• Journal of Microbiology and Biotechnology
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• v.13 no.5
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• pp.738-744
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• 2003

The gene ADH encoding NAD-dependent alcohol dehydrogenase from Bacillus stearothennophilus was cloned and overexpressed as a GST fusion protein at a high level in Escherichia coli. The expressed fusion protein was purified simply by glutathione affinity chromatography. GST fusion protein was then cleaved by thrombin, while soluble enzyme was further purified by glutathione affinity chromatography. The recombinant enzyme had the same elctrophoretic mobility as the native enzyme from Bacillus stearothennophilus. The recombinant enzyme catalyzed the oxidation of a number of alcohols and exhibited high activities towards secondary alcohols. The $K_m\;and\;V_{max}$ values of the recombinant enzyme for ethanol were 5.11 mM and 61.35 U/mg, respectively. Pyridine and imidazole notably inhibited the enzymatic activity. The activity of the recombinant enzyme optimally proceeded at pH 9.0 and $70^{\circ}C$. The midpoint of the temperature-stability curve for the recombinant enzyme was approximately $68^{\circ}C$, and the enzyme was not completely inactivated even at $85^{\circ}C$. The recombinant enzyme showed a high resistance towards denaturing agents (0.05% SDS, 0.1 M urea). Therefore, due to its stability and relatively broad substrate specificity, the recombinant enzyme could be utilized in bio-industrial processes and biosensors.

• Environmental Analysis Health and Toxicology
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• v.24 no.2
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• pp.107-117
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• 2009

Metal pollution of aquatic ecosystems is a problem of economic and health importance. Information regarding molecular responses to metal exposure is sorely needed in order to identify potential biomarkers. To determine the effects of heavy metals on chironomids, the full-length cDNA of alcohol dehydrogenase (ADH3) from Chironomus riparius was determined through molecular cloning and rapid amplification of cDNA ends (RACE). The expression of ADH3 was analyzed under various cadmium and copper concentrations. A comparative and phylogenetic study among different orders of insects and vertebrates was carried out through analysis of sequence databases. The complete cDNA sequence of the ADH3 gene was 1134 bp in length. The sequence of C. riparius ADH3 shows a low degree of amino acid identity (around 70%) with homologous sequences in other insects. After exposure of C. riparius to various concentrations of copper, ADH3 gene expression significantly decreased within 1 hour. The ADH3 gene expression was also suppressed in C. riparius after cadmium exposure for 24 hour. However, the effect of cadmium on ADH3 gene expression was transient in C. riparius. The results show that the suppression of ADH3 gene by copper exposure could be used as a possible biomarker in aquatic environmental monitoring and imply differential toxicity to copper and cadmium in C. riparius larvae.

• Korean Journal of Microbiology
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• v.24 no.4
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• pp.357-363
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• 1986

Synthesis of threonine and methionine in yeast, Saccharomyces cerevisiae shares a common pathway from aspartate via homoserine. HOM6 gene encodes homoserine dehydrogenase (HSDH) which catalyzes the inter-conversion of beta-aspartate semialdehyde and homoserine. The level of HSDH is under methionine specific control. A recombinant plasmid (pEK1: 13.3kb), containing HOM6 gene, has been isolated and cloned into E. coli by complenemtary transformation of a homoserine auxotrophic yeast strain M-20-20D (hom6, trp1, ura3) to a prototrophic M20-20D/pEK1, using a library of yeast genomic DNA fragments in a yeast centromeric plasmid, YCp50(8.0kb). Isolation of HOM6has been primarily confirmed by retransformation of the original yeast strain M20-20D, using the recombinant plasmid DNA which was extracted from M20-20D/pEK1 and subsequently amplified in E. coli. Eleven cleavage sites in the insery (5.3kb) have been localized through fragment analysis for 8 restriction endonucleases; Bgl II(2 site), Bgl II(1), Cla I(3), Eco RI(1), Hind III(2), Kpn I (1), Pvu II(1) and Xho I(1).

