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K Kitamoto

Publications and source records attributed to K Kitamoto.

At least 73 records · Page 4Linked to original sources
PubMed1993

Cloning and nucleotide sequence of the acid protease-encoding gene (pepA) from Aspergillus oryzae.

We have cloned a genomic DNA sequence encoding the acid protease (PEPA) from Aspergillus oryzae using a 0.6-kb fragment as a probe. This fragment was amplified by the polymerase chain reaction (PCR) using oligonucleotide primers designed from the partial amino acid sequences of peptide fragments of the purified protein. Nucleotide sequencing analysis has shown that the cloned gene (designated pepA) encodes 404 amino acid residues and contains 3 putative introns ranging in length from 50 to 61 nucleotides. The deduced amino acid sequence of the A. oryzae PEPA has a high degree of homology (67%) to the A. awamori PEPA. Comparison with the amino acid sequence of A. awamori PEPA suggests that the A. oryzae PEPA may consist of a 78 amino acid prepro-peptide and 326 amino acid mature protein. The amino acid composition of the mature protein was almost consistent with that of the acid protease purified from A. oryzae reported previously. Southern hybridization analyses showed that the pepA gene exists as a single copy in the A. oryzae chromosome. The cloned gene was found to be functional, since transformants of A. oryzae containing multiple copies of the pepA gene showed a 2-6 fold increase in acid protease activity compared with the recipient strain.

Amino Acid Sequence↗
PubMed1993

Serum myeloperoxidase and serum cytokines in anti-myeloperoxidase antibody-associated glomerulonephritis.

Anti-MPO antibodies, serum MPO and cytokines were examined in 106 patients with glomerulonephritis. Twenty-three patients had anti-MPO antibodies and crescent formation; 17 had crescentic glomerulonephritis (CRGN) and the remaining 6 had focal segmental necrosis with less than 50% crescent formation in the observed glomeruli. Pauci-immune CRGN accounted for 28 of 43 CRGN cases. Anti-MPO antibody titers were significantly higher in the cellular crescent stage and their titers correlated well with the number of crescents. Clinical observation revealed that 11 of 18 patients with anti-MPO antibody-associated CRGN had a respiratory tract infection prior to the onset of overt glomerulonephritis. Serum MPO was detected in 20 of 23 patients with anti-MPO antibodies and the amounts of MPO were especially high in the cellular crescent stage and correlated with anti-MPO antibodies. TNF-alpha and IL-6 were also detected in the sera in parallel with the anti-MPO antibody titers. These data suggest that anti-MPO antibodies, TNF-alpha and IL-6, induced by the infection, may activate neutrophils and MPO itself released from neutrophils, may play an important pathogenetic role in glomerular capillary necrosis leading to CRGN.

Antibodies, Antineutrophil Cytoplasmic↗
PubMed1993

[Clinical significance of microalbuminuria in patients with rheumatoid arthritis].

In order to make an estimate of clinical significance of microalbuminuria (MAU) in patients with rheumatoid arthritis (RA), we studied MAU in 138 patients with RA without macroalbuminuria. MAU was assayed by double-antibody RIA in the ambulatory urine. Moreover, urinary (U) beta 2-microglobulin (BMG) and N-acetyl-beta-D-glucosaminidase (NAG) were simultaneously measured. The values for MAU/U-creatinine (U-Alb index) in patients with RA, osteoarthropathy (OA) and normal controls were 25.7 +/- 38.2, 11.4 +/- 11.5 and 7.7 +/- 3.5, respectively, and U-Alb indices in patients with RA were significantly higher than U-Alb indices in patients with OA and normal controls. Especially, in patients with RA receiving lovenzarit disodium and gold sodium thiomalate (GST), U-Alb indices were elevated. U-Alb indices were not correlated with clinical findings in RA. Also, U-Alb indices were not correlated with U-BMG indices and U-NAG indices in patients with RA. In serial measurements of U-Alb index, U-BMG index and U-NAG index in a patient with RA who developed massive macroalbuminuria during GST therapy, it was found that U-Alb index was elevated first, followed by U-NAG index and finally U-BMG index was elevated. These results indicate that U-Alb indices are elevated in patients with RA without macroalbuminuria, and serial measurements of MAU in patients with RA, especially receiving disease modifying antirheumatic drugs, are useful for the detection of subclinical glomerular injury.

