Home About us Contact
|
Home About us Contact | ||
|
|
||
Electrophoresis Analysis (electrophoresis + analysis)
Kinds of Electrophoresis Analysis Selected AbstractsCover Picture: Electrophoresis 23'2008ELECTROPHORESIS, Issue 23 2008Article first published online: 3 DEC 200 Issue 23 is a paper symposium on "DNA sequencing and genotyping". It consists of 22 contributions covering a wide spectrum of investigations in the area of DNA sequencing by capillary electrophoresis and the latest technologies for DNA sequencing and genotyping on microfluidic devices. Ten contributions deal with microchip-based electrophoresis analyses of DNA, an indication that miniaturized devices may surge to the fore for medical sequencing applications on individual DNA samples, as well as for forensic applications. [source] Structural and functional responses of river biofilm communities to the nonsteroidal anti-inflammatory diclofenacENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 4 2007John R. Lawrence Abstract Diclofenac is a nonsteroidal anti-inflammatory drug (NSAID) that has been detected widely in surface waters in North America and Europe. The impact of diclofenac on river biofilm communities was investigated at exposures of 10 and 100 ,g L,1 of diclofenac or its molar equivalent in carbon and nutrients. Experiments were carried out with river water during spring and summer using rotating annular reactors as model systems. Diclofenac or nutrients at 10 ,g L,1 were observed to have no significant effect on algal, bacterial, and cyanobacterial biomass in spring, whereas in the summer the nutrient equivalent reduced algal biomass and diclofenac reduced cyanobacterial biomass relative to control biofilms (p < 0.05). In contrast, at 100 ,g L,1 diclofenac or nutrients, the result was increased cyanobacterial and bacterial biomass, respectively, relative to control biofilms in spring. In summer, 100 ,g L,1 diclofenac significantly increased bacterial biomass and the nutrient treatment had no significant effect (p < 0.05); both treatments resulted in increased biofilm thickness. The glycoconjugate composition of the exopolysaccharide matrix was influenced differentially by the treatments in both seasons. Biolog assessments of carbon use indicated that 100 ,g L,1 diclofenac or nutrients resulted in significant depressions in the use of carbon sources in summer and significant increases in spring. Impacts on protozoan and micrometazoan populations also were assessed. Denaturing gradient gel electrophoresis analyses of community DNA and fluorescent in situ hybridization studies indicated that diclofenac had significant effects on the nature of the bacterial community in comparison with control and nutrient-treated river biofilm communities. [source] Affinity monolith preconcentrators for polymer microchip capillary electrophoresisELECTROPHORESIS, Issue 16 2008Weichun Yang Abstract Developments in biology are increasing demands for rapid, inexpensive, and sensitive biomolecular analysis. In this study, polymer microdevices with monolithic columns and electrophoretic channels were used for biological separations. Glycidyl methacrylate- co -ethylene dimethacrylate monolithic columns were formed within poly(methyl methacrylate) microchannels by in situ photopolymerization. Flow experiments in these columns demonstrated retention and then elution of amino acids under conditions optimized for sample preconcentration. To enhance analyte selectivity, antibodies were immobilized on monoliths, and subsequent lysozyme treatment blocked nonspecific adsorption. The enrichment capability and selectivity of these affinity monoliths were evaluated by purifying fluorescently tagged amino acids from a mixture containing green fluorescent protein (GFP). Twenty-fold enrichment and 91% recovery were achieved for the labeled amino acids, with a >25,000-fold reduction in GFP concentration, as indicated by microchip electrophoresis analysis. These devices should provide a simple, inexpensive, and effective platform for trace analysis in complex biological samples. [source] Capillary electrophoresis analysis of glucooligosaccharide regioisomersELECTROPHORESIS, Issue 6 2004Gilles Joucla Abstract Complex gluco-oligosaccharide mixtures of two regioisomer series were successfully separated by CE. The gluco-oligosaccharide series were synthesized, employing a dextransucrase from Leuconostoc mesenteroides NRRL B-512F, by successive glucopyranosyl transfers from sucrose to the acceptor glucose or maltose. The glucosyl transfer to both acceptors, occurring through the formation of ,1,6 linkages, differed for the two series only in the glucosidic bond to the reducing end namely ,1,6 or ,1,4 bond for glucose or maltose acceptor, respectively. Thus, the combination of the two series results in mixed pairs of gluco-oligosaccharide regioisomers with different degrees of polymerization (DP). These regioisomer series were first derivatized by reductive amination with 9-aminopyrene-1,4,6-trisulfonate (APTS). Under acidic conditions using triethyl ammonium acetate as electrolyte, the APTS-gluco-oligosaccharides of each series were separated enabling unambiguous size determination by coupling CE to electrospray-mass spectrometry. However, neither these acidic conditions nor alkaline buffer systems could be adapted for the separation of the gluco-oligosaccharide regioisomers arising from the two combined series. By contrast, increased resolution was observed in an alkaline borate buffer, using differential complexation of the regioisomers with the borate anions. Such conditions were also successfully applied to the separation of glucodisaccharide regioisomers composed of ,1,2, ,1,3, ,1,4, and ,1,6 linkages commonly synthesized by glucansucrase enzymes. [source] Proteomic analysis of rat brain tissue: Comparison of protocols for two-dimensional gel electrophoresis analysis based on different solubilizing agentsELECTROPHORESIS, Issue 24 2002Lucia Carboni Abstract The present study reports a comparison of recently described solubilizing methods, to set up a simple protocol for obtaining two-dimensional (2-D) gel electrophoresis maps of brain tissue. Different protocols were used for preparing rat brain homogenates and the resulting maps were compared by image analysis. Three different detergents, two delipidation methods, and introduction of a fractionation step based on different protein solubility in surfactants, were evaluated. When using efficient zwitterionic detergents (3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonate, CHAPS; amidosulfobetaine 14, ASB-14), the patterns obtained by direct loading of total extracts were qualitatively overlapping with patterns obtained from fractionated samples. In contrast, a weaker nonionic agent (Nonidet P-40, NP-40) produced a different protein pattern in the collected fractions. Delipidation did not improve the results for all the different extraction methods. Immunoblots performed with antibodies recognizing cytosolic and membrane-spanning proteins, which were detected as nondegraded spots, showed that membrane proteins with intermediate molecular mass could be recovered. We suggest, as a simple and efficient method for preparing rat brain maps, the homogenization in a solution containing an efficient zwitterionic surfactant, which allows to solubilize cytosolic and membrane proteins in a single step. Alternatively, a fractionation can be carried out on samples homogenized by a weak solubilizing agent, a more labor-intensive effort resulting in a larger number of proteins on two maps. [source] Accessory gland secretory proteins in relation to fitness parameters of Drosophila ananassae and D. variansENTOMOLOGICAL RESEARCH, Issue 5 2010Raghavendra B. HIREMANI Abstract Developmental morphometry, qualitative and quantitative analysis of the accessory gland secretory proteins, fecundity and productivity in relation to protein ejected during subsequent (first to fourth-time) matings have been studied in Drosophila ananassae Doleschall and Drosophila varians Bock. In both species, size and secretion of accessory glands increases from 1 to 8 days and the stored secretion ejected from males to the female genital tract during subsequent mating varies. The maximum number of eggs and flies are produced from the females mated with bachelor males and it is a minimum when virgin females are mated with fourth-time mated males. Sodium dodecylsulfate,polyacrylamide gel electrophoresis analysis of accessory gland secretory protein patterns and their glycosylation differs in both the species. Correlation coefficient analysis between gland size and quantity of secretion, percentage of secretory protein transferred per mating, and eggs and flies that emerged showed a highly significant, positive relationship. Among different matings, the number of eggs laid and flies that emerged per female between subsequent (first to fourth-time) matings of males was found to be highly significant and the difference between fecundity and productivity between the two species was highly significant. [source] Growth, activity and temperature responses of ammonia-oxidizing archaea and bacteria in soil microcosmsENVIRONMENTAL MICROBIOLOGY, Issue 5 2008Maria Tourna Summary Ammonia oxidation, as the first step in the nitrification process, plays a central role in the global cycling of nitrogen. Although bacteria are traditionally considered to be responsible for ammonia oxidation, a role for archaea has been suggested by data from metagenomic studies and by the isolation of a marine, autotrophic, ammonia-oxidizing, non-thermophilic crenarchaeon. Evidence for ammonia oxidation by non-thermophilic crenarchaea in marine and terrestrial environments is largely based on abundance of bacterial and archaeal ammonia monooxygenase (amo) genes, rather than activity. In this study, we have determined the influence of temperature on the response of ammonia-oxidizing bacteria and archaea in nitrifying soil microcosms using two approaches, involving analysis of transcriptional activity of 16S rRNA genes and of a key functional gene, amoA, which encodes ammonia monooxygenase subunit A. There was little evidence of changes in relative abundance or transcriptional activity of ammonia-oxidizing bacteria during nitrification. In contrast, denaturing gradient gel electrophoresis analysis of crenarchaeal 16S rRNA and crenarchaeal amoA genes provided strong evidence of changes in community structure of active archaeal ammonia oxidizers. Community structure changes were similar during incubation at different temperatures and much of the activity was due to a group of non-thermophilic crenarchaea associated with subsurface and marine environments, rather than soil. The findings suggest a role for crenarchaea in soil nitrification and that further information is required on their biogeography. [source] Anaerobic redox cycling of iron by freshwater sediment microorganismsENVIRONMENTAL MICROBIOLOGY, Issue 1 2006Karrie A. Weber Summary The potential for microbially mediated anaerobic redox cycling of iron (Fe) was examined in a first-generation enrichment culture of freshwater wetland sediment microorganisms. Most probable number enumerations revealed the presence of significant populations of Fe(III)-reducing (approximately 108 cells ml,1) and Fe(II)-oxidizing, nitrate-reducing organisms (approximately 105 cells ml,1) in the freshwater sediment used to inoculate the enrichment cultures. Nitrate reduction commenced immediately following inoculation of acetate-containing (approximately 1 mM) medium with a small quantity (1% v/v) of wetland sediment, and resulted in the transient accumulation of NO2, and production of a mixture of gaseous end-products (N2O and N2) and NH4+. Fe(III) oxide (high surface area goethite) reduction took place after NO3, was depleted and continued until all the acetate was utilized. Addition of NO3, after Fe(III) reduction ceased resulted in the immediate oxidation of Fe(II) coupled to reduction of NO3, to NH4+. No significant NO2, accumulation was observed during nitrate-dependent Fe(II) oxidation. No Fe(II) oxidation occurred in pasteurized controls. Microbial community structure in the enrichment was monitored by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified 16S rDNA and reverse transcription polymerase chain reaction-amplified 16S rRNA, as well as by construction of 16S rDNA clone libraries for four different time points during the experiment. Strong similarities in dominant members of the microbial community were observed in the Fe(III) reduction and nitrate-dependent Fe(II) oxidation phases of the experiment, specifically the common presence of organisms closely related (, 95% sequence similarity) to the genera Geobacter and Dechloromonas. These results indicate that the wetland sediments contained organisms such as Geobacter sp. which are capable of both dissimilatory Fe(III) reduction and oxidation of Fe(II) with reduction of NO3, to NH4+. Our findings suggest that microbially catalysed nitrate-dependent Fe(II) oxidation has the potential