Biomedical subjects

Tomoyuki Nakagawa

Publications and source records attributed to Tomoyuki Nakagawa.

15 recordsLinked to original sources

PubMed2025

Duganella hordei sp. nov., Duganella caerulea sp. nov., and Duganella rhizosphaerae sp. nov., isolated from barley rhizosphere.

Duganella sp. strains R1T, R57T, and R64T, isolated from barley roots in Japan, are Gram-stain-negative, motile, rod-shaped bacteria. Duganella species abundantly colonized barley roots. Strains R1T, R57T, and R64T were capable of growth at 4 °C, suggesting adaptation to colonize winter barley roots. Strains R57T and R64T formed purple colonies, indicating violacein production, while strain R1T did not. Based on 16S rRNA gene sequence similarities, strains R1T, R57T, and R64T were most closely related to D. violaceipulchra HSC-15S17T (99.10%), D. vulcania FT81WT (99.45%), and D. violaceipulchra HSC-15S17T (99.86%), respectively. Their genome sizes ranged from 7.05 to 7.38 Mbp, and their genomic G+C contents were 64.2-64.7%. The average nucleotide identity and digital DNA-DNA hybridization values between R1T and D. violaceipulchra HSC-15S17T, R57T and D. vulcania FT81WT, R64T and D. violaceipulchra HSC-15S17T were 86.0% and 33.2%, 95.7% and 67.9%, and 92.7% and 52.6%, respectively. Their fatty acids were predominantly composed of C16:0, C17:0 cyclo, and summed feature 3 (C16:1 ω7c and/or C16:1 ω6c). Based on their distinct genetic and phenotypic characteristics, and supported by chemotaxonomic analyses, we propose that strains R1T, R57T, and R64T represent novel species within the Duganella genus, for which the names Duganella hordei (type strain R1T = NBRC 115982 T = DSM 115069 T), Duganella caerulea (type strain R57T = NBRC 115983 T = DSM 115070 T), and Duganella rhizosphaerae (type strain R64T = NBRC 115984 T = DSM 115071 T) are proposed.

Hordeum↗

PubMed2006

Molecular characterization of the Tim9 homologue from the methylotrophic yeast Pichia methanolica.

In this paper we describe molecular characterization of the TIM9 gene encoding the essential mitochondrial inner-membrane protein in the methylotrophic yeast Pichia methanolica. PmTIM9 contains two exons corresponding to a gene product of 89 amino acid residues and a 140 bp intron. The deduced amino acid sequence exhibited high identity to those of other yeast Tim9ps, and possessed two CX(3)C motifs that contained two cysteine residues conserved among small Tim family proteins. Moreover, PmTIM9 had the ability to partially suppress the temperature sensitivity of Saccharomyces cerevisiae strain tim9-3, suggesting that PmTIM9 is a functional homologue of the ScTIM9 gene.

Amino Acid Sequence↗

PubMed2006

Purification and molecular characterization of cold-active beta-galactosidase from Arthrobacter psychrolactophilus strain F2.

In this study, we purified and molecularly characterized a cold-active beta-galactosidase from Arthrobacter psychrolactophilus strain F2. The purified beta-galactosidase from strain F2 exhibited high activity at 0 degrees C, and its optimum temperature and pH were 10 degrees C and 8.0, respectively. It was possible to inactivate the beta-galactosidase rapidly at 45 degrees C in 5 min. The enzyme was able to hydrolyze lactose as a substrate, as well as o-nitrophenyl-beta-D-galactopyranoside (ONPG), the Km values with ONPG and lactose being calculated to be 2.8 mM and 50 mM, respectively, at 10 degrees C. Moreover, the bglA gene encoding the beta-galactosidase of strain F2 was cloned and analyzed. The bglA gene consists of a 3,084-bp open reading frame corresponding to a protein of 1,028 amino acid residues. BglAp, the gene product derived from bglA, had several conserved regions for glycosyl hydrolase family 2, e.g., the glycosyl hydrolase 2 (GH2) sugar binding domain, GH2 acid-base catalyst, GH2 triosephosphate isomerase barrel domain, GH2 signature 1, and several other GH2 conserved regions. From these facts, we conclude that the beta-galactosidase from A. psychrolactophilus strain F2, which is a new member of glycosyl hydrolase family 2, is a cold-active enzyme that is extremely heat labile and could have advantageous applications in the food industry.

Amino Acid Sequence↗

PubMed2006

Regulation of two distinct alcohol oxidase promoters in the methylotrophic yeast Pichia methanolica.