Acetylglucosaminidase↗
PubMed1992

High level expression of the synthetic human lysozyme gene in Aspergillus oryzae.

Aspergillus oryzae was transformed with a synthetic gene consisting of a chicken lysozyme signal sequence and a mature human lysozyme (HLY) sequence. The transformants secreted active HLY (about 1.2 mg/l) when the HLY gene was expressed under the control of the Taka-amylase A gene (amyB) promoter. Western blot analysis suggested that the secreted protein was immunoreactive with anti-human lysozyme antibody and the signal peptide was correctly cleavaged off in the A. oryzae transformants. The transcriptional level of the HLY gene was investigated by Northern blot analysis using a probe that was equivalently specific to both the HLY gene and the amyB gene. The HLY gene was expressed at a higher level compared with the amyB gene because of its multi-copy integration. The efficient transcription of the HLY gene suggested that A. oryzae is a promising host for production of heterologous proteins from higher eukaryotes.

Amylases↗
PubMed1992

Functional elements of the promoter region of the Aspergillus oryzae glaA gene encoding glucoamylase.

Analysis was made of the promoter region of the Aspergillus oryzae glaA gene encoding glucoamylase. Northern blots using a glucoamylase cDNA as a probe indicated that the amount of mRNA corresponding to the glaA gene increased when expression was induced by starch or maltose. The promoter region of the glaA gene was fused to the Escherichia coli uidA gene, encoding beta-glucuronidase (GUS), and the resultant plasmid was introduced into A. oryzae. Expression of GUS protein in the A. oryzae transformants was induced by maltose, indicating that the glaA-GUS gene was regulated at the level of transcription in the presence of maltose. The nucleotide sequence 1.1 kb upstream of the glaA coding region was determined. A comparison of the nucleotide sequence of the A. oryzae glaA promoter with those of A. oryzae amyB, encoding alpha-amylase, and A. niger glaA showed two regions with similar sequences. Deletion and site-specific mutation analysis of these homologous regions indicated that both are essential for direct high-level expression when grown on maltose.

Aspergillus niger↗
PubMed1992

Deletion analysis of the Taka-amylase A gene promoter using a homologous transformation system in Aspergillus oryzae.

The Taka-amylase A gene (amyB) of Aspergillus oryzae is induced by starch or maltose. The molecular mechanism of the induction was investigated using a fusion of the amyB promoter and the Escherichia coli uidA gene encoding beta-glucuronidase (GUS). To identify the region responsible for high-level expression and regulation within the amyB promoter, a series of deletion promoters was constructed and introduced into the A. oryzae met locus by homologous recombination. Deletion of the region between -377 to -290 (the number indicates the distance in base pairs from the translation initiation point (+1) to the deletion end point) significantly reduced of the GUS activity, but slight reduction of the GUS activity was observed in deletions up to -377. Northern blot analysis showed that reduction of the GUS activity depended upon the expression level of the GUS gene. The region between -377 to -290 is suggested to include the sequence required directly for high-level expression and regulation of the amyB gene.

Aspergillus oryzae↗
PubMed1992

Site-directed mutagenesis of catalytic active-site residues of Taka-amylase A.

The cDNA encoding Taka-amylase A (EC.3.2.1.1, TAA) was isolated to identify functional amino acid residues of TAA by protein engineering. The putative catalytic active-site residues and the substrate binding residue of TAA were altered by site-directed mutagenesis: aspartic acid-206, glutamic acid-230, aspartic acid-297, and lysine-209 were replaced with asparagine or glutamic acid, glutamine or aspartic acid, asparagine or glutamic acid, and phenylalanine or arginine, respectively. Saccharomyces cerevisiae strain YPH 250 was transformed with the expression plasmids containing the altered cDNA of the TAA gene. All the transformants with an expression vector containing the altered cDNA produced mutant TAAs that cross-reacted with the TAA antibody. The mutant TAA with alteration of Asp206, Glu230, or Asp297 in the putative catalytic site had no alpha-amylase activity, while that with alteration of Lys209 in the putative binding site to Arg or Phe had reduced activity.

