascorbate had no effect on the onset of either I kappa B alpha degradation or the nuclear translocation of NF-kappa B, but it delayed the recovery of I kappa B alpha. The prolonged degradation of I kappa B alpha caused by ascorbate in LPS- and IFN-gamma-activated cells paralleled elevated NF-kappa B binding to DNA, which led to an increase in the iNOS protein level. Ascorbate alone did not induce I kappa B alpha degradation or NF-kappa B activation. Furthermore, ascorbate exerted no effect on the expression of I kappa B alpha and ubiquitin genes in the activated cells. Ascorbate could modulate NF-kappa B DNA binding activity in response to combined LPS and IFN-gamma activation, which increases NO production in activated macrophages.

Animals↗
PubMed1998

Cloning of a thermostable ascorbate oxidase gene from Acremonium sp. HI-25 and modification of the azide sensitivity of the enzyme by site-directed mutagenesis.

A gene encoding a thermostable ascorbate oxidase (ASOM) was cloned from Acremonium sp. HI-25 and sequenced. The gene comprised 1709 bp and was interrupted by a single intron of 57 bp. ASOM consisted of 551 amino acids including a signal peptide with a molecular mass of 61200, and contained four histidine-rich regions with high sequence homology to the corresponding regions of other multicopper oxidases. The ASOM gene was expressed in Aspergillus nidulans under the Aspergillus oryzae Taka-amylase A gene promoter. The recombinant enzyme (An-ASOM) exhibited almost the same enzymatic properties as ASOM. The ASOM gene was mutated by site-directed mutagenesis with reference to the amino acid sequences of plant enzymes to generate enzymes with altered azide sensitivity. Site-directed mutagenesis at the trinuclear active copper site resulted in an increase in azide resistance; the Ala465Leu and Phe463Trp/Ala465Leu mutants exhibited approximately 10 and 20% increases in azide resistance, respectively.

Acremonium↗
PubMed1998

Ascorbate-dependent enhancement of nitric oxide formation in activated macrophages.

Macrophages activated with bacterial lipopolysaccharide (LPS) and cytokines produce nitric oxide through the induction of iNOS gene expression. Ascorbate increased NOx (nitrite and nitrate) formation by approximately 40% in a mouse macrophage-like cell line, J774.1, activated with LPS and interferon-gamma. Ascorbate alone exhibited no inductive activity toward NO formation. N(G)-Monomethyl-L-arginine inhibited nitrite formation in cells activated in the presence or absence of ascorbate. Northern and Western blotting analyses showed that both iNOS mRNA and protein steady-state levels were increased approximately twofold in cells activated in the presence of ascorbate compared to in cells activated only with the inducers. These data suggest that ascorbate increased NO production by increasing the amount of iNOS in the activated macrophages.

Animals↗
PubMed1998

Utilization of the TEF1-alpha gene (TEF1) promoter for expression of polygalacturonase genes, pgaA and pgaB, in Aspergillus oryzae.

For the development of an efficient gene expression system in a shoyu koji mold Aspergillus oryzae KBN616, the TEF1 gene, encoding translation-elongation factor 1 alpha, was cloned from the same strain and used for expression of polygalacturonase genes. The TEF1 gene comprised 1647 bp with three introns. The TEF1-alpha protein consisted of 460 amino acids possessing high identify to other fungal TEF proteins. Two nucleotide sequences homologous to the upstream activation sequence, characterized for the ribosomal protein genes in Saccharomyces cerevisiae, as well as the pyrimidine-rich sequences were present in the TEF1 gene promoter region, suggesting that the A, oryzae TEF1 gene has a strong promoter activity. Two expression vectors, pTFGA300 and pTFGB200 for production of polygalacturonases A and B respectively, were constructed by using the TEF1 gene promoter. A polygalacturonase (PGB) gene cloned from the same strain comprised 1226 bp with two introns and encoded a protein of 367 amino acids with high similarity to other fungal polygalacturonases. PGA and PGB were secreted at approximately 100 mg/l in glucose medium and purified to homogeneity. PGA had a molecular mass of 41 kDa, a pH optimum of 5.0 and temperature optimum of 45 degrees C. PGB had a molecular mass of 39 kDa, a pH optimum of 5.0 and temperature optimum of 55 degrees C.

Amino Acid Sequence↗
PubMed1998

Repression of the expression of genes encoding xylanolytic enzymes in Aspergillus oryzae by introduction of multiple copies of the xynF1 promoter.

