Cloning and analysis of laccases from acinetobacter baumannii isolates

Laccases (benzenediol : oxygen oxi doreductases; EC 1.10.3.2) are wide spread i n nature. They are usually found in higher plants and fungi (Thurston 19 94; Mayer and Staples 2002), but recently some bacterial laccases have also been found . The first laccase studied was from Rhus vernicifera in 1883, a Japanese lacquer tree, fr om which the name laccase was derived (Yoshida , 1883). These enzymes belong to the group of bl ue multi - copper oxidases (MCOs) . They usually contain four copper atoms located in three distinct sites. Each site reacts differently to light. The Type 1 (T1) site copper atom absorbs intensely at 600 nm and emits the blue light , the Type 2 (T2) site copper atom is not visible in the absorption spectr um and last, the Type 3 (T3) site has two c opper atoms and absorbs at 330 nm ( Santhanam et al . , 2011; Quintanar et al . , 2007 ) . The protei n structure acts as a complex ligand for the catalytic coppers, providing them the right structure where changes between the reduction states are thermodynamically possible (Dub é , 2008 ) . These enzymes oxidize a surprisingly wide variety of organic and inorganic compounds like, diphenols, polyphenols, substituted phenols, diamines and a romatic amines, with concomitant reduction of molecular oxygen to water (Thurston , 1

A key to selected rockfishes (Sebastes spp.) based on mitochondrial DNA restriction fragment analysis

Larval and juvenile rockfishes (Sebastes spp.) are difficult to identify using morphological characters. We developed a key based on sizes of restriction endonuclease fragments of the NADH dehydrogenase-3 and -4 (ND3/ND4) and 12S and 16S ribosomal RNA (12S/16S) mitochondrial regions. The key makes use of variation in the ND3/ND4 region. Restriction endonuclease Dde I variation can corroborate identifications, as can 12S/16S variation. The key, based on 71 species, includes most North American taxa, several Asian species, and Sebastolobus alascanus and Helicolenus hilgendorfi that are closely related to rockfishes. Fifty-eight of 71 rockfish species in our database can be distinguished unequivocally, using one to five restriction enzymes; identities of the remaining species are narrowed to small groups: 1) S. polyspinis, S. crameri, and S. ciliatus or variabilis (the two species could not be distinguished and were considered as a single species) ; 2) S. chlorostictus, S. eos, and S. rosenblatti; 3) S. entomelas and S. mystinus; 4)S. emphaeus, S. variegatus, and S. wilsoni; and 5) S. carnatus and S. chrysomelas.

Characterization of three fibrinogenolytic enzymes from Chinese green tree viper (Trimeresurus stejnegeri) venom

Rong Gao, Yun Zhang, Qing-Xiong Meng, Wen-Hui Lee, Dong-Sheng Li, Yu-liang Xiong and Wan-Yu Wang. Characterization of three fibrinogenolytic enzymes from Chinese green tree viper (Trimeresurus stejneger ) venom. Toxicon 36, 457-467, 1998.-From the venom of Chinese green tree viper (Trimeresurus stejnegeri), three distinct fibrinogenolytic enzymes: stejnefibrase-l, stejnefibrase-2 and stejnefibrase-3, were purified by gel filtration, ion-exchange chromatography and reverse-phase high-performance chromatograghy (HPLC). SDS-PAGE analysis of those three enzymes showed that they consisted of a single polypeptide chain with mel. wt of -50 000, 31 000 and 32 000, respectively. Like TSV-PA (a specific plasminogen activator) and stejnobin (a fibrinogen-clotting enzyme) purified from the same venom, stejnfibrase-1, -2 and -3 were able to hydrolyze several chromogenic substrate. On the other hand, different from TSV-PA. and stejnobin, stejnefibrase-l, -2 and -3 did not activate plasminogen and did not possess fibrinogen-clotting activity. The three purified enzymes directly degraded fibrinogen to small fragments and rendered it unclottable by thrombin. Stejnefibrase-2 degraded preferentially BE-chain while stejnefibrase-l and -3 cleaved concomitantly Ax and B beta-chains of fibrinogen. None of these proteases degraded the gamma-chain of fibrinogen. When correlated with the loss of clottability of fibrinogen, the most active enzyme was stejnefibrase-l. The activities of the three enzymes were inhibited by phenylmethylsulfonyl fluoride (PMSF) and p-nitrophenyl-p-guanidinobenzoate (NPGB), indicating that like TSV-PA and stejnobin, they are venom serine proteases. (C) 1998 Elsevier Science Ltd. All rights reserved.

