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Sulphate-polyacrylamide Gel Electrophoresis (sulphate-polyacrylamide + gel_electrophoresis)

Distribution by Scientific Domains
Life Sciences52%
Medical Sciences17%
Chemistry17%

Kinds of Sulphate-polyacrylamide Gel Electrophoresis

  • dodecyl sulphate-polyacrylamide gel electrophoresis
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  • sodium dodecyl sulphate-polyacrylamide gel electrophoresis
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    Selected Abstracts

    Endochitinase activity in the apoplastic fluid of Phellinus weirii -infected Douglas-fir and its association with over wintering and antifreeze activity

    FOREST PATHOLOGY, Issue 5 2003
    A. Zamani
    Summary Extracellular proteins were extracted from Phellinus weirii infected Douglas-fir (Pseudotsuga menziesii var. menziesii) roots and needles to examine endochitinase activity. Chitinases have been associated with the plant's defence response against fungal attack because they hydrolyse chitin, a structural component of fungal cell walls. Protein separation using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western immunoblot analysis using a polyclonal antibody specific to an endochitinase-like protein (ECP) resulted in the detection of up to three polypeptides between 27 and 30 kDa in size. Two-dimensional gel electrophoresis (2-D PAGE) followed by Western immunoblot analysis revealed that the apoplastic fluid contained multiple ECP isoforms with isoelectric points (pIs) ranging from 5.3 to 5.8 and molecular masses of 27,30 kDa. Chitinase activity in needle and root tissues was measured spectrophotometrically using a colorimetric assay. A gel overlay technique using glycol chitin as a substrate for endochitinase was applied to confirm that the ECP antibody detected an enzymatically active protein. The apoplastic fluid collected from P. weirii -infected winter Douglas-fir needles showed anti-freeze activity and seasonal analysis of needle tissue showed some evidence of ECP accumulation in winter months. ECP was distributed systemically throughout the tree. Increased levels of endochitinase activity in the region of P. weirii infection supports a physiological role for ECP in the plant defence response. Résumé Les protéines extra-cellulaires ont été extraites des racines et aiguilles de douglas (Pseudotsuga menziesii var menziesii) infectés par Phellinus weirii (Murr.) Gilbn., pour étudier l'activité endochitinase. Les chitinases ont été associées aux réactions de défense des plantes contre les attaques fongiques parce-qu'elles hydrolysent la chitine, un composant de la paroi des cellules fongiques. La séparation des protéines, réalisée par électrophorèse en gel de polyacrylamide avec sodium dodecyl sulfate (SDS-PAGE), suivie par une analyse par Western immunoblot en utilisant un anticorps polyclonal spécifique d'une protéine de type endochitinase (ECP), a permis la détection de 3 polypeptides de taille comprise entre 27 et 30 kDa. Une électrophorèse sur gel en 2-dimensions (2-D PAGE) suivie par une analyse par Western immunoblot a révélé que le fluide apoplastique contient de multiples isoformes d'ECP avec des pI dans une gamme de 5.3 à 5.8 et des masses moléculaires de 27 à 30 kDa. L'activité chitinase dans les aiguilles et tissus racinaires a été mesurée par spectrophotométrie par une méthode colorimétrique. Une technique d'overlay utilisant de la chitine glycol comme substrat de l'endochitinase a été appliquée pour confirmer que l'anticorps ECP avait détecté une protéine active du point de vue enzymatique. Le fluide apoplastique d'aiguilles récoltées en hiver sur des douglas infectés par P. weirii a montré une activité antigel et l'analyse saisonnière des tissus foliaires a montré une certaine accumulation d'ECP pendant l'hiver. L'ECP est répartie de façon systémique dans l'ensemble de l'arbre. Les niveaux accrus d'activité endochitinase dans la zone infectée par P. weirii suggère un rôle physiologique de l'ECP dans les réactions de défense de la plante. Zusammenfassung Aus Wurzeln und Nadeln von mit Phellinus weirii infizierten Douglasien (Pseudotsuga menziesii var. menziesii) wurden extrazelluläre Proteine extrahiert, um die Endochitinase-Aktivität zu bestimmen. Chitinasen werden mit der pflanzlichen Abwehrreaktion auf Pilzinfektionen in Verbindung gebracht, da sie Chitin, eine Strukturkomponente der pilzlichen Zellwand, hydrolysieren. Die Proteine wurden mit Natrium-Dodecyl-Sulfat-Polyacrylamid-Gelelektrophorese (SDS-PAGE) getrennt, gefolgt von einer Western Immunoblot-Analyse mit einem gegen ein Endochitinase-ähnliches Protein (ECP) spezifischen polyklonalen Antikörper. Hiermit liessen sich bis zu drei Polypeptide zwischen 27-30 kDa nachweisen. Eine zweidimensionale Gelelektrophorese (2-D PAGE) mit anschliessender Western Immunoblot-Analyse ergab, dass die Apoplastenflüssigkeit multiple ECP-Isoformen enthielt (mit pIs von 5,3 bis 5,8 und Molekularmassen von 27 bis 30 kDa). Die Chitinase-Aktivität wurde auch im Nadel- und Wurzelgewebe spektrophotometrisch mit einer Farbreaktion gemessen. Um sicher zu stellen, dass der ECP-Antikörper ein enzymatisch aktives Protein nachwies, wurde eine Gel-Overlay-Methode verwendet, mit Glycolchitin als Substrat für die Endochitinase. Die Apoplastenflüssigkeit der Nadeln von mit P. weirii infizierten Douglasien zeigte in Winterzustand eine Antifrost-Aktivität, ihre Analyse während des gesamten Jahres ergab aber keine Hinweise auf eine ECP-Anreicherung während der Wintermonate. ECP war systemisch im gesamten Baum enthalten. Die erhöhte Endochitinase-Aktivität in Bereichen mit P. weirii -Infektion lässt auf eine physiologische Rolle von ECP in der Pflanzenabwehr schliessen. [source]

