Characterization of putative hydrophobic substrate binding site residues of a Delta class glutathione transferase from Anopheles dirus

To date, investigations of the hydrophobic substrate site of the insect Delta class glutathione transferase are limited in number. In the present study, putative hydrophobic site residues of AdGSTD4-4 have been proposed and characterized. These residues are Gln-112, Thr-174, Phe-212, Arg-214, Tyr-215 and Phe-216. It was found that Gln-112 does not contribute significantly to the catalytic properties of AdGSTD4-4. Arg-214, Tyr-215 and Phe-216 made contributions to catalytic properties and the rate-limiting step. Thr-174 and Phe-212 appeared to be important in enzymatic catalysis by stabilizing the active site β1-α1 loop on which the critical catalytic residue Ser-9 is located. The aromatic Phe-212 pi cloud appears to be important for interactions with its hydrophobic size representing an almost equally important factor. The data suggests that these residues are not directly involved in catalysis but exert their influence through secondary interactions. In addition, active site rearrangements occur to bring different residues into play even for conjugation through the same mechanisms. Therefore, due to the conformational rearrangements topologically equivalent residues observed in crystal structures may not perform equivalent roles in catalysis in different GST classes.     

Health Risk Assessment of Petrol Station Workers in the Inner City of Bangkok,Thailand, to the Exposure to BTEX and Carbonyl Compounds by Inhalation

The occupational health risk of petrol station workers from exposure to BTEX and carbonyl compounds via inhalation was estimated in the inner city of Bangkok. Personal sampling was performed within the workers’ breathing zone using 2,4 dinitrophenylhydrazine cartridges and charcoal glass tubes connected to a personal air pump during eight working hours at six petrol stations. BTEX and carbonyl compounds were quantitatively analyzed by GC/FID and HPLC/UV, respectively. Of all detectable BTEX and carbonyl compounds, the levels of the four most prevalent compounds (benzene, ethylbenzene, formaldehyde, and acetaldehyde) were used to assess the lifetime cancer risk and 95% confidence interval of the risk levels were found to be totally higher than acceptable criteria for benzene (1.82 × 10^–4–2.50 × 10^–4), formaldehyde (7.81 × 10^–6–1.04 × 10^–5), ethylbenzene (4.11 × 10^–6–5.52 × 10^–6), and acetaldehyde (1.39 × 10^–6–2.45 × 10^–6). Thus, petrol station workers in the inner city of Bangkok have a potentially high cancer risk through inhalation exposure. With respect to the noncarcinogenic agents, toluene, m,p-xylene, o-xylene, and propionaldehyde, all non-cancer health risk were within hazard quotients of 1 and of acceptable risk.     

Modulation of antioxidant defense by <Emphasis Type="Italic">Alpinia galanga</Emphasis> and <Emphasis Type="Italic">Curcuma aromatica</Emphasis> extracts correlates with their inhibition of UVA-induced melanogenesis

Ultraviolet A (UVA) irradiation is suggested to contribute to melanogenesis through promoting cellular oxidative stress and impairing antioxidant defenses. An overproduction of melanin can be associated with melanoma skin cancer and hyperpigmentation. Therefore, developing effective antimelanogenic agents is of importance. Alpinia galanga (AG) and Curcuma aromatica (CA) are traditional medicinal plants widely used for skin problems. Hence, this study investigated the antimelanogenic effects of AG and CA extracts (3.8–30 μg/ml) by assessing tyrosinase activity, tyrosinase mRNA levels, and melanin content in human melanoma cells (G361) exposed to UVA. The roles in protecting against melanogenesis were examined by evaluating their inhibitory effects on UVA-induced cellular oxidative stress and modulation of antioxidant defenses including antioxidant enzymes, catalase (CAT) and glutathione peroxidase (GPx), and intracellular glutathione (GSH). In addition, possible active compounds accountable for biological activities of the extracts were identified by thin layer chromatography (TLC)-densitometric analysis. Our study demonstrated that UVA (8 J/cm²) induced both tyrosinase activity and mRNA levels and UVA (16 J/cm²)-mediated melanin production were suppressed by the AG or CA extracts at noncytotoxic concentrations. Both extracts were able to protect against UVA-induced cellular oxidant formation and depletion of CAT and GPx activities and GSH content in a dose-dependent manner. Moreover, TLC-densitometric analysis detected the presence of eugenol and curcuminoids in AG and CA, respectively. This is the first report representing promising findings on AG and CA extract-derived antityrosinase properties correlated with their antioxidant potential. Inhibiting cellular oxidative stress and improving antioxidant defenses might be the mechanisms by which the extracts yield the protective effects on UVA-dependent melanogenesis.     

