Biomedical subjects

Keisuke Makino

Publications and source records attributed to Keisuke Makino.

At least 19 recordsLinked to original sources

PubMed2007

alpha-ketoglutaric semialdehyde dehydrogenase isozymes involved in metabolic pathways of D-glucarate, D-galactarate, and hydroxy-L-proline. Molecular and metabolic convergent evolution.

Azospirillum brasilense possesses an alternative pathway of l-arabinose metabolism in which alpha-ketoglutaric semialdehyde (alphaKGSA) dehydrogenase (KGSADH) is involved in the last step, the conversion of alphaKGSA to alpha-ketoglutarate. In the preceding studies, we identified a set of metabolic genes of the l-arabinose pathway including the KGSADH gene (Watanabe, S., Kodaki, T., and Makino, K. (2006) J. Biol. Chem. 281, 2612-2623; Watanabe, S., Kodaki, T., and Makino, K. (2006) J. Biol. Chem. 281, 28876-28888; Watanabe, S., Shimada, N., Tajima, K., Kodaki, T., and Makino, K. (2006) J. Biol. Chem. 281, 33521-33536). Here, we describe that A. brasilense possesses two different KGSADH isozymes from l-arabinose-related enzyme (KGSADH-I); that is, d-glucarate/d-galactarate-inducible KGSADH-II and hydroxy-l-proline-inducible KGSADH-III. They were purified homogeneously from A. brasilense cells grown on d-galactarate or hydroxy-l-proline, respectively. When compared with KGSADH-I, amino acid sequences of KGSADH-II and KGSADH-III were significantly similar but not totally identical. Physiological characterization using recombinant enzymes revealed that KGSADH-II and KGSADH-III showed similar high substrate specificity for alphaKGSA and different coenzyme specificity; that is, NAD(+)-dependent KGSADH-II and NADP(+)-dependent KGSADH-III. In the phylogenetic tree of the aldehyde dehydrogenase (ALDH) superfamily, KGSADH-II and KGSADH-III were poorly related to the known ALDH subclasses including KGSADH-I. On the other hand, ALDH-like ycbD protein involved in d-glucarate/d-galactarate operon from Bacillus subtilis is closely related to the methylmalonyl semialdehyde dehydrogenase subclass but not A. brasilense KGSADH isozymes. To estimate the correct function, the corresponding gene was expressed, purified, and characterized. Kinetic analysis revealed the physiological role as NADP(+)-dependent KGSADH. We conclude that three different types of KGSADH appeared in the bacterial evolutional stage convergently. Furthermore, even the same pathway such as l-arabinose and d-glucarate/d-galactarate metabolism also evolved by the independent involvement of KGSADH.

Aldehyde Oxidoreductases↗

PubMed2007

Effects of edaravone on singlet oxygen released from activated human neutrophils.

The effects of edaravone, a curative agent for acute brain infarction, on singlet oxygen ((1)O2) released from activated human neutrophils were examined, and the effects were compared to those of histidine, a (1)O2 singlet oxygen scavenger. The neutrophils, stimulated with opsonized zymosan, released (1)O2 that was detected by chemiluminescence using a (1)O2 specific probe, trans-1-(2'-methoxyvinyl)pyrene. Edaravone dose-dependently suppressed the (1)O2 release with an IC(50) of approximately 0.3 microM, while the IC(50) of histidine was approximately 1 mM. This (1)O2 scavenging activity of edaravone might be involved in its curative effects on acute brain infarction.

Adult↗

PubMed2007

Gene expression in enhanced apoptosis of human lymphoma U937 cells treated with the combination of different free radical generators and hyperthermia.

The effects of various free radicals derived from 6-formylpterin (6-FP), alpha-phenyl-tert-butyl nitrone (PBN) and 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH) combined with hyperthermia, on gene expression in similarly enhanced apoptosis of human lymphoma U937 cells were investigated using cDNA microarrays containing approximately 16,600 genes and computational gene expression analysis tools. When the cells were treated for 10 min at 44 degrees C (15% apoptosis level), 39 up-regulated and 3 down-regulated genes were identified. In the up-regulated genes, apoptosis- and unfolded protein response-associated genes were contained. The combined treatment with heat and either chemical enhanced apoptosis level (approximately 30%) and showed a chemical-specific gene expression pattern. Furthermore, the expression levels of selected genes were confirmed by a real-time quantitative PCR. The present results will provide a basis for further understanding the molecular mechanisms in enhancement of heat-induced apoptosis by different intracellular oxidative stress.

