These results indicated that quantitative analysis of proteome components is a feasible method for examining disease-associated proteins and clustering clinical strains of H. pylori.
Garlics were aged at 60, 70, 80, and 90 o C for 1, 3 and 6 days. Samples were analyzed for physico-chemical components and antioxidant activities, such as DPPH scavenging activity and reducing power of hot water and ethanol extracts. The Hunter L, a and b values were significantly lower in sample aged at higher temperature and for longer time. In initiation of aging, the share force was the lowest in sample aged at 90 o C (85.70±1.44 kg/cm 2 ) and it increased for 6 days to 411.30±13.90 kg/cm 2 in aging. The pH of garlic was acidified at increasing aged temperature and periods. In sample aged for 6 days at 60 o C and 90 o C, pH was 6.12 and 3.90, respectively. Contents of total phenolics and flavonoids also increased in sample aged at higher temperature and for longer time. Their contents increased about 3.5 and 9.1 times higher in sample aged for 6 days at 90 o C than sample aged for 6 days at 60 o C, respectively. Total pyruvate contents were fluctuated by aging temperature and periods. DPPH scavenging activity was increased in sample aged at higher temperature and longer time. The highest activity of DPPH scavenging showed 87.48±0.20% in sample aged for 6 days at 90 o C. Similar results were observed in reducing power activity. It was estimated that such increases in anti-oxidant activities in aged garlics may come from actions of phenolics, flavonoids and browning compounds in them.
An NADPH-dependent aldehyde reductase (ALR) isolated from a red yeast, Sporobolomyces salmonicolor, catalyzes the reduction of a variety of carbonyl compounds. To investigate its primary structure, we cloned and sequenced the cDNA coding for ALR. The aldehyde reductase gene (ALR) comprises 969 bp and encodes a polypeptide of 35,232 Da. The deduced amino acid sequence showed a high degree of similarity to other members of the aldo-keto reductase superfamily. Analysis of the genomic DNA sequence indicated that the ALR gene was interrupted by six introns (two in the 5 noncoding region and four in the coding region). Southern hybridization analysis of the genomic DNA from S. salmonicolor indicated that there was one copy of the gene. The ALR gene was expressed in Escherichia coli under the control of the tac promoter. The enzyme expressed in E. coli was purified to homogeneity and showed the same catalytic properties as did the enzyme from S. salmonicolor. Aldehyde reductase (ALR) (EC 1.1.1.2), aldose reductase (EC 1.1.1.21), and carbonyl reductase (EC 1.1.1.184) catalyze the NADPH-dependent reduction of a variety of carbonyl compounds and are widely distributed in mammalian and plant tissues. The amino acid sequences of aldose reductases and ALRs show significant similarity, but that of carbonyl reductase does not show similarity to the other sequences. These enzymes are members of the aldo-keto reductase superfamily (3), which includes prostaglandin F synthase (29),-crystalline (8), ␦-3-ketosteroid 5--reductase (17), a soybean reductase (30), and chlordecone reductase (34). The yeast GCY gene product (16), the p100/11E gene product of Leishmania major (21), and mouse androgen-dependent protein (19), whose functions are not known, also show a high degree of sequence similarity with members of the aldo-keto reductase superfamily. The physiological roles of the aldo-keto reductases have not been established. It is suggested that under physiological conditions aldose reductase participates in osmoregulation, but under hyperglycaemic conditions it contributes to the onset and development of severe complications in diabetes. We isolated an NADPH-dependent ALR from the red yeast Sporobolomyces salmonicolor, which in addition to catalyzing common substrates of ALRs (35) can catalyze the asymmetric reduction of ethyl 4-chloro-3-oxobutanoate to ethyl (R)-4chloro-3-hydroxybutanoate, a promising chiral building block for chemical synthesis of L-carnitine. L-Carnitine deficiency is a common secondary problem associated with many metabolic diseases, and oral administration of the compound produces a
Among 1590 ORFs in the Helicobacter pylori genome, >250 have been identified as authentic genes by proteomic analysis. Low-abundance proteins need to be enriched to a minimal amount for MALDI-TOF analysis and salt precipitation has generally been used for protein enrichment. Here, a whole-cell extract of H. pylori strain 26695 was subjected to protein fractionation with stepwise concentrations of ammonium sulfate and the proteins were displayed by 2-DE. The protein spots were quantified using PDQUEST software and identified by peptide fingerprinting. The 2-DE profiles and intensities of individual protein spots differed among the protein fractions. Out of the 98 identified proteins, 61 were found in the stepwise ammonium sulfate fractions but not in the whole-cell extract. Out of these, 37 proteins, including KdsA, were found exclusively in a single fraction. In contrast, GroEL, UreA, UreB, TrxA, NapA, and FldA were ubiquitously present in all fractions. Iron-containing proteins such as NapA, SodB, CeuE, and Pfr were found predominantly in the 100% saturated ammonium sulfate precipitate. Additionally, 29 proteins were newly identified in this study. These data will facilitate the preparation of significant H. pylori proteins, as well as provide information about low-abundance proteins.
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