A xylanase gene, xynF1, was cloned and characterized from a shoyu koji mould Aspergillus oryzae KBN616. The xynF1 gene was found to be comprised of 1484 bp with ten introns. The deduced amino acid sequence encodes a protein consisting of 327 amino acids (35,402 Da) which is very similar to the fungal family F xylanases such as Aspergillus nidulans XlnC, Aspergillus kawachii XynA and Penicillium chrysogenum XylP. The intron/exon organization of xynF1 is very similar to that of the fungal family F xylanase genes. Plasmid pXPR64, which contains 64 copies of the xynF1 promoter region (PxynF1) in the same direction, was constructed and introduced into A. oryzae. This led to reduced expression of both xylanase and beta-xylosidase genes in the transformants.

Amino Acid Sequence↗
PubMed1998

The Aspergillus nidulans CCAAT-binding factor AnCP/AnCF is a heteromeric protein analogous to the HAP complex of Saccharomyces cerevisiae.

The Aspergillus nidulans hapC gene was expressed as a fusion protein with MalE or glutathione-S-transferase (GST) in Escherichia coli, and used for the purification of HapC and the preparation of anti-HapC antiserum. The CCAAT-binding factor AnCP/AnCF contains a component with an approximate molecular mass of 32 kDa that cross-reacts with the antibody. The MalE-HapC fusion protein was able to replace authentic HapC in AnCP when incubated under appropriate conditions. Furthermore, reconstitution experiments with recombinant HapC, yHAP2 and yHAP5 polypeptides showed that all three polypeptides were required for the assembly of a complex capable of binding to CCAAT-containing taaG2 promoter DNA. The relationship between AnCP/AnCF and the Saccharomyces cerevisiae HAP complex is discussed.

Aspergillus nidulans↗
PubMed1998

Involvement of outer membrane protein TolC, a possible member of the mar-sox regulon, in maintenance and improvement of organic solvent tolerance of Escherichia coli K-12.

Escherichia coli mutants with improved organic solvent tolerance levels showed high levels of outer membrane protein TolC and inner membrane protein AcrA. The TolC level was regulated positively by MarA, Rob, or SoxS. A possible mar-rob-sox box sequence was found upstream of the tolC gene. These findings suggest that tolC is a member of the mar-sox regulon responsive to stress conditions. When a defective tolC gene was transferred to n-hexane- or cyclohexane-tolerant strains by P1 transduction, the organic solvent tolerance level was lowered dramatically to the decane-tolerant and nonane-sensitive level. The tolerance level was restored by transformation of the transductants with a wild-type tolC gene. Therefore, it is evident that TolC is essential for E. coli to maintain organic solvent tolerance.

Alkanes↗
PubMed1998

[Regulation of gene expression in Aspergilli].

The Aspergillus oryzae Taka-amylase A (TAA) gene has been used as a model gene to characterize the regulatory mechanisms of gene expression in Aspergilli. TAA gene contained a typical eukaryotic promoter with a TATA box and putative regulatory elements such as a CCAAT sequence in its 5'-noncoding region. A nuclear protein designated AnCP bound to the CCAAT sequence. Replacement of the CCAAT sequence with CGTAA was found to abolish the binding of AnCP and to have an inhibitory effect on taa promoter activity. Although AnCP was not purified to homogeneity, AnCP appeared to have an apparent molecular mass of approximately 120 kDa. In Saccharomyces cerevisiae, the CCAAT-binding HAP complex is a heteromeric protein comprising at least four subunits (yHAP2, yHAP3, yHAP4 and yHAP5) and is involved in the regulation of genes responsible for oxidative phosphorylation. A gene designated hapC with significant homology to yHAP3 has been isolated from A. nidulans by Hynes et al. We expressed hapC gene as a fusion protein with MalE in E. coli, purified HapC protein and prepared anti-HapC antiserum. The MalE-HapC fusion protein was able to substitute for the authentic HapC in AnCP. Furthermore, addition of the anti-HapC antiserum to the DNA binding reaction mixture retarded the mobility of the shifted band. These indicate clearly that HapC protein acts as a subunit of AnCP and that AnCP is a counterpart of the yeast Hap complex.

Aspergillus↗
PubMed1997

An Aspergillus nidulans nuclear protein, AnCP, involved in enhancement of Taka-amylase A gene expression, binds to the CCAAT-containing taaG2, amdS, and gatA promoters.

Using AnCP (Aspergillus nidulans CCAAT-binding protein) as a CCAAT-specific binding factor model, the possibility that one factor is able to recognize CCAAT sequences in several different genes in A. nidulans was examined. DNase I protection analysis showed that AnCP specifically bound to CCAAT sequence-containing regions comprising 21 to 36 bp of the taa, amdS and gatA genes. Furthermore, replacement of the CCAAT sequence with CGTAA was found to abolish the binding of AnCP and to have an inhibitory effect on taa promoter activity. This clearly demonstrates a positive function of the CCAAT element. However, amylase was induced by starch and repressed by glucose in a CCAAT-box disruptant, as in wild-type cells.

Amidohydrolases↗
PubMed1997

Ascorbate-dependent expression of ubiquitin genes in guinea pigs.