Amidines↗

PubMed2006

Reactive oxygen species generation through NADH oxidation by 6-formylpterin derivatives in the dark.

6-formylpterin (6FP) has been reported to produce reactive oxygen species (ROS) such as *O2- and H2O2 from O2 in the presence of NADH under light condition. In the present study, we prepared a variety of 6FP derivatives and found that 2-(N,N-dimethylaminomethyleneamino)-6-formyl-3-pivaloylpteridin-4-one and 2-(N,N-dimethylaminomethyleneamino)-6-formyl-3-methylpteridin-4-one, in which the 2-amino groups are modified by a dimethylaminomethylene group and the 3-positions by pivaloyl and methyl groups and 2-amino-6-formyl-3-methylpteridin-4-one in which the amino group at the 2-position is free and the 3-position is modified by a methyl group generated H2O2 from O2 on oxidation of NADH to NAD+ in the dark. However, 6FP and 2-(N,N-dimethylaminomethyleneamino)-6-formylpteridin-4-one, in which the 3-position is free did not yield H2O2. These results indicate that modification of the 3-position is essential to make the activities of 6FP available in the dark and would be suggestive for designing pharmaceutical compounds that generate appropriate and controllable amounts of ROS in vivo.

Darkness↗

PubMed2006

Development of an efficient amine-functionalized glass platform by additional silanization treatment with alkylsilane.

Aminosilane-treated molecular layers on glass surfaces are frequently used as functional platforms for biosensor preparation. All the amino groups present on the surface are not available in reactive forms, because surface amino groups interact with remaining unreacted surface silanol groups. Such nonspecific interactions might reduce the efficiency of chemical immobilization of biomolecules such as DNA, enzymes, antibodies, etc., in biosensor fabrication. To improve immobilization efficiency we have used additional surface silanization with alkylsilane (capping) to convert the remaining silanol groups into Si-O-Si linkages, thereby liberating the amino groups from nonspecific interaction with the silanol groups. We prepared different types of capped amine surface and evaluated the effect of capping on immobilization efficiency by investigating the fluorescence intensity of Cy3-NHS (N-hydroxysuccinimide) dye that reacted with amino groups. The results indicate that most of the capped amine surfaces resulted in enhanced efficiency of immobilization of Cy3-NHS compared with the untreated control amine surface. We found a trend that trialkoxysilanes had greater capping effects on immobilization efficiency than monoalkoxysilanes. It was also found that the aliphatic chain of alkylsilane, which does not participate in the capping of the silanol, had an important function in enhancing immobilization efficiency. These results would be useful for preparation of an amine-modified surface platform, with enhanced immobilization efficiency, which is essential for developing many kinds of biosensors on a silica matrix.

Alkylation↗

PubMed2006

Identification and characterization of L-arabonate dehydratase, L-2-keto-3-deoxyarabonate dehydratase, and L-arabinolactonase involved in an alternative pathway of L-arabinose metabolism. Novel evolutionary insight into sugar metabolism.