GENETIC DIVERSITY IN THE CHINESE PANGOLIN (MANIS-PENTADACTYLA) - INFERRED FROM RESTRICTION ENZYME ANALYSIS OF MITOCHONDRIAL DNAS

Two different forms of Chinese pangolins can be recognized according to the color of their scales, i.e., brown and dusky. We analyzed mitochondrial DNA (mtDNA) purified from the livers of seven dusky and six brown Chinese pangolins from the same locality, using cleavage patterns from 19 restriction enzymes. From the 19 6-bp recognition enzymes used, 51-56 sites were observed. By combining the cleavage patterns for each enzyme, the 13 samples were classified into four restriction types: two in dusky and two in brown Chinese pangolins. The estimated number of nucleotide substitutions per site in dusky and brown types is 0.002, and that between dusky and brown types is 0.012. Divergence between brown and dusky forms began 0.6 Myr ago, provided the mean rate of sequence divergence is 0.02 per Myr in mtDNA. Our results suggest that there is considerable divergence in Chinese pangolins, and brown and dusky Chinese pangolins may be quite different forms or, at least, belong to different maternal groups.

Identification, characteristic and phylogenetic analysis of type II DNA topoisomerase gene in Giardia lamblia

The genes encoding type II DNA topoisomerases were investigated in Giardia lamblia genome, and a type IIA gene, GlTop 2 was identified. It is a single copy gene with a 4476 by long ORF without intron. The deduced amino acid sequence shows strong homology to eukaryotic DNA Top 2. However, some distortions were found, such as six insertions in the ATPase domain and the central domain, a similar to 100 as longer central domain; a similar to 200 as shorter C-terminal domain containing rich charged residues. These features revealed by comparing with Top 2 of the host, human, might be helpful in exploiting drug selectivity for antigiardial therapy. Phylogenetic analysis of eukaryotic enzymes showed that kinetoplastids, plants, fungi, and animals were monophyletic groups, and the animal and fungi lineages shared a more recent common ancestor than either did with the plant lineage; microsporidia grouped with fungi. However, unlike many previous phylogenetic analyses, the "amitochondriate" G. lamblia was not the earliest branch but diverged after mitochondriate kinetoplastids in our trees. Both the finding of typical eukaryotic type IIA topoisomerase and the phylogenetic analysis suggest G. lamblia is not possibly as primitive as was regarded before and might diverge after the acquisition of mitochondria. This is consistent with the recent discovery of mitochondrial remnant organelles in G. lamblia.

Induction of hepatic enzymes and oxidative stress in Chinese rare minnow (Gobiocypris rarus) exposed to waterborne hexabromocyclododecane (HBCDD)