In this study, two Pichia methanolica alcohol oxidase (AOD) promoters, P(MOD1) and P(MOD2), were evaluated in a promoter assay system utilizing the acid phosphatase (AP) gene from Saccharomyces cerevisiae (ScPHO5) as a reporter. Heterologous gene expression driven by the P(MOD1) and P(MOD2) promoters was found to be strong and tightly regulated by carbon source at the transcriptional level. P(MOD1) was induced not only by methanol but also by glycerol. P(MOD2) was induced only by methanol, although it was not repressed on the addition of glycerol to a methanol medium, suggesting that P(MOD2) is regulated in a manner distinct from that of other AOD-gene promoters. On the other hand, methanol and oxygen level-influenced gene expression mediated by P(MOD1) and P(MOD2). P(MOD1) expression was optimal at low methanol concentrations, whereas P(MOD2) was predominantly expressed at high methanol and high oxygen concentrations. Based on these results, both P(MOD2) and P(MOD1) should be useful tools for controlling heterologous gene expression in P. methanolica. In particular, it should be possible to differentially control the production phases of two heterologous proteins, using P(MOD1) and P(MOD2) in the same host cell and in the same flask.

Acid Phosphatase↗

PubMed2006

Effect of ciprofloxacin on levels of lipopolysaccharide and cytokines in experimentally induced gram-negative bacterial pneumonia in mice.

This study was performed to examine the ability of ciprofloxacin (CPFX) to suppress the inflammation associated with lipopolysaccharide (LPS) and an inflammatory cytokine in gram-negative bacterial pneumonia. For this purpose we measured viable cell counts in bronchoalveolar lavage fluid (BALF), LPS concentrations in BALF, and BALF levels of tumor necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10) in mice exposed to Klebsiella pneumoniae. We used three groups of mice: controls, treated with physiological saline; mice treated with CPFX; and mice treated with ceftadizime (CAZ). The viable cell count in BALF was low in both the CAZ and CPFX groups. LPS values in BALF were significantly lower in the CPFX group than in the CAZ group at 48 and 72 h after K. pneumoniae exposure (48 h, P < 0.001; 72 h, P < 0.05). The BALF TNF-alpha level was significantly higher in the CAZ group at 24, 48, and 72 h compared to levels in the control and CPFX groups (P < 0.05). In conclusion, these results suggest that CPFX inhibited increases of LPS and TNF-alpha in gram-negative bacterial pneumonia, thereby suppressing the lung inflammation that accompanies pneumonia.

Animals↗

PubMed2005

Cold-active acid beta-galactosidase activity of isolated psychrophilic-basidiomycetous yeast Guehomyces pullulans.

In the present study, psychrophilic yeasts, which grow on lactose as a sole carbon source at low temperature and under acidic conditions, were isolated from soil from Hokkaido, Japan. The phenotypes and sequences of 28S rDNA of the isolated strains indicated a taxonomic affiliation to Guehomyces pullulans. The isolated strains were able to grow on lactose at below 5 degrees C, and showed cold-active acid beta-galactosidase activity even at 0 degrees C and pH 4.0 in the extracellular fractions. Moreover, K(m) of beta-galactosidase activity for lactose in the extracellular fraction from strain R1 was found to be 50.5 mM at 10 degrees C, and the activity could hydrolyze lactose in milk at 10 degrees C. The findings in this study indicate the possibility that the isolated strains produce novel acid beta-galactosidases that are able to hydrolyze lactose at low temperature.

Base Sequence↗

PubMed2005

Molecular characterization of maize acetylcholinesterase: a novel enzyme family in the plant kingdom.

Acetylcholinesterase (AChE) has been increasingly recognized in plants by indirect evidence of its activity. Here, we report purification and cloning of AChE from maize (Zea mays), thus providing to our knowledge the first direct evidence of the AChE molecule in plants. AChE was identified as a mixture of disulfide- and noncovalently linked 88-kD homodimers consisting of 42- to 44-kD polypeptides. The AChE hydrolyzed acetylthiocholine and propyonylthiocholine, but not S-butyrylthiocholine, and the AChE-specific inhibitor neostigmine bromide competitively inhibited its activity, implying that maize AChE functions in a similar manner as the animal enzyme. However, kinetic analyses indicated that maize AChE showed a lower affinity to substrates and inhibitors than animal AChE. The full-length cDNA of maize AChE gene is 1,471 nucleotides, which encode a protein having 394 residues, including a signal peptide. The deduced amino acid sequence exhibited no apparent similarity with that of the animal enzyme, although the catalytic triad was the same as in the animal AChE. In silico screening indicated that maize AChE homologs are widely distributed in plants but not in animals. These findings lead us to propose that the AChE family, as found here, comprises a novel family of the enzymes that is specifically distributed in the plant kingdom.