Amylases↗
PubMed1992

A case of Goodpasture's syndrome associated with anti-myeloperoxidase antibodies.

A case of Goodpasture's syndrome with anti-myeloperoxidase (MPO) antibodies is reported. Histological examination revealed crescentic glomerulonephritis and alveolar hemorrhage with linear deposition of IgG along the glomerular capillary walls and alveolar capillary walls by immunofluorescence microscopy. Not only anti-glomerular basement membrane (GBM) antibodies but also anti-MPO antibodies, an anti-neutrophil cytoplasmic antibody, were simultaneously detected in the serum. Although it is generally accepted that crescentic glomerulonephritis in Goodpasture's syndrome is mediated by anti-GBM antibodies, this case suggested that anti-MPO antibodies might also participate in the pathogenesis of crescentic glomerulonephritis and probably alveolar hemorrhage of Goodpasture's syndrome, especially with vasculitis.

Anti-Glomerular Basement Membrane Disease↗
PubMed1992

[A case of microscopic polyarteritis nodosa associated with myeloperoxidase-antineutrophil cytoplasmic autoantibodies (MPO-ANCA)].

We report a case of microscopic polyarteritis nodosa associated with myeloperoxidase-antineutrophil cytoplasmic autoantibodies (MPO-ANCA). A 38 year-old female was admitted to our hospital, because of proteinuria, recurrent pyrexia, polyarthralgia, abdominal pain and purpura. She had a history of severe pulmonary hemorrhage and 4 kg weight loss for 8 months. On admission perinuclear ANCA without cytoplasmic ANCA was detected by indirect immunofluorescence assay and MPO-ANCA was detected by enzyme linked immunosorbent assay. But anti-nuclear antibodies, immune complexes and anti-glomerular basement membrane antibodies were not detected. Renal biopsy showed necrotizing crescentic glomerulonephritis without immune deposits. Skin biopsy revealed leukocytoclastic vasculitis. Diagnosis of microscopic polyarteritis nodosa was made by these clinical and histological evidence of vasculitis. As renal failure progressed after admission, corticosteroid and cyclophosphamide administration were started. Renal function and other symptoms improved paralleled with decreased MPO-ANCA titer to normal values. It is suggested that MPO-ANCA may be closely related to the pathogenesis of microscopic polyarteritis nodosa and it may be a good serological marker for diagnosis and disease activity of this disease.

Adult↗
PubMed1992

[Alterations in phosphatidylcholine and phospholipase A2 of the renal tissue in gentamicin nephrotoxicity: comparative study with buthionine sulfoximine administered model].

We investigated to confirm the biochemical mechanisms of the hypothesis that lipid peroxidation participates in the pathogenesis of aminoglycoside-induced nephrotoxicity. Male Sprague-Dawley rats were injected with gentamicin (GM), 300mg/kg per day. Twenty-four hours after the injection the rats were killed and the renal cortex was processed for glutathione (GSH), malondialdehyde (MDA), phospholipase A2 (PLA2), phosphatidylcholine (PC), sphingomyelin (SPH) and phospholipids (PL). And we also studied the GSH reduced rats by buthionine sulfoximine (BSO) administration, to compare the biochemical differences with these parameters in two groups. GM induced a significant decrease of PLA2, SHP/PC ratio and GSH. Marked elevation of MDA (lipid peroxidation) and PC were observed after a single injection of GM. In contrast, BSO injected rats were not showed increment of tissue MDA, in spite of marked reduction of renal GSH. These data support the conclusion that accelerated lipid peroxidation occurs early in the course of GM administration and inhibition of lysosomal PLA2 activity involved in the degradation of lysosomal membrane which consisted of phospholipids.

Acute Kidney Injury↗
PubMed1992

[Clinical investigation of cis-platinum nephrotoxicity in 244 cases of primary lung cancer].