The objective of this study was to evaluate the sub-lethal toxicity of hexabromocyclododecane (HBCDD) in fish. Adult Chinese rare minnows as in vivo models were exposed to waterborne HBCDD from 1 to 500 mu g/l for 14, 28 and 42 days. Hepatic CYP1A1 (ethoxyresorufin-O-deethylase, EROD) and CYP2B1 (pentaoxyresorufin-O-depentylase, PROD) activities were measured. At the same time, molecular biomarkers of oxidative stress were also assayed in the brain, including reactive oxygen species (ROS), lipid peroxidation products (thiobarbituric acid-reactive substances, TBARS), DNA damage and protein carbonyl, as well as superoxide dismutase (SOD) activity and glutathione (GSH) content. DNA damage was evaluated using the Comet assay on erythrocytes. Besides, the content of HBCDD in whole fish was determined after 42 days exposure. The results show that HBCDD could induce EROD and PROD at 500 mu g/l after 28 days exposure, and at 100 to 500 mu g/l after 42 days exposure (P < 0.05), respectively. ROS formation in fish brain was observed to be increased in both time- and dose-dependent manner due to HBCDD exposure. The significant increases in TBARS and protein carbonyl contents occurred in fish brain after 28 and 42 days exposure (P < 0.05). Significant DNA damage in erythrocytes by Comet assay was also found in the 100-500 mu g/l exposure groups (P < 0.05) after 42 days exposure. Moreover, significant depletion in brain GSH content occurred in all treated groups (P < 0.05) and apparent inhibition in SOD activity in brain was observed in the groups of 10-500 mu g/l concentrations during 42 days exposure. The results demonstrate that increasing duration of HBCDD exposure induced EROD and PROD activities, caused excess ROS formation, finally resulted in oxidative damage to lipids, proteins and DNA and decreased antioxidant capacities in fish. Chemical analysis of HBCDD in whole fish showed accumulation up to 654 mu g/g wet weight. (c) 2007 Elsevier B.V. All rights reserved.

PCR-RFLP analysis of mitochondrial DNA ND5/6 region among 3 subspecies of common carp (Cyprinus carpio L.) and its application to genetic discrimination of subspecies

Mitochondrial DNA ND5/6 region was studied by PCR-RFLP analysis among ten representative strains belonging to three subspecies (Cyprinus carpio carpio, Cyprinus carpio haematopterus and Cyprinus carpio rubrofuscus) of common carp (Cyprinus carpio L.). A total of 2.4 kb fragment was amplified and subjected to restriction endonuclease analysis with nine restriction endonucleases subsequently. The results indicated that each subspecies owned one hyplotype and four restriction enzymes (Dde I, HaeIII, Taq I and Mbo I) produced diagnostic restriction sites which could be used for discriminating the three subspecies and as molecular genetic markers for assistant selective breeding of common carp.

Excretion pattern investigation of urinary normal and modified nucleosides of breast cancer patients by RP-HPLC and factor analysis method

Modified nucleosides, formed post-transcriptionally in RNA by a number of modification enzymes, are excreted in abnormal levels in the urine of patients with malignant tumors. To test their usefulness as tumor markers, and to compare them with the conventional tumor markers, a reversed-phase high-performance liquid chromatographic (RP-HPLC) method and a factor analysis method have been used to study the excretion pattern of nucleosides of breast cancer patients. A clear cut differentiation of the breast cancer group and the healthy individuals in two clusters without overlapping was obtained. Copyright (C) 2000 John Wiley & Sons, Ltd.

H-1 NMR analysis for metabolites in serum and urine from rats administrated chronically with La(NO3)(3)

H-1 NMR spectroscopy has been used to assess long-term toxicological effects of a rare earth. Male Wistar rats were administrated orally with La(NO3)(3) at doses of 0.1, 0.2, 2.0, 10, and 20 mg/kg body wt, resp., for 3-6 months. Urine was collected at 1, 2, and 3 months and serum samples were taken after 6 months. Numerous low-M-r metabolites in rats serum and rats urine, including creatinine, citrate, glucose, ketone bodies, trimethylamine N-oxide (TMAO), and various amino acids, were identified on 400- and 500-MHz H-1 NMR spectra. La3+-induced renal and liver damage is characterized by an increase in the amounts of the excreted ketone bodies, amino acids, lactate, ethanol, succinate, TMAO, dimethylamine, and taurine and a decrease in citrate, glucose, urea, and allantoin. Information on the molecular basis of the long-term toxicity of La(NO3)(3) was derived from the abnormal patterns of metabolite excretions. An assay of some biochemical indexes and analysis of some enzymes in plasma supported NMR results.