• Journal of Preventive Medicine and Public Health
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• v.3 no.1
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• pp.111-119
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• 1970

Alterations of H-and M-isozymes of Lactic Dehydrogenase(LDH) were observed in the various tissues after exposing the rats to 50ppm and 250ppm of sulfur dioxide. These isozymes of the respective tissue were separated by Diethlaminoethyl (DTAE)-cellulose from the tissue homogenates of brain, lung and muscle, presenting the activities by rate of reduction of nicotinamide-adenine-dinucleotide ($NAD^+$). Pure LDH and the coenzyme ($NAD^+$) were directly treated with sulfur dioxide in vitro in order to find out the direct to sulfur dioxide on LDH and $NAD^+$ and the results were as follows. 1. In the normal tissues, the H-isozyme activity was dominant in the brain and heart, and the M-isozyme in the muscle. 2. In the lung tissue of normal rats, there was no difference between the activity of H-and M-type of LDH. 3. When rats inhale sulfur dioxide gas in concentration of 50ppm and 250ppm, it appeared that the H-type tend to be suppressed in aerobic tissues and the M-type in anaerobic tissues. 4. In the lung tissue exposed to sulfur dioxide, both the LDH activities were suppressed. 5. It seems that LDH and the coenzyme ($NAD^+$) are not directly affected by exposing in sulfur dioxide gas.

• Korean Journal of Pharmacognosy
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• v.22 no.4
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• pp.225-232
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• 1991

The hypoglycemic and metabolic effects of Commelina communis L. extract were investigated in alloxan-diabetic rats. The increased blood glucose level in the diabetic rats was significantly lowered and the loss of body weight in the diabetic rats was recovered with the treatments of the crude extract. Administration of the extract elicited the significant increase of glucose-6-phosphate dehydrogenase activity and liver weight which were decreased in the alloxan-treated rat serum and liver. On the other hand, the kidney weight and glucose-6-phosphate dehydrogenase activity were increased in the alloxan treated rat kidney and were potentiated by the treatment of the extract. In both liver and kidney, together with serum, alkaline phosphatase and ATPase activities were increased in the alloxan diabetic rats and were not recovered, rather potentiated by the administration of the extract.

• Journal of Life Science
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• v.15 no.1 s.68
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• pp.100-105
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• 2005

The lactate dehydrogenase (EC 1.1.1.27, LDH) inhibitors were isolated from the LDH-free crude mitochondrial fraction of skeletal muscle in Syrian hamster (Mesocricetus auratus) and Korean native cattle (Bos taurus coreanae). The LDH inhibitor in skeletal muscle of M. auratus was successfully isolated by the treatment with 175 mM NaCl and ultrasonic. The LDH inhibitor in skeletal muscle of B. taurus coreanae was highly stable to heat and LDH fu isozyme was largely inhibited by the LDH inhibitor. The molecular weight of inhibitor was 22 kDa. Inhibitor played an important role in the binding of LDH with the mitochondria in tissues of skeletal muscle, kidney and liver except heart.

• Archives of Pharmacal Research
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• v.17 no.5
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• pp.343-347
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• 1994

Effects of betaine on glutamate-induced neurotoxicity were examined on primary culturs of chicken embryonic brain cells and on rat cortical cultures. Betaine was found to attenuate glutamate-induced neurotoxicity both morphologically and biochemically. A 30 min exposure of chicken embryonic brain cells cultured for 12 days to 500 .mu.M glutamate produced wide-spread acute neuronal swelling and neurtic fragmentation. A 2-h pretreatment of cultured chicken embryonic brain cells with i mM betaine prior to a 30 min exposure to 500 , mu, M glutamate significantly raised the survival rate of neurons in the culture. When chicken embryonic brain cells were pretreated for 2 h with i mM betaine followed by exposure to 100 .mu.M glutamate for 42 h, lactate dehydrogenase levels within the cells remained at 62% of .mu.M untreated control values while glutamate-treated control fell to 0% lactate dehydrogenase. Betaine also exerted attenuating effects on N-methyl-D-asparte-, kainate-and quisqualate-induced neurotoxicity in a similar manner to that observed with glutamate. Similar neuroprotective effects of betaine with rat cortical cultures.