Azospirillum brasiliense possesses an alternative pathway of L-arabinose metabolism, different from the known bacterial and fungal pathways. In the preceding articles, we identified and characterized L-arabinose-1-dehydrogenase and alpha-ketoglutaric semialdehyde dehydrogenase, which catalyzes the first and final reaction steps in this pathway, respectively (Watanabe, S., Kodaki, T., and Makino, K. (2006) J. Biol. Chem. 281, 2612-2623 and Watanabe, S., Kodaki, T., and Makino, K. (2006) J. Biol. Chem. 281, 28876-28888). We here report the remaining three enzymes, L-arabonate dehydratase, L-2-keto-3-deoxyarabonate (L-KDA) dehydratase, and L-arabinolactonase. N-terminal amino acid sequences of L-arabonate dehydratase and L-KDA dehydratase purified from A. brasiliense cells corresponded to those of AraC and AraD genes, which form a single transcriptional unit together with the L-arabinose-1-dehydrogenase gene. Furthermore, the L-arabinolactonase gene (AraB) was also identified as a component of the gene cluster. Genetic characterization of the alternative L-arabinose pathway suggested a significant evolutional relationship with the known sugar metabolic pathways, including the Entner-Doudoroff (ED) pathway and the several modified versions. L-arabonate dehydratase belongs to the ILVD/EDD family and spectrophotometric and electron paramagnetic resonance analysis revealed it to contain a [4Fe-4S](2+) cluster. Site-directed mutagenesis identified three cysteine ligands essential for cluster coordination. L-KDA dehydratase was sequentially similar to DHDPS/NAL family proteins. D-2-Keto-3-deoxygluconate aldolase, a member of the DHDPS/NAL family, catalyzes the equivalent reaction to L-KDA aldolase involved in another alternative L-arabinose pathway, probably associating a unique evolutional event between the two alternative L-arabinose pathways by mutation(s) of a common ancestral enzyme. Site-directed mutagenesis revealed a unique catalytic amino acid residue in L-KDA dehydratase, which may be a candidate for such a natural mutation.

Amino Acid Sequence↗

PubMed2006

A novel alpha-ketoglutaric semialdehyde dehydrogenase: evolutionary insight into an alternative pathway of bacterial L-arabinose metabolism.

Azospirillum brasilense possesses an alternative pathway of l-arabinose metabolism, which is different from the known bacterial and fungal pathways. In a previous paper (Watanabe, S., Kodaki, T., and Makino, K. (2006) J. Biol. Chem. 281, 2612-2623), we identified and characterized l-arabinose 1-dehydrogenase, which catalyzes the first reaction step in this pathway, and we cloned the corresponding gene. Here we focused on the fifth enzyme, alpha-ketoglutaric semialdehyde (alphaKGSA) dehydrogenase, catalyzing the conversion of alphaKGSA to alpha-ketoglutarate. alphaKGSA dehydrogenase was purified tentatively as a NAD(+)-preferring aldehyde dehydrogenase (ALDH) with high activity for glutaraldehyde. The gene encoding this enzyme was cloned and shown to be located on the genome of A. brasilense separately from a gene cluster containing the l-arabinose 1-dehydrogenase gene, in contrast with Burkholderia thailandensis in which both genes are located in the same gene cluster. Higher catalytic efficiency of ALDH was found with alphaKGSA and succinic semialdehyde among the tested aldehyde substrates. In zymogram staining analysis with the cell-free extract, a single active band was found at the same position as the purified enzyme. Furthermore, a disruptant of the gene did not grow on l-arabinose. These results indicated that this ALDH gene was the only gene of the NAD(+)-preferring alphaKGSA dehydrogenase in A. brasilense. In the phylogenetic tree of the ALDH family, alphaKGSA dehydrogenase from A. brasilense falls into the succinic semialdehyde dehydrogenase (SSALDH) subfamily. Several putative alphaKGSA dehydrogenases from other bacteria belong to a different ALDH subfamily from SSALDH, suggesting strongly that their substrate specificities for alphaKGSA are acquired independently during the evolutionary stage. This is the first evidence of unique "convergent evolution" in the ALDH family.

Aldehyde Oxidoreductases↗

PubMed2006

Novel 6-formylpterin derivatives: chemical synthesis and O2 to ROS conversion activities.

