A New DNA‐Intercalative Cytotoxic Allylic Xanthone from Swertia corymbosa

Phytochemical investigation of the CHCl₃ fraction of Swertia corymbosa resulted in the isolation of a new 3‐allyl‐2,8‐dihydroxy‐1,6‐dimethoxy‐9H‐xanthen‐9‐one ( 1 ), along with four known xanthones, gentiacaulein ( 3 ), norswertianin ( 4 ), 1,3,6,8‐tetrahydroxyxanthone ( 5 ), and 1,3‐dihydroxyxanthone ( 6 ). Structure of compound 1 was elucidated with the aid of IR, UV, NMR, and MS data, and chemical transformation via new allyloxy xanthone derivative ( 2 ). Compounds 1 – 6 exhibited various levels of antioxidant and anti‐α‐glucosidase activities. Absorption and fluorescence spectroscopic studies on 1 – 6 indicated that these compounds could interact with calf thymus DNA (CT‐DNA) through intercalation and with bovine serum albumin (BSA) in a static quenching process. Compound 1 was found to be significantly cytotoxic against human cancer cell lines HeLa, HCT116, and AGS, and weakly active against normal NIH 3T3 cell line.     

Isolation and identification of a novel valienamine-producing bacterium

AIMS: To isolate, identify and assess valienamine production by a soil bacterial isolate from a wheat field in Hangzhou, China. METHODS AND RESULTS: A validamycin A-degrading bacterial strain, numbered ZJB-041, was isolated and identified as Stenotrophomonas maltophilia, based on morphology, physiological tests, ATB system (ID32 GN), and 16S rDNA analysis. The strain was capable of producing valienamine by decomposing validamycin A. After fermentation in shaking flasks at 30 degrees C for 7 days, 96.0% of 34.49 mmol l(-1) of validamycin A was degraded and 2.65 mmol l(-1) of valienamine was obtained. The resting cells of this strain also produced valienamine by degrading validamycin A. After 72 h of incubation in 0.2 mol l(-1) of phosphate buffer (pH 7.5), 90.2% of 17.16 mmol l(-1) of validamycin A was degraded, and 1.77 mmol l(-1) of valienamine was obtained. CONCLUSIONS: Our data suggested that S. maltophilia ZJB-041, a bacterial isolate, has the potential for validamycin A degradation and valienamine production. SIGNIFICANCE AND IMPACT OF THE STUDY: The validamycin A-degrading bacterium could potentially be utilized in the disposal of validamycin residues and in the production of valienamine.     

Insight into glucosidase II from the red marine microalga Porphyridium sp. (Rhodophyta)

N‐glycosylation of proteins is one of the most important post‐translational modifications that occur in various organisms, and is of utmost importance for protein function, stability, secretion, and loca‐lization. Although the N‐linked glycosylation pathway of proteins has been extensively characterized in mammals and plants, not much information is available regarding the N‐glycosylation pathway in algae. We studied the α 1,3‐glucosidase glucosidase II (GANAB) glycoenzyme in a red marine microalga Porphyridium sp. (Rhodophyta) using bioinformatic and biochemical approaches. The GANAB‐gene was found to be highly conserved evolutionarily (compo‐sed of all the common features of α and β subunits) and to exhibit similar motifs consistent with that of homolog eukaryotes GANAB genes. Phylogenetic analysis revealed its wide distribution across an evolutionarily vast range of organisms; while the α subunit is highly conserved and its phylogenic tree is similar to the taxon evolutionary tree, the β subunit is less conserved and its pattern somewhat differs from the taxon tree. In addition, the activity of the red microalgal GANAB enzyme was studied, including functional and biochemical characterization using a bioassay, indicating that the enzyme is similar to other eukaryotes ortholog GANAB enzymes. A correlation between polysaccharide production and GANAB activity, indicating its involvement in polysaccharide biosynthesis, is also demonstrated. This study represents a valuable contribution toward understanding the N‐glycosylation and polysaccharide biosynthesis pathways in red microalgae.     