Cisplatin (CDDP) is used widely in the treatment of a large number of carcinomas. The clinical use of cisplatin, however, can be complicated by myelotoxicity, intestinal toxicity and nephrotoxicity. We reviewed CDDP nephrotoxicity in 244 cases with primary lung cancer retrospectively. The enzyme histochemically localized in proximal tubular cells, N-acetyl-beta-D-glucosaminidase (NAG) and beta 2-microglobulin (BMG), a low molecular weight peptide normally reabsorbed by the renal tubular cells that has been used as an indicator for renal proximal tubular damage, were measured. And fractional excretion of Na (FENa%) and serum magnesium (Mg) levels were also measured before and after CDDP administration serially. The following results were obtained; 1) Over 45% of patients with lung cancer showed transient hyperexcretion of urinary NAG and BMG after CDDP administration. And peak excretion of NAG and BMG appeared to occur within 36 hours after administration of CDDP. 2) Almost all cases with persistent azotemia after CDDP administration showed high values of FENa (%), in spite of gradual normalization of urinary NAG and BMG excretion. 3) Hypomagnesemia was a common complication of CDDP nephrotoxicity that might be caused by a defect in renal Mg reabsorption. CDDP-induced nephrotoxicity seemed to be initiated by an acute, mainly proximal tubular impairment that reflects alterations in excretion of urinary enzymes and low molecular weight protein. In cases with persistent azotemia after CDDP administration depressed renal function might be attributed to the impairment of proximal as well as distal tubular reabsorptive functions.

Acetylglucosaminidase↗
PubMed1992

[A case report of lupus nephritis associated with minimal change nephrotic syndrome--comparison of various histological types of 67 cases with lupus nephritis].

A 22 year old male of lupus nephritis associated with minimal change nephrotic syndrome was described. The patient was well until 4 years earlier, when Raynaud's phenomenon and photosensitivity developed. One week before admission, edema appeared suddenly and proteinuria was pointed out on July 28, 1990. On admission, his legs, ankles, and eyelids were edematous. There was no sclerodactylia, although Raynaud's phenomenon was positive when his hand disclosed to the cold. Urinalysis showed heavy albuminuria (10.4g/day), but urinary sediment showed no abnormality. Immunological examination showed positive antinuclear antibody, determination at a titer of 1: 160 with a speckled pattern. Anti-RNP and anti-Sm antibody were positive. However, neither anti-DNA antibody nor hypocomplementemia was detected. There was high concentration of serum IgE (2564IU/ml). Renal biopsy was performed. Light microscopic study showed slight increase of mesangial cells and matrix. Immunofluorescence study showed mesangial localization of IgG and C3. Electron microscopic study showed electron dense deposits only in the mesangial area. The diagnosis of lupus nephritis associated with minimal change nephrotic syndrome (MCNS) was made and administration of PSL was started. Proteinuria disappeared after 3 weeks and nephrotic syndrome remitted completely. The case of lupus nephritis associated with MCNS is very rare. Therefore, the relationship between amounts of proteinuria and various histological types of 67 cases with lupus nephritis which we experienced at our hospital was evaluated. Results showed that nephrotic range proteinuria was not present in mesangial lupus nephritis. So, it was concluded that if heavy albuminuria was found in mesangial lupus nephritis, we should consider the possibility of lupus nephritis associated with MCNS.

Adult↗
PubMed1991

Cloning and molecular characterization of the acetamidase-encoding gene (amdS) from Aspergillus oryzae.

We have isolated an acetamidase-encoding gene (amdS) from Aspergillus oryzae by heterologous hybridization using the corresponding Aspergillus nidulans gene as a probe. The gene is located on a 3.5-kb SacI fragment and its nucleotide (nt) sequence was determined. Compared with the A. nidulans amdS gene, the coding region of A. oryzae gene consists of seven exons interrupted by six introns and encodes 545 amino acid (aa) residues. The deduced aa sequence has a high degree of homology with that of the A. nidulans acetamidase protein. Three introns (IVS-1, IVS-2, and IVS-4) exist at the same positions as those of A. nidulans amdS, whilst three additional introns (IVS-3, IVS-5, and IVS-6) are also present. There is no preference in its codon usage (G + C content in the third position of codons is 51%). Gene disruption experiments demonstrate that the resulting mutants show significantly reduced growth on acetamide-containing medium, indicating that the A. oryzae amdS gene encodes a functional acetamidase that is required for acetamide utilization. Transcriptional analysis by Northern blot reveals a 1.8-kb transcript in RNA extracted from mycelium grown in medium containing acetamide or acetate plus beta-alanine as the sole carbon and nitrogen sources.