The Immunoreactive Exo-1,3-β-Glucanase from the Pathogenic Oomycete Pythium insidiosum Is Temperature Regulated and Exhibits Glycoside Hydrolase Activity

The oomycete organism, Pythium insidiosum, is the etiologic agent of the life-threatening infectious disease called “pythiosis”. Diagnosis and treatment of pythiosis is difficult and challenging. Novel methods for early diagnosis and effective treatment are urgently needed. Recently, we reported a 74-kDa immunodominant protein of P. insidiosum, which could be a diagnostic target, vaccine candidate, and virulence factor. The protein was identified as a putative exo-1,3-ß-glucanase (Exo1). This study reports on genetic, immunological, and biochemical characteristics of Exo1. The full-length exo1 coding sequence (2,229 bases) was cloned. Phylogenetic analysis showed that exo1 is grouped with glucanase-encoding genes of other oomycetes, and is far different from glucanase-encoding genes of fungi. exo1 was up-regulated upon exposure to body temperature, and its gene product is predicted to contain BglC and X8 domains, which are involved in carbohydrate transport, binding, and metabolism. Based on its sequence, Exo1 belongs to the Glycoside Hydrolase family 5 (GH5). Exo1, expressed in E. coli, exhibited ß-glucanase and cellulase activities. Exo1 is a major intracellular immunoreactive protein that can trigger host immune responses during infection. Since GH5 enzyme-encoding genes are not present in human genomes, Exo1 could be a useful target for drug and vaccine development against this pathogen.     

A stress-induced rice (Oryza sativa L.) beta-glucosidase represents a new subfamily of glycosyl hydrolase family 5 containing a fascin-like domain

GH5BG, the cDNA for a stress-induced GH5 (glycosyl hydrolase family 5) beta-glucosidase, was cloned from rice (Oryza sativa L.) seedlings. The GH5BG cDNA encodes a 510-amino-acid precursor protein that comprises 19 amino acids of prepeptide and 491 amino acids of mature protein. The protein was predicted to be extracellular. The mature protein is a member of a plant-specific subgroup of the GH5 exoglucanase subfamily that contains two major domains, a beta-1,3-exoglucanase-like domain and a fascin-like domain that is not commonly found in plant enzymes. The GH5BG mRNA is highly expressed in the shoot during germination and in leaf sheaths of mature plants. The GH5BG was up-regulated in response to salt stress, submergence stress, methyl jasmonate and abscisic acid in rice seedlings. A GUS (glucuronidase) reporter tagged at the C-terminus of GH5BG was found to be secreted to the apoplast when expressed in onion (Allium cepa) cells. A thioredoxin fusion protein produced from the GH5BG cDNA in Escherichia coli hydrolysed various pNP (p-nitrophenyl) glycosides, including beta-D-glucoside, alpha-L-arabinoside, beta-D-fucoside, beta-D-galactoside, beta-D-xyloside and beta-D-cellobioside, as well as beta-(1,4)-linked glucose oligosaccharides and beta-(1,3)-linked disaccharide (laminaribiose). The catalytic efficiency (kcat/K(m)) for hydrolysis of beta-(1,4)-linked oligosaccharides by the enzyme remained constant as the DP (degree of polymerization) increased from 3 to 5. This substrate specificity is significantly different from fungal GH5 exoglucanases, such as the exo-beta-(1,3)-glucanase of the yeast Candida albicans, which may correlate with a marked reduction in a loop that makes up the active-site wall in the Candida enzyme.     

Analysis of rice glycosyl hydrolase family 1 and expression of Os4bglu12 β-glucosidase

Background

Glycosyl hydrolase family 1 (GH1) β-glucosidases have been implicated in physiologically important processes in plants, such as response to biotic and abiotic stresses, defense against herbivores, activation of phytohormones, lignification, and cell wall remodeling. Plant GH1 β-glucosidases are encoded by a multigene family, so we predicted the structures of the genes and the properties of their protein products, and characterized their phylogenetic relationship to other plant GH1 members, their expression and the activity of one of them, to begin to decipher their roles in rice.

Results

Forty GH1 genes could be identified in rice databases, including 2 possible endophyte genes, 2 likely pseudogenes, 2 gene fragments, and 34 apparently competent rice glycosidase genes. Phylogenetic analysis revealed that GH1 members with closely related sequences have similar gene structures and are often clustered together on the same chromosome. Most of the genes appear to have been derived from duplications that occurred after the divergence of rice and Arabidopsis thaliana lineages from their common ancestor, and the two plants share only 8 common gene lineages. At least 31 GH1 genes are expressed in a range of organs and stages of rice, based on the cDNA and EST sequences in public databases. The cDNA of the Os4bglu12 gene, which encodes a protein identical at 40 of 44 amino acid residues with the N-terminal sequence of a cell wall-bound enzyme previously purified from germinating rice, was isolated by RT-PCR from rice seedlings. A thioredoxin-Os4bglu12 fusion protein expressed in Escherichia coli efficiently hydrolyzed β-(1,4)-linked oligosaccharides of 3–6 glucose residues and laminaribiose.

Conclusion

Careful analysis of the database sequences produced more reliable rice GH1 gene structure and protein product predictions. Since most of these genes diverged after the divergence of the ancestors of rice and Arabidopsis thaliana, only a few of their functions could be implied from those of GH1 enzymes from Arabidopsis and other dicots. This implies that analysis of GH1 enzymes in monocots is necessary to understand their function in the major grain crops. To begin this analysis, Os4bglu12 β-glucosidase was characterized and found to have high exoglucanase activity, consistent with a role in cell wall metabolism.