• Biomolecules & Therapeutics
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• v.18 no.1
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• pp.16-23
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• 2010

Adipocyte differentiation in human bone marrow mesenchymal stem cells (hBM-MSCs) is not as efficient as that in murine pre-adipocytes when induced by adipogenic agents including insulin, dexamethasone, and 3-isobutyl-1-methylxanthine (IDX condition). Therefore, the promotion of adipocyte differentiation in hBM-MSCs has been used as a cell culture model to evaluate insulin sensitivity for anti-diabetic drugs. In hBM-MSCs, $PPAR{\gamma}$ agonists or sulfonylurea anti-diabetic drugs have been added to IDX conditions to promote adipocyte differentiation. Here we show that troglitazone, a peroxisome proliferator-activated receptor-gamma ($PPAR{\gamma}$) agonist, significantly reduced the levels of anti-adipogenic $PGE_2$ in IDX-conditioned hBM-MSC culture supernatants when compared to $PGE_2$ levels in the absence of $PPAR{\gamma}$ agonist. However, there was no difference in the mRNA levels of cyclooxygenases (COXs) and the activities of COXs and prostaglandin synthases during adipocyte differentiation in hBM-MSCs with or without troglitazone. In hBM-MSCs, troglitazone significantly increased the mRNA level of 15-hydroxyprostaglandin dehydrogenase (HPGD) which can act to decrease $PGE_2$ levels in culture. These results suggest that the role of $PPAR{\gamma}$ activation in promoting adipocyte differentiation in hBM-MSCs is to reduce anti-adipogenic $PGE_2$ levels through the up-regulation of HPGD expression.

• Biotechnology and Bioprocess Engineering:BBE
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• v.6 no.3
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• pp.173-178
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• 2001

Food yeast, Saccharomyces cerevisiae, is a safe organism with a long history of use for the production of biomass rich in high quality proteins and vitamins. AmA1, a seed storage albumin from Amaranthus hypochondriacus, has a well-balanced amino acid composition and high levels of essential amino acids and offers the possibility of further improving food animal feed additives. In order to find an effective means of expressing AmA1 in yeast, the gene was cloned into an episomal shuttle vector. Four different promoters were tested: the glyceraldehyde-3-phosphate dehydrogenase promoter, galactose dehydrogenase 10 promoter, alcohol dehydrogenase II promoter, and a hybrid ADH2-GPD promoter. The recombinant AmA1 genes were then introduced into the yeast Saccharomyces cerevisiae 2805. Northern and Western blot analyses of the yeast under appropriate conditions revealed that AmA1 was expressed by all four promoters at varying levels. An enzyme-linked immunosorbent assay demonstrated that the amount of AmA1 protein in the recombinant yeast was 1.3-4.3% of the total soluble proteins. The highest expression level was obtained from the hybrid ADH2-GPD promoter.

• Journal of Microbiology and Biotechnology
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• v.15 no.1
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• pp.118-124
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• 2005

In this research, we examined the effect of NaCl on the growth, energy metabolism, and proton motive force of Halomonas salina, and the effect of compatible solutes on the bacterium growing in the high salinity environment. H. salina was isolated from seawater and identified by 16srDNA sequencing. The growth of H. salina was not enhanced by the addition of external compatible solutes (choline and betaine) in the high salinity environment. The resting cells of H. salina absorbed more glucose in the presence of 2.0 M NaCl than in its absence. H. salina did not grow in the medium with either KCl, RbCl, CsCl, $Na_2SO_4$, or $NaNO_3$, in place of NaCl. The optimal concentration of NaCl for the growth of H. salina ranged from 1.4 M to 2.5 M, and the growth yield was decreased in the presence of NaCl below 1.4M and above 2.5M. The activity of isocitrate dehydrogenase, pyruvate dehydrogenase, and malate dehydrogenase of H. salina was not inhibited by NaCl in in vitro test. The proton translocation of H. salina was detected in the presence of NaCl only. These results indicate that NaCl is absolutely required for the normal growth and energy metabolism of H. salina, but the bacterial growth is not enhanced by the compatible solutes added to the growth medium.