In an attempt to characterize a gene(s), of which the expression is ascorbate-dependent, a cDNA fragment encoding ubiquitin was isolated from a subtracted cDNA library constructed from spleen RNAs of ascorbate-deficient or -replete guinea pigs. On Northern blot analysis, three transcripts (1.8 kb ubiX, 1.3 kb ubiY and 0.7 kb ubiZ) were detected. The ubiY encodes four direct repeats of the 76 amino acid ubiquitin sequence with seven additional amino acids, V-Y-A-S-P-I-F, at the C-terminus. The transcript ubiX appears to comprise more than five repeats of the ubiquitin-encoding unit. The ubiZ encodes a ubiquitin monomer fused to an 80 amino acid extension exhibiting 100% amino acid sequence identity to the human homolog, HUMUBA80R. The ubiX gene was expressed animal-dependently. The ubiY mRNA levels decreased under ascorbate-deficient conditions, and increased under ascorbate-replete conditions, whereas ubiZ mRNA remained unaltered at low levels under the feeding conditions used here.

Amino Acid Sequence↗
PubMed1996

Synergistic effect of BMP-2 and ascorbate on the phenotypic expression of osteoblastic MC3T3-E1 cells.

Of TGF-beta superfamily proteins, BMP-2 enhanced alkaline phosphatase (ALP) activity in cultured osteoblastic cells, MC3T3-E1, to the same level promoted by ascorbate, whereas TGF-beta s (beta 1, beta 2, beta 3) reduced ALP activity and altered cell morphology under the same conditions. Activin appeared to have no distinct effect on ALP synthesis. Ascorbate dependent increase in ALP synthesis was markedly stimulated in the presence of BMP-2. The synergistic effect of ascorbate on ALP synthesis was replaced by type I collagen coated on the culture dish. However, BMP-2 appeared not to bind to type I collagen. These findings indicate that BMP-2 acts directly on osteoblastic cells through its receptors and collagenous matrix can neither recruit BMP-2 nor modulate directly the action of BMP-2 in the pericellular area. Treatment of cells grown in ascorbate media with TGF-beta s decreased rapidly the cellular ALP activity indicating that TGF-beta s direct cells to the dedifferentiated stage.

Activins↗
PubMed1996

Demonstration of alkaline phosphatase participation in the mineralization of osteoblasts by antisense RNA approach.

MC3T3-E1 cells grown with ascorbic acid express sequentially osteoblastic marker proteins such as alkaline phosphatase (ALPase) and then form a mineralized extracellular matrix (ECM) as a consequence of osteoblastic differentiation. To explore the functional roles of ALPase in the process of osteoblastic maturation, an inducible expression vector for antisense ALPase RNA was constructed and stably transfected into MC3T3-E1 cells. The expression of antisense ALPase RNA in the differentiated MC3T3-E1 transfectants reduced markedly the ALPase activity, which resulted in a significant decrease in the deposition of minerals upon prolonged culture. These findings demonstrated directly that ALPase participated in the mineralization of ECM.

Alkaline Phosphatase↗
PubMed1996

Molecular cloning, purification and characterization of two endo-1,4-beta-glucanases from Aspergillus oryzae KBN616.

Two endo-1,4-beta-glucanase genes, designated celA and celB, from a shoyu koji mold Aspergillus oryzae KBN616, were cloned and characterized. The celA gene comprised 877 bp with two introns. The CelA protein consisted of 239 amino acids and was assigned to the cellulase family H. The celB gene comprised 1248 bp with no introns. The CelB protein consisted of 416 amino acids and was assigned to the cellulase family C. Both genes were overexpressed under the promoter of the A. oryzae taka-amylase A gene for purification and enzymatic characterization of CelA and CelB. CelA had a molecular mass of 31 kDa, a pH optimum of 5.0 and temperature optimum of 55 degrees C, whereas CelB had a molecular mass of 53 kDa, a pH optimum of 4.0 and temperature optimum of 45 degrees C.

Amino Acid Sequence↗
PubMed1996

Sequence-specific binding sites in the Taka-amylase A G2 promoter for the CreA repressor mediating carbon catabolite repression.

The N-terminal part of the CreA protein encompassing two zinc fingers was expressed in Escherichia coli as a fusion protein with the maltose binding protein (MalE) of E. coli. Our results show that CreA binds to the promoter of the Taa-G2 gene encoding Taka-amylase A of Aspergillus oryzae. DNase I footprinting experiments showed that CreA bound to three sites with high affinity and to one site with low affinity within the first 401-bp region upstream of the transcription initiation site. All of the sites contained sequences related to the CreA consensus binding site (5'-SYGGRG-3'), and are suggested to participate in repression of the Taa-G2 gene in response to glucose.

Amino Acid Sequence↗