to contribute to a dynamic anaerobic Fe redox cycle in freshwater sediments. [source] PRECLINICAL STUDY: Proteomic analysis of methamphetamine-induced reinforcement processes within the mesolimbic dopamine systemADDICTION BIOLOGY, Issue 3-4 2008Moon Hee Yang ABSTRACT Methamphetamine (MAP) is a commonly used, addictive drug, and a powerful stimulant that dramatically affects the central nervous system. In this study, we used the conditioned place preference (CPP) paradigm in order to study the reinforcing properties of MAP and the herewith associated changes in proteins within the mesolimbic dopamine system. A CPP was induced by MAP after three intermittent intraperitoneal injections (1 mg/kg) in rats and protein profiles in the nucleus accumbens, striatum, prefrontal cortex, cingulate cortex and hippocampus were compared with a saline-treated control group. In addition, a group of animals was run through extinction and protein profiles were compared with a non-extinguished group. Protein screening was conducted using two-dimensional electrophoresis analysis which identified 27 proteins in the group that showed MAP-induced CPP. Some of the proteins were confirmed by Western lot analysis. Identified proteins had functions related to the cytoskeleton, transport/endocytosis or exocytosis (e.g. profilin-2 and syntaxin-binding protein), and signal transduction, among others. [source] Fungal endophytes in potato roots studied by traditional isolation and cultivation-independent DNA-based methodsFEMS MICROBIOLOGY ECOLOGY, Issue 3 2006Monika Götz Abstract The composition and relative abundance of endophytic fungi in roots of field-grown transgenic T4-lysozyme producing potatoes and the parental line were assessed by classical isolation from root segments and cultivation-independent techniques to test the hypothesis that endophytic fungi are affected by T4-lysozyme. Fungi were isolated from the majority of root segments of both lines and at least 63 morphological groups were obtained with Verticillium dahliae, Cylindrocarpon destructans, Colletotrichum coccodes and Plectosporium tabacinum as the most frequently isolated species. Dominant bands in the fungal fingerprints obtained by denaturing gradient gel electrophoresis analysis of 18S rRNA gene fragments amplified from total community DNA corresponded to the electrophoretic mobility of the 18S rRNA gene fragments of the three most abundant fungal isolates, V. dahliae, C. destructans and Col. coccodes, but not to P. tabacinum. The assignment of the bands to these isolates was confirmed for V. dahliae and Col. coccodes by sequencing of clones. Verticillium dahliae was the most abundant endophytic fungus in the roots of healthy potato plants. Differences in the relative abundance of endophytic fungi colonizing the roots of T4-lysozyme producing potatoes and the parental line could be detected by both methods. [source] How elevated pCO2 modifies total and metabolically active bacterial communities in the rhizosphere of two perennial grasses grown under field conditionsFEMS MICROBIOLOGY ECOLOGY, Issue 3 2006Maryline Jossi Abstract The response of total (DNA-based analysis) and active (RNA-based analysis) bacterial communities to a pCO2 increase under field conditions was assessed using two perennial grasses: the nitrophilic Lolium perenne and the oligonitrophilic Molinia coerulea. PCR- and reverse transcriptase-PCR denaturing gradient gel electrophoresis analysis of 16S rRNA genes generated contrasting profiles. The pCO2 increase influenced mainly the active and root-associated component of the bacterial community. Bacterial groups responsive to the pCO2 increase were identified by sequencing of corresponding denaturing gradient gel electrophoresis bands. About 50% of retrieved sequences were affiliated to Proteobacteria. Our data suggest that Actinobacteria in soil and Myxococcales (Deltaproteobacteria) in root are stimulated under elevated pCO2. [source] Cloning and characterization of CmGPD1, the Candida magnoliae homologue of glycerol-3-phosphate dehydrogenaseFEMS YEAST RESEARCH, Issue 8 2008Dae-Hee Lee Abstract Glycerol-3-phosphate dehydrogenase (GPDH) plays a central role in glycerol metabolism. A genomic CmGPD1 gene encoding NADH-dependent GPDH was isolated from Candida magnoliae producing a significant amount of glycerol. The gene encodes a polypeptide of 360 amino acids, which shows high homology with known NADH-dependent GPDHs of other species. The CmGPD1 gene was expressed in recombinant Escherichia coli with the maltose-binding protein (MBP) fusion system and purified to homogeneity using simple affinity chromatography. The purified CmGpd1p without the MBP fusion displayed an apparent molecular mass of 40 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The CmGpd1p enzyme exhibited a Kcat/Km value of 195 min,1 mM,1 for dihydroxyacetone phosphate whereas Kcat/Km for glycerol-3-phosphate is 0.385 min,1 mM,1. In a complementation study, CmGpd1p rescued the ability of glycerol synthesis and salt tolerance in a Saccharomyces cerevisiae GPD1,GPD2, mutant strain. The overall results indicated that CmGPD1 encodes a functional homologue of S. cerevisiae GPDH. [source] Autoantibodies against stress-induced phosphoprotein-1 as a novel biomarker candidate for ovarian cancerGENES, CHROMOSOMES AND CANCER, Issue 7 2010Sunghoon Kim Detection of autoantibodies against tumor-associated antigens (TAA) has recently been shown to be a powerful tool for early detection of various cancers. The aim of this study was to investigate the possibility of using autoantibodies against TAA as novel biomarkers by a proteomics-based approach in patients with ovarian cancer. We used two-dimensional differential gel electrophoresis analysis of immuno-precipitated tumor antigens (2D-DITA) to compare the levels of autoandibodies in pretreatment and posttreatment sera of patients with ovarian cancers. The identified autoantibodies were validated by SYBR Green real-time polymerase chain reaction (PCR) and immunohistochemistry (IHC). We further evaluated the level of autoantibody in sera of 68 ovarian cancer patients by an enzyme-linked immunosorbent assay (ELISA). The autoantibody directed against stress-induced phosphoprotein-1 (STIP-1) emerged as a novel biomarker candidate for ovarian cancer. SYBR Green PCR and IHC confirmed that the STIP-1 mRNA and protein expression levels were significantly up-regulated in ovarian cancers compared with normal and benign tumors (P = 0.003 and P < 0.001, respectively). A preliminary ELISA study