Exotic grasses and nitrate enrichment alter soil carbon cycling along an urban–rural tropical forest gradient

Urban areas are expanding rapidly in tropical regions, with potential to alter ecosystem dynamics. In particular, exotic grasses and atmospheric nitrogen (N) deposition simultaneously affect tropical urbanized landscapes, with unknown effects on properties like soil carbon (C) storage. We hypothesized that (H1) soil nitrate (NO₃^?) is elevated nearer to the urban core, reflecting N deposition gradients. (H2) Exotic grasslands have elevated soil NO₃^? and decreased soil C relative to secondary forests, with higher N promoting decomposer activity. (H3) Exotic grasslands have greater seasonality in soil NO₃^? vs. secondary forests, due to higher sensitivity of grassland soil moisture to rainfall. We predicted that NO₃^? would be positively related to dissolved organic C (DOC) production via changes in decomposer activity. We measured six paired grassland/secondary forest sites along a tropical urban‐to‐rural gradient during the three dominant seasons (hurricane, dry, and early wet). We found that (1) soil NO₃^? was generally elevated nearer to the urban core, with particularly clear spatial trends for grasslands. (2) Exotic grasslands had lower soil C than secondary forests, which was related to elevated decomposer enzyme activities and soil respiration. Unexpectedly, soil NO₃^? was negatively related to enzyme activities, and was lower in grasslands than forests. (3) Grasslands had greater soil NO₃^? seasonality vs. forests, but this was not strongly linked to shifts in soil moisture or DOC. Our results suggest that exotic grasses in tropical regions are likely to drastically reduce soil C storage, but that N deposition may have an opposite effect via suppression of enzyme activities. However, soil NO₃^? accumulation here was higher in urban forests than grasslands, potentially related to of aboveground N interception. Net urban effects on C storage across tropical landscapes will likely vary depending on the mosaic of grass cover, rates of N deposition, and responses by local decomposer communities.     

Degradation of rice bran hemicellulose by <Emphasis Type="Italic">Paenibacillus</Emphasis> sp. strain HC1: gene cloning,characterization and function of β-D-glucosidase as an enzyme involved in degradation

A bacterium (strain HC1) capable of assimilating rice bran hemicellulose was isolated from a soil and identified as belonging to the genus Paenibacillus through taxonomical and 16S rDNA sequence analysis. Strain HC1 cells grown on rice bran hemicellulose as a sole carbon source inducibly produced extracellular xylanase and intracellular glycosidases such as β-d-glucosidase and β-d-arabinosidase. One of them, β-d-glucosidase was further analyzed. A genomic DNA library of the bacterium was constructed in Escherichia coli and gene coding for β-d-glucosidase was cloned by screening for β-d-glucoside-degrading phenotype in E. coli cells. Nucleotide sequence determination indicated that the gene for the enzyme contained an open reading frame consisting of 1,347 bp coding for a polypeptide with a molecular mass of 51.4 kDa. The polypeptide exhibits significant homology with other bacterial β-d-glucosidases and belongs to glycoside hydrolase family 1. β-d-Glucosidase purified from E. coli cells was a monomeric enzyme with a molecular mass of 50 kDa most active at around pH 7.0 and 37°C. Strain HC1 glycosidases responsible for degradation of rice bran hemicellulose are expected to be useful for structurally determining and molecularly modifying rice bran hemicellulose and its derivatives.     

Glycogen storage in multiple muscles of old GSD-II mice can be rapidly cleared after a single intravenous injection with a modified adenoviral vector expressing hGAA

BACKGROUND: Glycogen storage disease II (GSD-II) is an autosomal recessive lysosomal storage disease, due to acid-alpha-glucosidase (GAA) deficiency. The disease is characterized by massive glycogen accumulation in the cardiac and skeletal muscles. There is early onset (infantile, also known as Pompe disease) as well as late onset (juvenile and adult) forms of GSD-II. Few studies have been published to date that have explored the consequences of delivering a potential therapy to either late onset GSD-II subjects, and/or early onset patients with long-established muscle pathology. One recent report utilizing GAA-KO mice transgenically expressing human GAA (hGAA) suggested that long-established disease in both cardiac and skeletal muscle is likely to prove resistant to therapies. To investigate the potential for disease reversibility in old GSD-II mice, we studied their responsiveness to exogenous hGAA exposure via a gene therapy approach that we have previously shown to be efficacious in young GAA-KO mice. METHODS: An [E1-, polymerase-] adenoviral vector encoding hGAA was intravenously injected into two groups of aged GAA-KO mice; GAA expression and tissue glycogen reduction were evaluated. RESULTS: After vector injection, we found that extremely high amounts of hepatically secreted hGAA could be produced, and subsequently taken up by multiple muscle tissues in the old GAA-KO mice by 17 days post-injection (dpi). As a result, all muscle groups tested in the old GAA-KO mice showed significant glycogen reductions by 17 dpi, relative to that of age-matched, but mock-injected GAA-KO mice. For example, glycogen reduction in heart was 84%, in quadriceps 46%, and in diaphragm 73%. Our data also showed that the uptake and the subsequent intracellular processing of virally expressed hGAA were not impaired in older muscles. CONCLUSIONS: Overall, the previously reported 'resistance' of old GAA-KO muscles to exogenous hGAA replacement approaches can be rapidly overcome after a single intravenous injection with a modified adenoviral vector expressing hGAA.     