Amidohydrolases↗
PubMed1991

Nucleotide sequence and expression of the glucoamylase-encoding gene (glaA) from Aspergillus oryzae.

The glucoamylase-encoding gene (glaA) from Aspergillus oryzae was cloned using its cDNA as a probe, which had been isolated previously. From comparison of nucleotide (nt) sequences of genomic clones with its cDNA, the glaA gene was found to contain four short putative introns, 45-56 nt in length. The A. oryzae glaA gene shared 62% homology at the nt level with the A. niger glaA gene with the four introns located at the same position. The 5'-flanking region contained a TATA box at nt-72 from the start codon, and two putative CAAT sequences at nt-87 and -331. Genomic Southern analysis and physical mapping showed that the glaA gene is located on the smallest chromosome (3.4 Mb) of six separated bands of chromosomes. Clones containing the glaA gene, when re-introduced intro A. oryzae, resulted in a three- to eightfold increase in glucoamylase activity.

Amino Acid Sequence↗
PubMed1991

Construction of a fusion gene comprising the Taka-amylase A promoter and the Escherichia coli beta-glucuronidase gene and analysis of its expression in Aspergillus oryzae.

Northern blot analysis of glucose-grown and starch-grown mycelia of Aspergillus oryzae RIB40 was conducted using the cloned Taka-amylase A (TAA) gene as a probe. The amount of mRNA homologous to the TAA gene was increased when this fungus was grown with starch as a sole carbon source. In order to analyze the induction mechanism, we inserted the Escherichia coli uidA gene encoding beta-glucuronidase (GUS) down-stream of the TAA promoter and introduced the resultant fusion gene into the A. oryzae genome. Production of a functional GUS protein was induced by starch, but not by glucose. When the effects of various sugars on expression of the fusion gene were examined, the results suggested that the expression of the fusion gene was under control of the TAA gene promoter.

Aspergillus oryzae↗
PubMed1991

Cloning and nucleotide sequence of the genomic ribonuclease T2 gene (rntB) from Aspergillus oryzae.

Using synthetic oligonucleotide probes, we have cloned a genomic DNA sequence encoding a ribonuclease (RNase) T2 gene (rntB) from Aspergillus oryzae on a 4.8 kb HindIII fragment. DNA sequence analysis of the RNase T2 revealed the following: (1) The gene is arranged as five exons and four introns; (2) The deduced amino acid sequence contains 239 amino acid residues of the mature enzyme. In addition, there exist 17 amino acid residues thought to be a signal peptide sequence at the N-terminus and 20 amino acid residues at the C-terminus; (3) The nucleotide sequence of the rntB gene is homologous to those of the RNase Rh gene from Rhizopus niveus and the S2 stylar glycoprotein gene of Nicotiana alata with degree of about 51% and 47%, respectively; (4) A. oryzae and A. nidulans transformed with the cloned rntB gene had much higher ribonuclease T2 activity than wild-type strains.

Amino Acid Sequence↗
PubMed1991

Genetic engineering of a sake yeast producing no urea by successive disruption of arginase gene.

Urea is reported to be a main precursor of ethyl carbamate (ECA), which is suspected to be a carcinogen, in wine and sake. In order to minimize production of urea, arginase-deficient mutants (delta car1/delta car1) were constructed from a diploid sake yeast, Kyokai no. 9, by successive disruption of the two copies of the CAR1 gene. First, the yeast strain was transformed with plasmid pCAT2 (delta car1 SMR1), and strains heterozygous for CAR1 gene were isolated on sulfometuron methyl plates. Successively, the other CAR1 gene was disrupted by transformation with plasmid pCAT1 (delta car1 G418r) and the resulting car1 mutants were isolated on a G418 plate. Arginase assay of the total cell lysate of the mutants showed that 70% of transformants isolated on G418 plates had no detectable enzyme activity, possibly as a result of the disruption of the two copies of the CAR1 gene. Further genomic Southern analysis confirmed this result. We could brew sake containing no urea with the delta car1/delta car1 homozygous mutant. It is of additional interest that no ECA was detected in the resulting sake, even after storage for 5 months at 30 degrees C. This molecular biological study suggests that ECA in sake originates mainly from urea that is produced by the arginase.

Alcoholic Beverages↗