Comparative Analysis of Fatty Acid Desaturases in Cyanobacterial Genomes

Fatty acid desaturases are enzymes that introduce double bonds into the hydrocarbon chains of fatty acids. The fatty acid desaturases from 37 cyanobacterial genomes were identified and classified based upon their conserved histidine-rich motifs and phylogenetic analysis, which help to determine the amounts and distributions of desaturases in cyanobacterial species. The filamentous or N-2-fixing cyanobacteria usually possess more types of fatty acid desaturases than that of unicellular species. The pathway of acyl-lipid desaturation for unicellular marine cyanobacteria Synechococcus and Prochlorococcus differs from that of other cyanobacteria, indicating different phylogenetic histories of the two genera from other cyanobacteria isolated from freshwater, soil, or symbiont. Strain Gloeobacter violaceus PCC 7421 was isolated from calcareous rock and lacks thylakoid membranes. The types and amounts of desaturases of this strain are distinct to those of other cyanobacteria, reflecting the earliest divergence of it from the cyanobacterial line. Three thermophilic unicellular strains, Thermosynechococcus elongatus BP-1 and two Synechococcus Yellowstone species, lack highly unsaturated fatty acids in lipids and contain only one Delta 9 desaturase in contrast with mesophilic strains, which is probably due to their thermic habitats. Thus, the amounts and types of fatty acid desaturases are various among different cyanobacterial species, which may result from the adaption to environments in evolution. Copyright (c) 2008 Xiaoyuan Chi et al.

Molecular cloning and functional analysis of cathepsin B in nutrient metabolism during larval development in Meretrix meretrix

Seed rearing is an important part in large scale clam culture industry. Since the nutritional history affects early development in bivalve, the condition of larval nutrition plays a key role in successful seed rearing. So far, the molecular mechanism of nutrient uptake in bivalve larvae is unclear. As one of the important proteolytic enzymes, cathepsin B of several organisms has been reported to be involved in digestion. We intended to analyze whether cathepsin B is involved in larval nutrient metabolism in the economic bivalve, clam Meretrix meretrix. The full length of M. meretrix cathepsin B (MmeCB) cDNA was cloned, which is 1647 bp with an open reading frame of 1014 bp. The deduced amino acid sequence encoded a preproenzyme of 337 residues with Cys-114, His-282 and Asn-302 composing cathepsin B activity center. The temporal and spatial expressions of MmeCB mRNA were examined from trochophore to post larva stages by whole mount in situ hybridization. In trochophore stage, no detectable signal was found. In the later three stages, MmeCB mRNA was detected in the digestive gland, suggesting a possible role of MmeCB in digestion. Moreover, MmeCB mRNA was also observed in the epidermal cells in D-veligers. Cathepsin B specific inhibitor (CA074 methyl ester) was applied to block the activity of cathepsin B in unfed larvae. The average shell lengths of treated larvae were smaller than that in control groups. The results of mRNA epidermal distribution and inhibitor treatment in D-veligers indicated that MmeCB may be also associated with other pathway of nutrient metabolism in larval epidermis. The overall results in this paper revealed that MmeCB might play a role in larval nutrient metabolism. (C) 2008 Elsevier B.V. All rights reserved.

Molecular cloning, characterization and evolutionary analysis of phytoene desaturase (PDS) gene from Haematococcus pluvialis

Phytoene desaturase is one of the most important enzymes necessary for the biosynthesis of carotenoids in some cyanobacteria, green algae and plants. In this study, genomic DNA and cDNA of pds were cloned from unicellular green alga Haematococcus pluvialis strain323 using PCR and RT-PCR methods. The cDNA was cloned into plasmid pET-28a and efficiently expressed in Escherichia coli BL21. The complete genomic PDS gene of H. pluvialis, 3.3 kb in size, included eight exons and seven introns. To locate transcriptional regulation elements, an approximate 1 kb of 5'-flanking region was isolated by genome-walking method. Results of bioinformatic analysis showed several putative cis-elements e.g. the ABRE motif (abscisic acid responsive element), the C-repeat/DRE (dehydration responsive element) motif and the GCN4 motif were located in 5'-flanking region of pds. Results of phylogenetic analyses reveal that different sources of PDS genes form a separate clade, respectively, with 100% bootstrap support. Moreover, a maximum likelihood approach was employed to detect evidence of positive selection in the evolution of PDS genes. Results of branch-site model analysis suggest that 7.9% of sites along the green algal branch are under positive selection, and the PDS gene in green algae exhibits a different evolutionary pattern from its counterparts in cyanobacteria and plants.