6-Formylpterin (6FP) has been demonstrated to have strong neuroprotective effects against transient ischemia-reperfusion injury in gerbils. Also it has been shown that in rats, 6FP protected retinal neurons even when it was administered after the ischemic insult. Since there is a significant need for such a compound that effectively suppresses the events caused by the lack of oxygen supply, 6FP has attracted further investigation. Unfortunately, however, 6FP is hardly soluble in water at neutral pH and in organic solvents because of its self-assembling ability. Although a several mM solution of 6FP is available in alkaline water, it is unstable. In the present study, a novel chemical derivatization of 6FP has been developed which maintains the formyl group on the 6-position of 6FP, which is essential for the physiological activities of 6FP, and increases solubility in water and organic solvents. In the method, the 2- and 3-positions of 6FP were modified by a three component coupling reaction: 6FP was subjected to the reaction with acid chloride and N,N-dimethylformamide. The derivatives synthesized here, 2-(N,N-dimethylaminomethyleneamino)-6-formyl-3-pivaloylpteridine-4-one 1, 2-(N,N-dimethylaminomethyleneamino)-6-formyl-3-isobutyrylpteridine-4-one 2, and 2-(N,N-dimethylaminomethyleneamino)-6-formyl-3-o-toluoylpteridine-4-one 3, showed high solubility in water (1.0-5.6 mM) and organic solvents. The O(2) conversion property has also been determined for the derivative 1. Using an oxygen electrode, it has been found that O(2) is consumed in the presence of 1 and NADH at around pH 7.4 and that the rate of O(2) consumption is enhanced by UV-A irradiation. Electron paramagnetic resonance (EPR) analysis coupled with DMPO spin trapping has also revealed that in the presence of NADH, 1 converts O(2) to O(2)(-), which is further reduced to OH. By UV-A illumination in the analogous systems, (1)O(2) formation was observed. These results are similar to those reported previously for 6FP.

Electrodes↗

PubMed2006

Synthesis and characterization of a practically better DEPMPO-type spin trap, 5-(2,2-dimethyl-1,3-propoxy cyclophosphoryl)-5-methyl-1-pyrroline N-oxide (CYPMPO).

5-(2,2-dimethyl-1,3-propoxy cyclophosphoryl)-5-methyl-1-pyrroline N-oxide (CYPMPO), a new cyclic DEPMPO-type nitrone was evaluated for spin-trapping capabilities toward hydroxyl and superoxide radicals. CYPMPO is colorless crystalline and freely soluble in water. Both the solid and diluted aqueous solution did not develop electron spin resonance (ESR) signal for at least 1 month at ambient conditions. CYPMPO can spin-trap superoxide and hydroxyl radicals in both chemical and biological systems, and the ESR spectra are readily assignable. Half life for the superoxide adduct of CYPMPO produced in UV-illuminated hydrogen peroxide solution was approximately 15 min, and in biological systems such as hypoxanthine (HX)/xanthine oxidase (XOD) the half-life of the superoxide adduct was approximately 50 min. In UV-illuminated hydrogen peroxide solution, there was no conversion from the superoxide adduct to the hydroxyl adduct. Although overall spin-trapping capabilities of CYPMPO are similar to DEPMPO, its high melting point, low hygroscopic property, and the long shelf-life would be highly advantageous for the practical use.

Cyclic N-Oxides↗

PubMed2006

Structural property and enzymatic response of oxanine in DNA strands.

Oxanine (Oxa, O), one of the major products generated from guanine (Gua) by nitrosative oxidation has been expected as mutagenic lesion involved in NO- or HNO(2) -induced genotoxicity. Here, to elucidate the biological meaning of Oxa in DNA strands, several kinds of Oxa-containing oligodeoxynucleotides (Oxa-ODNs) were synthesized and applied to biophysical and biochemical characterization. CD and NMR analyses revealed that the conformations of all the Oxa-containing duplexes are basically B-type without causing any severe distortion in the whole DNA structure. It was also determined that restriction endonucleases recognize and cleave the specific base-sequence even when Gua was substituted by Oxa in the sequence. When Oxa-ODN was testified as substrates for other DNA-relevant enzymes, the enzymatic functions were not largely affected by Oxa.

Alkaline Phosphatase↗

PubMed2006

Synthesis and application of new amine-modified oligonucleotides using H-phosphonate chemistry.