A comparative study of activity and apparent inhibition of fungal β‐glucosidases

β‐Glucosidases (BGs) from Aspergillus fumigates, Aspergillus niger, Aspergillus oryzae, Chaetomium globosum, Emericella nidulans, Magnaporthe grisea, Neurospora crassa, and Penicillium brasilianum were purified to homogeneity, and analyzed by isothermal titration calorimetry with respect to their hydrolytic activity and its sensitivity to glucose (product) using cellobiose as substrate. Global non‐linear regression of several reactions, with or without added glucose, to a product inhibition equation enabled the concurrent derivation of the kinetic parameters k_cat, K_m, and the apparent product inhibition constant _appK_i for each of the enzymes. A more simple fit is not advisable to use as the determined _appK_i are in the same range as their K_m for some of the tested BGs and produced glucose would in these cases interfere. The highest value for k_cat was determined for A. fumigatus (768 s^?1) and the lowest was a factor 9 less. K_m varied by a factor of 3 with the lowest value determined for C. globosum (0.95 mM). The measured _appK_i varied a factor of 15; the hydrolytic activity of N. crassa being the most resistant to glucose with an apparent product inhibition constant of 10.1 mM. Determination of _appK_i using cellobiose as substrate is important as it reflects to what extent the different BGs are hydrolytically active under industrial conditions where natural substrates are hydrolyzed and the final glucose concentrations are high. Biotechnol. Bioeng. 2010;107: 943–952. © 2010 Wiley Periodicals, Inc.     

Evaluation of novel fluorogenic substrates for the detection of glycosidases in Escherichia coli and enterococci

AIMS: Enzyme substrates based on 4-methylumbelliferone are widely used for the detection of Escherichia coli and enterococci in water, by detection of beta-glucuronidase and beta-glucosidase activity respectively. This study aimed to synthesize and evaluate novel umbelliferone-based substrates with improved sensitivity for these two enzymes. METHODS AND RESULTS: A novel beta-glucuronide derivative based on 6-chloro-4-methylumbelliferone (CMUG) was synthesized and compared with 4-methylumbelliferyl-beta-D-glucuronide (MUG) using 42 strains of E. coli in a modified membrane lauryl sulfate broth. Over 7 h of incubation, the fluorescence generated from the hydrolysis of CMUG by E. coli was over twice that from MUG, and all of the 38 glucuronidase-positive strains generated a higher fluorescence with CMUG compared with MUG. Neither substrate caused inhibition of bacterial growth in any of the tested strains. Four beta-glucosidase substrates were also synthesized and evaluated in comparison with 4-methylumbelliferyl-beta-D-glucoside (MU-GLU) using 42 strains of enterococci in glucose azide broth. The four substrates comprised beta-glucoside derivatives of umbelliferone-3-carboxylic acid and its methyl, ethyl and benzyl esters. Glucosides of the methyl, ethyl and benzyl esters of umbelliferone-3-carboxylic acid, were found to be superior to MU-GLU for the detection of enterococci, especially after 18 h of incubation, while umbelliferone-3-carboxylic acid-beta-D-glucoside was inferior. However, the variability in detectable beta-glucosidase activity among the different strains of enterococci in short-term assays using the three carboxylate esters (7 h incubation) may compromise their use for rapid detection and enumeration of these faecal indicator bacteria. CONCLUSIONS: The beta-glucuronidase substrate CMUG appears to be a more promising detection system than the various beta-glucosidase substrates tested. SIGNIFICANCE AND IMPACT OF THE STUDY: The novel substrate CMUG showed enhanced sensitivity for the detection of beta-glucuronidase-producing bacteria such as E. coli, with a clear potential for application in rapid assays for the detection of this indicator organism in natural water and other environmental samples.     

Seminal plasma zinc concentration and α-glucosidase activity with respect to semen quality

This study was conducted to examine the relationship between the concentration of zinc and neutral α-glucosidase (NAG) with semen quality. Semen samples from 75 male partners of couples who were attending the Obstetrics and Gynecology Department were analyzed for semen quality. Based on sperm count, the subjects were divided into three groups. Zinc and neutral α-glucosidase activity were estimated in seminal plasma. Results showed that mean the α-glucosidase activity was lowest among the azoospermic group with respect to oligozoospermic and normozoospermic groups. Mean zinc levels were also lower among azoospermics compared to oligozoospermic and normospermic groups. The difference was statistically significant (p<0.05). A significant positive correlation was observed between zinc levels and sperm count (r=0.29, p<0.05) and zinc and α-glucosidase activity (r=0.31, p<0.05) in seminal plasma. These results suggest that zinc and neutral α-glucosidase seem to play an important role in human reproduction.