Acetylcholinesterase↗

PubMed2005

A cold-active pectin lyase from the psychrophilic and basidiomycetous yeast Cystofilobasidium capitatum strain PPY-1.

In the present study we purified a cold-active PNL (pectin lyase) from the extracellular fraction of the PPY (pectinolytic and psychrophilic yeast) Cystofilobasidium capitatum strain PPY-1. The purified PNL has a molecular mass of approx. 42 kDa, and its N-terminal amino acid sequence is ATGVTGSAYGFATGTTGGGSATPAY, which exhibits 72% identity with that of PNL F from Aspergillus niger. The purified PNL exhibited high activity at 10 degrees C, although its optimum temperature was 40 degrees C. Moreover, Km and Vmax for pectin as a substrate were found to have values 36.6 mg/ml and 3000 units/mg respectively. These findings may indicate that this enzyme from strain PPY-1 is a cold-active PNL that is able to degrade pectin compounds at low temperature.

Amino Acid Sequence↗

PubMed2005

Pectin utilization by the methylotrophic yeast Pichia methanolica.

The methylotrophic yeast Pichia methanolica was able to grow on pectic compounds, pectin and polygalacturonate, as sole carbon sources. Under the growth conditions used, P. methanolica exhibited increased levels of pectin methylesterase, and pectin-depolymerizing and methanol-metabolizing enzyme activities. On the other hand, P. methanolica has two alcohol oxidase (AOD) genes, MOD1 and MOD2. On growth on pectin, the P. methanolica mod1Delta and mod1Deltamod2Delta strains showed a severe defect in the growth yield, although the mod2Delta strain could grow on polygalacturonate to the same extent as the wild-type strain. The expression of MOD1 was detected in pectin-grown cells, but the MOD2-gene expression detected by pectin was much lower than that of MOD1. Moreover, pectin could induce peroxisome proliferation in P. methanolica, like methanol and oleic acid. These findings showed that P. methanolica was able to utilize the methylester moiety of pectin by means of methanol-metabolic enzymes in peroxisomes, and that the functional AOD subunit for pectin utilization was Mod1p in P. methanolica.

Alcohol Oxidoreductases↗

PubMed2005

Cold-active polygalacturonase from psychrophilic-basidiomycetous yeast Cystofilobasidium capitatum strain PPY-1.

We purified and characterized a cold-active polygalacturonase (PG) from the extracellular fraction of Cystofilobasidium capitatum strain PPY-1. The purified PG from strain PPY-1 has a molecular mass of about 44 kDa, and exhibited high activity at 0 degrees C, although its optimum temperature was 45 degrees C. Although the Km value for polygalacturonate as a substrate at 45 degrees C was found to be 11.2 mg/ml, it decreased gradually with decreasing temperature, and it was 0.66 mg/ml at 0 degrees C. Moreover, its cleavage pattern was of the endo-type. These findings might indicate that PG from strain PPY-1 is a novel type of cold-active endo-PG that is able to degrade pectin compounds at low temperatures.

Amino Acid Sequence↗

PubMed2004

Molecular characterization of the glutathione-dependent formaldehyde dehydrogenase gene FLD1 from the methylotrophic yeast Pichia methanolica.

In this paper, we describe molecular characterization of the FLD1 gene, which encodes glutathione-dependent formaldehyde dehydrogenase (FLD), from the methylotrophic yeast Pichia methanolica. The P. methanolica FLD1 gene contains two exons corresponding to a gene product of 380 amino acid residues and a 225 bp intron, respectively, and its deduced amino acid sequence shows high similarity to those of Fld1ps from other methylotrophic yeasts (80-88%). In P. methanolica, FLD activity is mainly induced by methanol, and this induction is not completely repressed by glucose. Moreover, the expression of the PmFLD1 is strictly regulated, mainly at the mRNA level, its expression increasing with increasing methanol concentrations in the medium. These results suggest that FLD1 is involved in the detoxification of formaldehyde in methanol metabolism, and Fld1p coordinates the formaldehyde level in methanol-grown cells according to the methanol concentration on growth.

Aldehyde Oxidoreductases↗

PubMed2004

Antimicrobial therapy in community-acquired pneumonia among emergency patients in a university hospital in Japan.