Oligodeoxynucleotide probes tethered with amine linkers are commonly used for development of DNA based biosensor tools for studying gene expression and biological assays. However, the current preparation methods for synthesizing amine tethered probes often show defects during acid deprotection and during hydrophobic purification with the amine linker protecting groups. Here, we developed a prospective preparation method of using modified H-phosphonate chemistry to prepare amine tethered oligonucleotide probes. The present developed method will be helpful for preparing efficient amine tethered oligodeoxynucleotide probes that can be used in DNA biosensor applications.

Amines↗

PubMed2006

Development of DNA-arrayed column for sensitive and selective analysis of DNA.

Quantitative separation of target DNA molecules was performed by DNA-arrayed silica capillary column on the basis of base pairing interaction of nucleic acids. We prepared DNA-arrayed silica capillary column by conjugating 5'-amniohexyl oligonucleotide (probe) on inner surface of the capillary column pre-treated with 3-aminopropyltriethoxysilane (APS) and disuccinimidyl glutarate (DSG) cross-linker. Sufficient resolution was observed by controlling of target-DNAs concentration, salt gradients, and temperature gradients. Finally, we succeeded to separate two different DNA targets even with same melting temperature according to their own concentrations. These results would be useful for developing quantitative analysis of cDNA, which is related to mRNA levels in biological sample.

Chromatography, Affinity↗

PubMed2006

Construction of various mutants of xylose metabolizing enzymes for efficient conversion of biomass to ethanol.

We applied protein engineering to construct an efficient biomass-ethanol conversion system using Saccharomyces cerevisiae. Intercellular redox imbalance caused by the different coenzyme specificity of xylose reductase (XR) and xylitol dehydrogenase (XDH) has been thought to be one of the main factors of xylitol excretion. Introduction of NADH-dependant XR generated in this study reduced the xylitol excretion probably because of maintaining the intercellular redox balance. Ethanol fermentation was measured in batch culture under anaerobic conditions. The best strain R276H produced a maximum of 5.94 g/l ethanol with yield of 0.43 g/g from 5 g glucose/l plus 15 g xylose/l.

Aldehyde Reductase↗

PubMed2006

Site-directed mutagenesis of a yeast gene for improvement of enzyme thermostability.

Enzyme stability is one of the critical factors to construct an efficient biological conversion system. Xylitol dehydrogenase (XDH) from Pichia stipitis is one of the key enzymes for bio-ethanol fermentation system from xylose. Previously, we tried to improve thermostability of XDH by introduction of structural zinc into the enzyme and successfully obtained a mutant, named C4 mutant, with an increased unfolding temperature (J. Biol. Chem., 280:10340-10349, 2005). We focused on further improvement of the thermostability of XDH in this study and employed subsequent site directed mutagenesis in structural zinc binding region for stabilizing the structural zinc binding loop. Two variants (C4/F98R and C4/E101F) showed higher thermostability than C4 mutant judged by thermal inactivation of enzyme activity and thermal transition temperature.

Binding Sites↗

PubMed2006

Chemical natures and application of 6-formylpterin derivatives.

Pterin, an analog of guanine, is an electron transfer compound in biological systems. Among the analogs, 6-formylpterin (6FP) has been demonstrated to have many marked physiological and pharmacological activities. In vitro, 6FP converts molecular oxygen to reactive oxygen species (ROS) in the presence of NADH or NADPH under light illumination, with the oxidation of NADH is to NAD(+). In the present study, it has been elucidated that some of 6FP derivatives in which the 3-position of 6FP is modified have such unique activities even in the dark where the most of in vivo biological events occur.

Darkness↗

PubMed2005

Cloning, expression, and characterization of bacterial L-arabinose 1-dehydrogenase involved in an alternative pathway of L-arabinose metabolism.