showed that the serum levels of anti-STIP-1 autoantibodies were significantly elevated in ovarian cancer patients compared with healthy controls (P = 0.03). The results suggest that 2D-DITA is a useful tool to detect autoantibodies and that STIP-1 is a potential biomarker candidate for ovarian cancers. © 2010 Wiley-Liss, Inc. [source] Non- pylori Helicobacteraceae in the Upper Digestive Tract of Asymptomatic Venezuelan Subjects: Detection of Helicobacter cetorum- like and Candidatus Wolinella africanus -like DNAHELICOBACTER, Issue 5 2007M. Alexandra García-Amado Abstract Background: The spectrum of human non- pylori Helicobacter infections is expanding, with species such as H. heilmannii and H. felis occasionally being associated with gastritis. However, the existence of non- pylori Helicobacter colonization in asymptomatic subjects has not been evaluated. The aim of this study was to investigate whether Helicobacter species other than pylori are present in the upper digestive tract of asymptomatic human subjects. Materials and methods: A Helicobacteraceae-specific semi-nested polymerase chain reaction (PCR) assay was used to detect Helicobacter- like organisms in the upper digestive tract of 91 Venezuelan volunteers (aged 18,68 years, 41 females, 50 males). Species were identified by denaturing gradient gel electrophoresis analysis and sequencing of the PCR products. Results: We detected DNA sharing 99,100% sequence identity in over 300,400 bp with the 16S rRNA genes of H. pylori, H. cetorum, and Candidatus Wolinella africanus in 76%, 16%, and 15% of the subjects, respectively. Multiple colonization was documented in 10% of the subjects: H. cetorum and Candidatus W. africanus (4%), H. pylori and Candidatus W. africanus (4%), and H. pylori and H. cetorum (2%). Conclusions: Our results suggest that non- pylori Helicobacteraceae colonization is relatively common in the Venezuelan asymptomatic population. This is the first report documenting the presence of H. cetorum DNA in the human upper digestive tract, and the second report of the recently discovered Candidatus W. africanus. [source] Protective effect of non-mitogenic human acidic fibroblast growth factor on hepatocyte injuryHEPATOLOGY RESEARCH, Issue 10 2007Hua Xu Aim:, To study whether non-mitogenic human acidic fibroblast growth factor (nm-haFGF) has protective effects on H2O2 -induced hepatocyte injury in vitro and CCl4 -induced hepatocyte injury in vivo. Methods:, (i) HL-7702 hepatocytes were incubated with different concentrations of nm-haFGF for 12 h, and then the activity of lactate dehydrogenase (LDH) in culture medium was detected, and genomic DNA electrophoresis analysis was observed after being exposed to H2O2 (8 mmol/L) for 4 h. Proximately, apoptotic rates and protein expressions of Bcl-2 and Bax of HL-7702 cell were detected after being exposed to H2O2 (0.2 mmol/L) for 20 h. (ii) Being injected intraperitoneally with nm-haFGF, mice were treated with CCl4 intraperitoneally to induce hepatic injury. Twenty-four hours later, serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured and histopathologic changes were evaluated. Results:, (i) In vitro tests: LDH activities and apoptotic rates decreased, the protein expression of Bcl-2 increased and Baxdecreased in nm-haFGF-treated groups at the concentrations of 100 150 and 200 ng/mL, compared with that in the model control group, which was treated with H2O2 alone. The genomic DNA remained nearly intact at the concentrations of 150 and 200 ng/mL. (ii) In vivo tests: serum ALT and AST in nm-haFGF-treated groups (10 ,g/kg and 20 ,g/kg) were much lower as compared to the model control group, which was treated with CCl4 alone. Histological examination showed that nm-haFGF markedly ameliorated hepatocytes vacuolation, cloudy swelling and inflammatory cells infiltration induced by CCl4. Conclusion:, nm-haFGF had protective effects against H2O2 -induced hepatocyte injury in vitro and CCl4 -induced acute liver injury in vivo. [source] Encapsulation efficiency and release behaviors of bovine serum albumin loaded in alginate microspheres prepared by sprayingJOURNAL OF APPLIED POLYMER SCIENCE, Issue 4 2008Jie Zhang Abstract Spraying and spraying with an electrostatic field (SEF) were employed to prepare alginate microspheres for delivering proteins, especially for intestinal digestive enzymes and cytokines. The encapsulation efficiency (EE) of a model protein [bovine serum albumin (BSA)] at a pH value lower than the isoelectric point was 20% higher than that at a natural pH. Moreover, for the microspheres prepared by SEF, EE improved significantly with increasing electric voltage. The interactions between BSA and the alginate microspheres were identified with Fourier transform infrared spectroscopy. The release profiles in vitro showed a controlled and pH-responsive release manner for the encapsulated BSA. A first-order release equation was postulated and modified to describe the release kinetics with an obviously initial burst release related to the eroded porous matrix. The equation fit the release data well when the pH value and composition of the release media were changed. The analysis of the release kinetics indicated that the drug release rate was in an inverse ratio to the diameter of the microspheres. Increasing the gas flow rate or electric voltage decreased both the mean diameter and size distribution of the microspheres significantly and enhanced the release rate of loaded drugs from alginate microspheres. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis analysis revealed that BSA kept its structural integrity during the encapsulation and release process. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008 [source] Proteomics analysis of liver samples from puffer fish Takifugu rubripes exposed to excessive fluoride: An insight into molecular response to fluorosisJOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 1 2010Jian Lu Abstract Comparative proteomics was performed to identify proteins in the liver of Takifugu rubripes in response to excessive fluoride exposure. Sixteen fish were randomly divided into a control group and an experimental group. The control group was raised in soft water alone (F, = 0.4 mg/L), and the experimental group was raised in the same water with sodium fluoride at a high concentration of 35 mg/L. After 3 days, proteins were extracted from the fish livers and then subjected to two-dimensional polyacrylamide gel electrophoresis analysis. The matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was applied to identify the proteins that were differentially expressed from the two groups of fish. Among an average of 816 and 918 proteins detected in the control and treated groups, respectively, 16 proteins were upregulated and 35 were downregulated (P < 0.01) in the fluoride-treated group as compared with those in the control group. Twenty-four highly differentially expressed proteins were further analyzed by MALDI-TOF/TOF-MS, and eight were identified by Mascot. These eight proteins include disulfide isomerase ER-60, 4SNc-Tudor domain protein, SMC3 protein, Cyclin D1, and mitogen-activated protein kinase 10, as well as three unknown proteins. Consistent with their previously known functions, these identified proteins seem to be involved in apoptosis and other functions associated with fluorosis. These results will greatly contribute to our understanding of the effects of fluoride exposure on the physiological and biochemical functions of Takifugu and the toxicological mechanism of fluoride causing fluorosis in both fish and human. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 24:21,28, 2010; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20308 [source] Long-standing environmental conditions, geographic isolation and host,symbiont specificity influence the relative ecological dominance and genetic diversification of coral endosymbionts in the genus SymbiodiniumJOURNAL OF BIOGEOGRAPHY, Issue 5 2010Todd C. LaJeunesse Abstract Aim, This study examines the importance of geographic proximity, host life history and regional and local differences in environment (temperature and water clarity) in driving the ecological and evolutionary processes underpinning the global patterns of diversity and distribution of symbiotic dinoflagellates. By comparing and contrasting coral,algal symbioses from isolated regions with differing environmental conditions, we may assess the potential of coral communities to respond to significant changes in climate. Location, Indian Ocean. Methods, Community assemblages of obligate symbiotic invertebrates were sampled at numerous sites from two regions, the north-eastern Indian Ocean (Andaman Sea, western Thailand) and the western Indian Ocean (Zanzibar, Tanzania). Molecular genetic methods, including denaturing gradient gel electrophoresis analysis of the ribosomal internal transcribed spacers, DNA sequencing and microsatellite genotyping, were used to characterize the ,species' diversity and evolutionary relationships of symbiotic dinoflagellates (genus Symbiodinium). Host,symbiont specificity, geographic isolation and local and regional environmental factors were evaluated in terms of their importance in governing the distribution and prevalence of certain symbiont taxa. Results, Host-generalist symbionts (C3u and D1-4, formerly D1a now designated Symbiodinium trenchi) frequently occurred alone and sometimes together in hosts with horizontal modes of symbiont acquisition. However, the majority of Symbiodinium diversity consisted of apparently host-specific ,species'. Clade C Symbiodinium were diverse and dominated host assemblages from sites sampled in the western Indian Ocean, a pattern analogous to symbiont communities on the Great Barrier Reef with similar environmental conditions. Clade D Symbiodinium were diverse and occurred frequently in hosts from the north-eastern Indian Ocean, especially at inshore locations, where temperatures are warmer, water turbidity is high and large tidal exchanges commonly expose coral populations to aerial desiccation. Main conclusions, Regional and local differences in cnidarian,algal combinations indicate that these symbioses are ecologically and evolutionarily responsive and can thrive under various environmental conditions. The high temperatures and turbid conditions of the north-eastern Indian Ocean partly explain the ecological success of Clade D Symbiodinium relative to Clade C. Phylogenetic, ecological and population genetic data further indicate that Clade D has undergone an adaptive radiation, especially in regions around Southeast Asia, during the Pleistocene. [source] Aging induces cardiac diastolic dysfunction, oxidative stress, accumulation of advanced glycation endproducts and protein modificationAGING CELL, Issue 2 2005Shi-Yan Li Summary Evidence suggests that aging, per se, is a major risk factor for cardiac dysfunction. Oxidative modification of cardiac proteins by non-enzymatic glycation, i.e. advanced glycation endproducts (AGEs), has been implicated as a causal factor in the aging process. This study was designed to examine the role of aging on cardiomyocyte contractile function, cardiac protein oxidation and oxidative modification. Mechanical properties were evaluated in ventricular myocytes from young (2-month) and aged (24,26-month) mice using a MyoCam® system. The mechanical indices evaluated were peak shortening (PS), time-to-PS (TPS), time-to-90% relengthening (TR90) and maximal velocity of shortening/relengthening (± dL/dt). Oxidative stress and protein damage were evaluated by glutathione and glutathione disulfide (GSH/GSSG) ratio and protein carbonyl content, respectively. Activation of NAD(P)H oxidase was determined by immunoblotting. Aged myocytes displayed a larger cell cross-sectional area, prolonged TR90, and normal PS, ± dL/dt and TPS compared with young myocytes. Aged myocytes were less tolerant of high stimulus frequency (from 0.1 to 5 Hz) compared with young myocytes. Oxidative stress and protein oxidative damage were both elevated in the aging group associated with significantly enhanced p47phox but not gp91phox expression. In addition, level of cardiac AGEs was ,2.5-fold higher in aged hearts than young ones determined by AGEs-ELISA. A group of proteins with a molecular range between 50 and 75 kDa with pI of 4,7 was distinctively modified in aged heart using one- or two-dimension SDS gel electrophoresis analysis. These data demonstrate cardiac diastolic dysfunction and reduced stress tolerance in aged cardiac myocytes, which may be associated with enhanced cardiac oxidative damage, level of AGEs and protein modification by AGEs. [source] EFFECT OF SALTS AND POLYETHYLENE GLYCOLS ON THE PARTITIONING AND RECOVERY OF TRYPSIN FROM HYBRID CATFISH VISCERA IN AQUEOUS TWO-PHASE SYSTEMSJOURNAL OF FOOD BIOCHEMISTRY, Issue 4 2010SAPPASITH KLOMKLAO ABSTRACT The partitioning behavior of trypsin from hybrid catfish viscera in aqueous two-phase systems (ATPS) was studied. Factors such as polyethylene glycol (PEG) molecular mass and concentration, as well as types and concentration of salts, affected protein separation. Trypsin partitioned mainly in the top PEG-rich phase. ATPS formed by PEG of molecular weight 4,000 (20%, w/w) and NaH2PO4 (20%, w/w) showed the best capability for trypsin purification from hybrid catfish viscera. Under such conditions, the highest specific activity (30.05 units/µg protein) and purification (27.3-fold), were obtained. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that the enzyme after ATPS separation was near homogeneity and based on the activity staining, the band intensity of enzyme in ATPS fraction increased, indicating the greater specific activity of the viscera extract. The partitioned enzyme displayed optimal activity at pH 9.0 and 50C, respectively. The enzyme was stable up to 40C and within the pH range of 8,12. The enzyme exhibited a progressive decrease in activity with increasing NaCl concentration. PRACTICAL APPLICATIONS This paper describes the separation and recovery of trypsin from hybrid catfish viscera in ATPS and its properties. ATPS provides an efficient and attractive method for partitioning and recovery of trypsin from hybrid catfish viscera. Trypsins from various sources catalyze the hydrolysis of peptide bonds on the carboxyl sides of arginine and lysine. Therefore, it is expected that like other trypsins, trypsin after ATPS separation from hybrid catfish viscera could be useful in the biomedical, food and beverage industries. [source] PROTEOLYTIC ACTIVITY OF LACTOBACILLUS SAKEI, LACTOBACILLUS FARCIMINIS AND LACTOBACILLUS PLANTARUM ON SARCOPLASMIC PROTEINS OF PORK LEANJOURNAL OF FOOD BIOCHEMISTRY, Issue 3 2004ANNA LISA BASSO The aim of this study was to assess the proteolytic activity of Lactobacillus sakei (DSM 6333), L. plantarum (B21), and to a lesser extent, L. farciminis (DSM 20184) on meat sarcoplasmic proteins. The protein composition was assayed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and capillary electrophoresis after incubation of meat extract inoculated with bacteria. All strains showed proteolytic activity: a band about 94 kDa disappeared in samples inoculated with L. farciminis and L. plantarum and strongly decreased in those inoculated with L. sakei. The intensity of the bands with a molecular weight between 94 and 38 kDa decreased in all samples. Capillary electrophoresis analysis ascertained the disappearance of the fractions corresponding to 8.64 and 8.66 min retention time in all samples. The bands corresponding to 94 kDa and 38 kDa were, respectively, identified as glycogen phosphorylase muscle isoform and glyceraldehyde 3-phosphate dehydrogenase, by in situ digestion of protein gel bands and peptide map analysis using Matrix Assisted Laser Desorption/Ionization - Time of Flight Mass Spectrometry (MALDI-TOF MS). [source] EFFECT OF HIGH PRESSURE ON LACTOCOCCAL BACTERIOPHAGESJOURNAL OF FOOD SAFETY, Issue 1 2009M. DILEK ABSTRACT Four different host-specific lactococcal bacteriophages were subjected to high hydrostatic pressure and heat treatments. Pressure treatments were done at room temperature at 300 and 350 MPa for 5,40 min. Complete inactivation of bacteriophages was observed starting at 350 MPa for 20-min treatment at room temperature. The effect of heat on the bacteriophages was analyzed by heat treatment at 71.7C for predetermined lengths of time (1,5 min). Decrease in bacteriophage number was observed after 3 min of heat treatment at 71.7C. Pressure treatment at 350 MPa/5 min and heat treatment at 71.7C/3 min were both found to be effective for the inactivation of lactococcal bacteriophages. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis indicated that protein profiles of pressure-treated (350 MPa, 25 min) bacteriophages were altered. PRACTICAL APPLICATIONS Bacteriophages are still a problem for the production of fermented dairy products, as there has not been a process to eliminate them completely from the fermentation environment. Processes such as pasteurization are not adequate to eliminate bacteriophages. However, new food preservation methods have been developed, one of which is high hydrostatic pressure (HHP) processing. HHP has potential application for the inactivation of viruses. Here, we demonstrate the application of HHP to inactivate the bacteriophages of dairy starter culture Lactococcus in comparison with heat treatment. [source] Effects of Microbial Transglutaminase on the Wheat Proteins of Bread and Croissant DoughJOURNAL OF FOOD SCIENCE, Issue 6 2001J.A. Gerrard ABSTRACT: Transglutaminase is a crosslinking enzyme that is finding increasing use in foods, yet the molecular changes responsible for its effects are not fully understood. Proteins were extracted from bread and croissant doughs that had been treated with transglutaminase and compared to those from control doughs by size exclusion high performance liquid chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis. Transglutaminase increased the amount of protein extracted in the gliadin fraction in both bread and croissant doughs. In croissant doughs, a corresponding decrease in the protein extracted in the albumin to globulin fraction was seen. In each case, crosslinking of the high molecular weight glutenins was observed. The possible role of each of these changes on the functional properties of baked products is discussed. [source] Physiological and Biochemical Characteristics of Iranian Strains of Xanthomonas axonopodis pv. citri, the Causal Agent of Citrus bacterial Canker DiseaseJOURNAL OF PHYTOPATHOLOGY, Issue 2 2001M. Mohammadi Twenty-four strains of Xanthomonas axonopodis pv. citri (Xac), the causal agent of bacterial canker of citrus, isolated from Mexican lime (Citrus aurantifolia) and lemon (Citrus limon) in southern Iran, were characterized phenotypically. Strains were all pathogenic on C. aurantifolia. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis revealed slight differences in soluble protein profiles among the strains. Based on host range specificity and phenotypic characteristics, representative strains were differentiated into two groups of Asiatic (A) and atypical Asiatic (aA) forms. DNA fingerprinting analysis using EcoRI as the restriction endonuclease showed a negligible difference in restriction pattern between the two groups. On the basis of isozymic analysis, the two groups were distinct with respect to superoxide dismutase (SOD) and esterase (EST) banding patterns. Plasmid DNA profile analysis showed that the bacterial strains were different from each other in terms of plasmid number and molecular weight. Phage typing study revealed that most of group A strains were susceptible to Cp1 and/or Cp2 and some were resistant to both phage types including the strain in aA group. Bacteriocin production test indicated that there was a variation among Xac strains using different indicators for each bacteriocin producer. It is concluded that the Iranian strains of Xac are heterogeneous and constitute a subgroup(s) within the pathotype A. Physiologische und biochemische Merkmale iranischer Stämme von Xanthomonas axonopodis pv. citri, dem Erreger des bakteriellen Zitruskrebses Vierundzwanzig Stämme von Xanthomonas axonopodis pv. citri, dem Erreger des bakteriellen Zitruskrebses, wurden von mexikanischen Sauren Limetten (Citrus aurantifolia) und Zitronen (Citrus limon) im Südiran isoliert und phänotypisch charakterisiert. Alle Stämme waren für C. aurantifolia pathogen. Eine SDS-PAGE-Analyse zeigte, daß zwischen den Stämmen geringfügige Unterschiede bei den Profilen der löslichen Proteine bestanden. Auf Grundlage der Spezifität des Wirtsspektrums und phänotypischer Merkmale wurden repräsentative Stämme in die zwei Gruppen asiatische (A) und atypische asiatische (aA) Formen eingeteilt. Eine Analyse mit DNA-Fingerabdrücken, wobei EcoRI als Restriktionsendonuclease diente, zeigte einen vernachlässigbar kleinen Unterschied bei den Restriktionsmustern der beiden Gruppen. Die Isoenzymanalyse ergab Unterschiede zwischen beiden Gruppen bezüglich der Bandenmuster von Superoxiddismutase (SOD) und Esterase (EST). Eine Analyse der Plasmid-DNA-Profile zeigte, daß die Bakterienstämme unterschiedliche Plasmidzahlen und verschiedene Molekülmassen aufwiesen. Eine Phagentypisierung ergab, daß die meisten Stämme der Gruppe A anfällig für Cp1 und/oder Cp2 waren; einige waren resistent gegen beide Phagentypen, darunter der Stamm in der aA-Gruppe. Ein Test der Bacteriocinproduktion ergab, daß die Xac -Stämme variierten; hier wurden verschiedene Indikatoren für jeden Bakteriocinbildner verwendet. Es wird gefolgert, daß die iranischen Stämme von Xac heterogen sind und eine oder mehrere Untergruppen innerhalb des Pathotyps A bilden. [source] Presence of sourdough lactic acid bacteria in commercial total mixed ration silage as revealed by denaturing gradient gel electrophoresis analysisLETTERS IN APPLIED MICROBIOLOGY, Issue 4 2010C. Wang Abstract Aims:, To characterize the bacterial communities in commercial total mixed ration (TMR) silage, which is known to have a long bunk life after silo opening. Methods and Results:, Samples were collected from four factories that produce TMR silage according to their own recipes. Three factories were sampled three times at 1-month intervals during the summer to characterize the differences between factories; one factory was sampled 12 times, three samples each during the summer, autumn, winter and spring, to determine seasonal changes. Bacterial communities were determined by culture-independent denaturing gradient gel electrophoresis. All silages contained lactic acid as the predominant acid, and the contents appeared stable regardless of factories and product seasons. Acetic acid and 1-propanol contents were different between factories and indicated seasonal changes, with increases in warm seasons compared to cool seasons. Both differences and similarities existed among the bacterial communities from each factory and product season. Lactobacillus parabuchneri was found in the products from three of four factories. Various sourdough lactic acid bacteria (LAB) were identified in commercial TMR silage; Lactobacillus panis, Lactobacillus hammesii, Lactobacillus mindensis, Lactobacillus pontis, Lactobacillus frumenti and Lactobacillus farciminis were detected in many products. Moreover, changes owing to product season were distinctive, and Lact. pontis and Lact. frumenti became detectable in summer products. Conclusion:, Sourdough LAB are involved in the ensiling of commercial TMR silage. Silage bacterial communities vary more by season than by factory. The LAB species Lact. parabuchneri was detected in the TMR silage but may not be essential to the product's long bunk life after silo opening. Significance and Impact of the Study:, Commercial TMR silage resembles sourdough with respect to bacterial communities and long shelf life. The roles of sourdough LAB in the ensiling process and aerobic stability are worth examining. [source] Bacteriophages induced from lysogenic root canal isolates of Enterococcus faecalisMOLECULAR ORAL MICROBIOLOGY, Issue 4 2009R. H. Stevens Introduction:, Bacterial viruses play crucial roles in the pathogenesis of many systemic diseases. They are known to inhabit the oral cavity, both as free virions and as prophages in lysogenic bacterial strains; however, there has been no report of bacteriophages in endodontic infections. In this study, we sought to detect, isolate, and describe temperate bacteriophages harbored by Enterococcus faecalis strains isolated from endodontic infections. Methods: Ten E. faecalis strains were isolated from root canals of teeth undergoing retreatment following unsuccessful endodontic therapy. Mitomycin C was used to induce any prophages present in the bacterial isolates. The induced phages were purified and examined using electron microscopy. The DNA extracted from one of the phage isolates was subjected to restriction endonuclease digestion and agarose electrophoresis analysis. Results:, Lysogeny was demonstrated in 4 of the 10 E. faecalis strains. Three of the lysogenic strains yielded phages exhibiting a Siphoviridae morphology, with long, non-contractile tails 130 nm in length, and spherical/icosahedral heads 41 nm in diameter. The virus induced from the fourth lysogenic E. faecalis strain had a contractile tail characteristic of Myoviridae. Restriction endonuclease analysis of NsiI and NdeI DNA fragments from one of the Siphoviridae phage isolates (phage ,Ef11) indicated a genome size of approximately 41 kbp. Conclusion:, This is the first report of lysogenic bacteria and their inducible viruses in infected root canals. [source] Bacterial community profiles of endodontic abscesses from Brazilian and USA subjects as compared by denaturing gradient gel electrophoresis analysisMOLECULAR ORAL MICROBIOLOGY, Issue 1 2007J. C. Machado de Oliveira This study compared the bacterial community profiles of the microbiota associated with acute apical abscesses from Brazilian and USA patients using denaturing gradient gel electrophoresis (DGGE). DNA was extracted from purulent exudate