As antimicrobial therapy for pneumonia has not been well established in Japan, this study was designed to obtain a more definitive standard for antimicrobial treatment of this condition. Two hundred and thirty-one emergency patients admitted to Kyorin University Hospital between January 1998 and December 2000 were retrospectively analyzed in respect to their age, underlying disease, causative organism, and primary treatment with antimicrobial agent. Furthermore, the severity and prognosis were analyzed for those patients who had not responded to initial treatment with antimicrobial agents. The majority of the patients were elderly (over 65 years old; mean overall age 66.7 +/- 15.2 years) and had severe pneumonia; underlying diseases were recognized at a high rate in patients with severe pneumonia (P < 0.05) and in those classified as elderly (P < 0.0001). The most common underlying conditions in elderly patients were respiratory, cardiovascular (P < 0.01), and cerebrovascular (P < 0.05) diseases. The most common causative organisms were Haemophilus influenzae, Staphylococcus aureus, Streptococcus pneumoniae, and Mycoplasma pneumoniae. In patients with severe pneumonia, S. aureus, Klebsiella pneumoniae, and Pseudomonas aeruginosa were identified as the most common causative organisms. Complications associated with antimicrobial treatment were observed in those patients with K. pneumoniae isolates who also had severe pneumonia and were frequently treated with penicillin. Furthermore, increased mortality rates were observed in patients not responding well to the initial treatment with antimicrobial agents. Thus, the selection of appropriate initial antimicrobial agents is an important factor affecting the prognosis of patients with community-acquired pneumonia.

Aged↗

PubMed2003

Molecular characterization of GroES and GroEL homologues from Clostridium botulinum.

We report novel findings of significant amounts of 60- and 10-kDa proteins on SDS-PAGE in a culture supernatant of the Clostridium botulinum type D strain 4947 (D-4947). The N-terminal amino acid sequences of the purified proteins were closely related to those of other bacterial GroEL and GroES proteins, and both positively cross-reacted with Escherichia coli GroEL and GroES antibodies. Native GroEL homologue as an oligomeric complex is a weak ATPase whose activity is inhibited by the presence of GroES homologue. The 2634-bp groESL operon of D-4947 was isolated by PCR and sequenced. The sequence included two complete open reading frames (282 and 1629 bp), which were homologous to the groES and groEL gene family of bacterial proteins. Southern and Northern blot analyses indicate that the groESL operon is encoded on the genomic DNA of D-4947 as a single copy, and not on that of its specific toxin-converting phage.

Amino Acid Sequence↗

PubMed2002

Physiological role of the second alcohol oxidase gene MOD2 in the methylotrophic growth of Pichia methanolica.

The methylotrophic yeast Pichia methanolica has nine multiple alcohol oxidase (AOD) isozymes, which can be detected on native electrophoretic polyacrylamide gel and are encoded by two genes, MOD1 and MOD2. The aim of this work is to reveal the physiological roles of these AOD subunits, especially that of Mod2p, encoded by the second AOD-encoding gene, MOD2. A strain expressing only MOD2 showed severe growth inhibition with a low concentration of methanol (0.1%), but its growth was restored with an increase in the methanol concentration (up to 3%). The expression of MOD2 using the CbAOD1 promoter in the Candida boidinii alcohol oxidase-depleted strain was more advantageous for methylotrophic growth with high methanol concentrations than that of MOD1. The expression of MOD2 was not observed under derepression conditions (0% methanol), and the expression level increased with an increase in the methanol concentration used for induction. The expression of MOD1 was observed under derepression conditions and was rather constant throughout the tested methanol concentration range. Therefore, the ratio of Mod2p to Mod1p in an active AOD octamer was proved to be mainly controlled by changes in the MOD2 mRNA level. These and other results show that Mod2p is a unique AOD subunit more adapted to methylotrophic growth with high methanol concentrations (3%) than Mod1p.

Alcohol Oxidoreductases↗

PubMed2002

Cold-active pectinolytic activity of psychrophilic-basidiomycetous yeast Cystofilobasidium capitatum strain PPY-1.

A pectinolytic and psychrophilic yeast was isolated from soil from Abashiri, Hokkaido, Japan. The phenotype and sequencing of the 28S rDNA of the isolated strain (PPY-1) indicated a taxonomic affiliation to the basidiomycetous yeast Cystofilobasidium capitatum. C. capitatum strain PPY-1 was able to grow on two pectic compounds, polygalacturonate and pectin, at below 5 degrees C. Moreover, the extracellular fraction of the strain exhibited pectin methylesterase, pectin lyase and polygalacturonase activities at 5 degrees C. Thus strain PPY-1 may produce novel enzymes that are able to degrade pectin at low temperature, although the strain has isozymes of these enzymes.

Journal Article↗