Gastroprotective potential of Buddleja scordioides Kunth: Effects into the modulation of antioxidant enzymes and inflammation markers in an in vivo model

Ethnopharmacological relevance: A common plant used to treat several gastric disorders is Buddleja scordioides Kunth,commonly known as salvilla. Aim of thes tudy: To detect inflammatory markers,in order to evaluate the gastroprotective potential of salvilla infusions,as this could have beneficial impact on the population exposed to gastric ulcers and colitis. Materials and methods: The present work attempted infusions were prepared with B. scordioides (1% w/w) lyophilized and stored.Total phenolic content and GC–MS analysis were performed. Wistar rats were divided into five groups a negative vehicle control,an indomethacin group,and three experimental groups,named preventive,curative,and suppressive. All rats were sacrificed under deep ether anesthesia(6h)after the last oral administration of indomethacin/infusion.The rat stomachs were promptly excised,weighed,and chilled in ice-cold and 0.9%NaCl.Histological analysis,nitrites quantification and immunodetection assays were done. Results: B.scordioides infusions markedly reduced the visible hemorrhagic lesions induced byindomethacin in rat stomachs,also showed down-regulation of COX2, IL-8 and TNFα and up-regulation of COX-1with a moderate down-regulation of NFkB and lower amount of nitrites.However,this behavior was dependent on the treatment,showing most down-regulation of COX-2,TNFα and IL-8 in the curative treatment;more down-regulation of NF-kB in the preventive treatment;and more up-regulation of COX-1 for the suppressor and preventive treatments. Conclusion: The anti-inflammatory potential of B. scordioides infusions could be related with the presence of polyphenols as quercetin in the infusion and how this one is consumed.

Molecular analysis of the anaerobic rumen fungus Orpinomyces - insights into an AT-rich genome

Matthew J. Nicholson, Michael K. Theodorou and Jayne L. Brookman. (2005). Molecular analysis of the anaerobic rumen fungus Orpinomyces - insights into an AT-rich genome. Microbiology, 151 (1), 121-133. Sponsorship: BBSRC RAE2008

Fundamental studies on the application of enzymes when brewing with unmalted oats and sorghum

The use of unmalted oats or sorghum in brewing has great potential for creating new beer types/flavors and saving costs. However, the substitution of barley malt with oat or sorghum adjunct is not only innovative but also challenging due to their specific grain characteristics. The overall objectives of this Ph.D. project were: 1) to investigate the impact of various types and levels of oats or sorghum on the quality/processability of mashes, worts, and beers; 2) to provide solutions as regards the application of industrial enzymes to overcome potential brewing problems. For these purposes, a highly precise rheological method using a controlled stress rheometer was developed and successfully applied as a tool for optimizing enzyme additions and process parameters. Further, eight different oat cultivars were compared in terms of their suitability as brewing adjuncts and two very promising types identified. In another study, the limitations of barley malt enzymes and the benefits of the application of industrial enzymes in high-gravity brewing with oats were determined. It is recommended to add enzymes to high-gravity mashes when substituting 30% or more barley malt with oats in order to prevent filtration and fermentation problems. Pilot-scale brewing trials using 10–40% unmalted oats revealed that the sensory quality of oat beers improved with increasing adjunct level. In addition, commercially available oat and sorghum flours were implemented into brewing. The use of up to 70% oat flour and 50% sorghum flour, respectively, is not only technically feasible but also economically beneficial. In a further study on sorghum was demonstrated that the optimization of industrial mashing enzymes has great potential for reducing beer production costs. A comparison of the brewing performance of red Italian and white Nigerian sorghum clearly showed that European grown sorghum is suitable for brewing purposes; 40% red sorghum beers were even found to be very low in gluten.