Azospirillum brasiliense converts L-arabinose to alpha-ketoglutarate via five hypothetical enzymatic steps. We purified and characterized L-arabinose 1-dehydrogenase (EC 1.1.1.46), catalyzing the conversion of L-arabinose to L-arabino-gamma-lactone as an enzyme responsible for the first step of this alternative pathway of L-arabinose metabolism. The purified enzyme preferred NADP+ to NAD+ as a coenzyme. Kinetic analysis revealed that the enzyme had high catalytic efficiency for both L-arabinose and D-galactose. The gene encoding L-arabinose 1-dehydrogenase was cloned using a partial peptide sequence of the purified enzyme and was overexpressed in Escherichia coli as a fully active enzyme. The enzyme consists of 308 amino acids and has a calculated molecular mass of 33,663.92 Da. The deduced amino acid sequence had some similarity to glucose-fructose oxidoreductase, D-xylose 1-dehydrogenase, and D-galactose 1-dehydrogenase. Site-directed mutagenesis revealed that the enzyme possesses unique catalytic amino acid residues. Northern blot analysis showed that this gene was induced by L-arabinose but not by D-galactose. Furthermore, a disruptant of the L-arabinose 1-dehydrogenase gene did not grow on L-arabinose but grew on D-galactose at the same growth rate as the wild-type strain. There was a partial gene for L-arabinose transport in the flanking region of the L-arabinose 1-dehydrogenase gene. These results indicated that the enzyme is involved in the metabolism of L-arabinose but not D-galactose. This is the first identification of a gene involved in an alternative pathway of L-arabinose metabolism in bacterium.

Amino Acid Sequence↗

PubMed2005

Chemical synthesis and thermodynamic characterization of oxanine-containing oligodeoxynucleotides.

Oxanine (Oxa, O), one of the major damaged bases from guanine generated by NO- or HNO2-induced nitrosative deamination, has been considered as a mutagen-potent lesion. For exploring more detailed properties of Oxa, large-scale preparation of Oxa-containing oligodeoxynucleotide (Oxa-ODN) with the desired base sequence is a prerequisite. In the present study, we have developed a chemical synthesis procedure of Oxa-ODNs and characterized thermodynamic properties of Oxa in DNA strands. First, 2'-deoxynucleoside of Oxa (dOxo) obtained from 2'-deoxyguanosine by HNO2-nitrosation was subjected to 5'-O-selective tritylation to give 5'-O-(4,4'-dimethoxytrityl)-dOxo (DMT-dOxo) with a maximum yield of 70%. Subsequently, DMT-dOxo was treated with conventional phosphoramidation, which resulted in DMT-dOxo-amidite monomer with a maximum yield of 72.5%. The amidite obtained was used for synthesizing Oxa-ODNs: the coupling yields for Oxa incorporation were over 93%. The prepared Oxa-ODNs were employed for analyzing the thermodynamic properties of DNA duplexes containing base-matches of O:N [N; C (cytosine), T (thymine), G (guanine) or A (adenine)]. Melting temperatures (Tm) and thermodynamic stability (DeltaG37(0)) were found to be lower by 6.83 approximately 13.41 degrees C and 2.643 approximately 6.047 kcal mol(-1), respectively, compared with those of oligodeoxynucleotides, which had the same base sequence except that O:N was replaced by G:C (wild type). It has also been found that Oxa-pairing with cytosine shows relatively high stability in DNA duplex compared with other base combinations. The orders of DeltaDeltaG37(0) were O:C > O:T > O:A > O:G. The chemical synthesis procedure and thermodynamic characteristics of Oxa-ODNs established here will be helpful for elucidating the biological significance of Oxa in relation to genotoxic and repair mechanisms.

Base Pairing↗

PubMed2005

A novel method for synthesis of silica nanoparticles.

A sequential method has been used, for the first time, to prepare monodisperse and uniform-size silica nanoparticles using ultrasonication by sol-gel process. The silica particles were obtained by hydrolysis of tetraethyl orthosilicate (TEOS) in ethanol medium and a detailed study was carried out on the effect of different reagents on particle sizes. Various-sized particles in the range 20-460 nm were synthesized. The reagents ammonia (2.8-28 mol L(-1)), ethanol (1-8 mol L(-1)), water (3-14 mol L(-1)), and TEOS (0.012-0.12 mol L(-1)) were used and particle size was examined under scanning electron microscopy and transmission electron microscopy. In addition to the above observations, the effect of temperature on particle size was studied. The results obtained in the present study are in agreement with the results observed for the electronic absorption behavior of silica particles, which was measured by UV-vis spectrophotometry.

Ammonia↗