aspirates and part of the 16S rRNA gene was amplified by polymerase chain reaction and separated by DGGE. The resulting banding patterns, which were representative of the bacterial community structures in samples from the two locations, were then compared. Distinct DGGE banding patterns were observed from different samples. Ninety-nine bands with distinct positions in the gels were detected, of which 27 were found only in the USA samples and 13 were exclusive to Brazilian samples. Four of the 59 shared bands showed very discrepant findings with regard to prevalence in the two locations. Cluster analysis of DGGE banding profiles showed a great variability in the bacterial populations associated with teeth with abscesses regardless of the geographical location. Two big clusters, one for each location, were observed. Other clusters contained a mixture of samples from the two locations. The results of the present study demonstrated a great variability in the bacterial community profiles among samples. This indicates that the bacterial communities of abscesses are unique for each individual in terms of diversity. The composition of the microbiota in some samples showed a geography-related pattern. Furthermore, several bands were exclusive for each location and others were shared by the two locations and showed great differences in prevalence. [source] Clonal variability among oral Candida albicans assessed by allozyme electrophoresis analysisMOLECULAR ORAL MICROBIOLOGY, Issue 6 2000A. L. Mata A total of 49 Candida albicans strains were isolated from the saliva of 11 healthy children in Piracicaba, Brazil and were analyzed according to their alloenzymatic patterns. Among eight loci assayed, seven were polymorphic and allowed to determine allelic and genotype frequencies, in order to establish the genetic variables for this fungal population. Some children showed just one genetic type, whereas other harbored two or more clones of such yeast, in a multiclonal manner of colonization by C. albicans. [source] Airborne viable fungi in Riyadh and allergenic response of their extractsMYCOSES, Issue 9-10 2001A. S. Al-Suwaini Luftbürtige Pilze; Allergenität; Antigenität; Prick-Test; Saudi-Arabien. Summary. The allergenicity and antigenicity of various airborne fungi isolated from the atmosphere of Riyadh were studied. Protein nitrogen contents were estimated and found to range from 0.9 mg ml,1 for Cladosporium to 2.1 mg ml,1 for Aspergillus extracts. Sodium dodecyl sulphate,polyacrylamide gel electrophoresis analysis for those extracts exhibited a number of protein bands of higher molecular weight between 13 and 80 kDa for Alternaria, Ulocladium, Penicillium, Aspergillus and Cladosporium. Extracts in both aqueous and lyophilized forms were sterilized and tested for diagnostic skin prick test in 100 consecutive patients having bronchial asthma and allergic rhinitis. Overall, 13% of patients reacted positively to fungal extracts, revealing allergic sensitization to these fungi. These findings necessitate further investigation as regards the purification and characterization of these local extracts for better diagnostic use in patients in Saudi Arabia. Zusammenfassung. Es wurde die Allergenität und Antigenität mehrerer luftbürtiger Pilze untersucht, die aus der Luft von Riad isoliert worden waren. Hierzu wurden Pilzextrakte hergestellt, deren Proteinstickstoffgehalt zwischen 0.9 mg ml,1 bei Cladosporium und 2.1 mg ml,1 bei Aspergillus lag. Die SDS,PAGE-Analyse zeigte eine Anzahl von Proteinbanden höheren Molekulargewichts zwischen 13 und 80 kDa für Alternaria, Ulocladium, Penicillium, Aspergillus und Cladosporium. Die sowohl wässrigen wie lyophilisierten Extrakte wurden sterilisiert und an 100 unausgewählten Patienten mit Bronchialasthma und allergischer Rhinitis im Pricktest getestet. Ingesamt 13% der Patienten reagierten auf die Extrakte positiv, was für eine allergische Sensibilisierung gegen diese Pilze spricht. [source] Strategic shotgun proteomics approach for efficient construction of an expression map of targeted protein families in hepatoma cell linesPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 12 2003Chih-Lei Lee Abstract An expression map of the most abundant proteins in human hepatoma HepG2 cells was established by a combination of complementary shotgun proteomics approaches. Two-dimensional liquid chromatography (LC)-nano electrospray ionization (ESI) tandem mass spectrometry (MS/MS) as well as one-dimensional LC-matrix-assisted laser desorption/ionization MS/MS were evaluated and shown that additional separation introduced at the peptide level was not as efficient as simple prefractionation of protein extracts in extending the range and total number of proteins identified. Direct LC-nanoESI MS/MS analyses of peptides from total solubilized fraction and the excised gel bands from one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis fractionated insolubilized fraction afforded the best combination in efficient construction of a nonredundant cell map. Compiling data from multiple variations of rapid shotgun proteomics analyses is nonetheless useful to increase sequence coverage and confidence of hits especially for those proteins identified primarily by a single or two peptide matches. While the returned hit score in general reflects the abundance of the respective proteins, it is not a reliable index for differential expression. Using another closely related hepatoma Hep3B as a comparative basis, 16 proteins with more than two-fold difference in expression level as defined by spot intensity in two-dimensional gel electrophoresis analysis were identified which notably include members of the heat shock protein (Hsp) and heterogeneous nuclear ribonucleoprotein (hnRPN) families. The observed higher expression level of hnRNP A2/B1 and Hsp90 in Hep3B led to a search for reported functional roles mediated in concert by both these multifunctional cellular chaperones. In agreement with the proposed model for telomerase and telomere bound proteins in promoting their interactions, data was obtained which demonstrated that the expression proteomics data could be correlated with longer telomeric length in tumorigenic Hep3B. This biological significance constitutes the basis for further delineation of the dynamic interactions and modifications of the two protein families and demonstrated how proteomic and biological investigation could be mutually substantiated in a productive cycle of hypothesis and pattern driven research. [source] | |||||||||||||||||||