    Lipid and protein changes in chilled sea salmon (Pseudopercis semifasciata): effect of previous rosemary extract (Rossmarinus officinalis L.) application

    INTERNATIONAL JOURNAL OF FOOD SCIENCE & TECHNOLOGY, Issue 6 2009
    Valeria Tironi
    Summary The aim of this work was to analyse the effect of rosemary extract application (200 and 500 ppm) on lipid oxidation, colour and protein modifications during the chilled storage (1.0 ± 0.7 °C) of sea salmon (Pseudopercis semifasciata). Lipid oxidation and ,3-22:6 fatty acid content modification were prevented by the addition of rosemary extract. Analysis of interaction between lipid oxidation products and proteins by fluorescence showed no relationship between their temporal changes in the aqueous phase and the lipid oxidation evolution since a similar behaviour was observed in both absence and presence of antioxidant. Protein extractability, sodium dodecyl sulphate-polyacrylamide gel electrophoresis, differential scanning calorimetry and lysine content determinations revealed no differences between muscle untreated or treated with rosemary. Fluorescent compounds evolution in organic phase would be in relation with the appearance of lipid oxidation products. In addition, rosemary extract partially prevented the loss of red colour in chilled muscle. Although protein alterations could not be prevented, rosemary extract shows to be a promissory antioxidant in sea salmon muscle. [source]

    Morphological and biochemical analyses of otoliths of the ice-fish Chionodraco hamatus confirm a common origin with red-blooded species

    JOURNAL OF ANATOMY, Issue 1 2009
    Chiara Maria Motta
    Abstract The morphology and composition of the three otoliths of the Antarctic ice-fish Chionodraco hamatus were studied by scanning electron microscopy and X-ray diffraction. The composition of the sagitta, lapillus and asteriscus protein matrices was also analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, western blots and confocal laser scanning microscopy to reveal the presence of and to localize the calcium-binding proteins calmodulin, calbindin and S-100. Morphological results indicated that the otoliths in this ice-fish were similar to those of Trematomus bernacchii, a red-blooded Antarctic species [B. Avallone et al. (2003) J. Submicrosc. Cytol. Pathol. 35, 69,76], but rather different from those of other teleosts. These two Antarctic species possessed a completely vateritic asteriscus, whereas their sagitta and lapillus were made mostly of aragonite. Parallel analysis of protein patterns in C. hamatus and T. bernacchii revealed that the sagitta significantly differed from the lapillus and asteriscus in both species. The sagitta did not contain the S-100 protein and showed calmodulin and calbindin located in discontinuous or incremental zones, respectively. These results demonstrate that the otoliths of C. hamatus and T. bernacchii share more resemblances than differences and support the idea of a common origin of these species. [source]

    Relevance of incubation temperature for Vibrio salmonicida vaccine production

    JOURNAL OF APPLIED MICROBIOLOGY, Issue 6 2002
    D.J. Colquhoun
    Aims:,To investigate the relationships between water temperature, bacterial growth, virulence and antigen expression in Vibrio salmonicida, the causal agent of cold water vibriosis in Atlantic salmon (Salmo salar L.). Methods and Results:,The significance of sea temperature was investigated using historical clinical and oceanographic data. An upper threshold for disease of approx. 10°C was established. The effects of culture temperature and media type on bacterial growth were studied on solid and in liquid media. The highest rates of cell division were identified at 15°C on solid media and 10°C in liquid media. Outer membrane protein (OMP) expression and serological response in Atlantic salmon were studied using sodium dodecyl sulphate-polyacrylamide gel electrophoresis, Western blotting and enzyme-linked immunosorbent assay. A novel 76-kDa OMP produced in unshaken cultures at 10°C was not found to stimulate a specific humoral response. Conclusions:,Diagnostic agar plate-based incubation of suspected V. salmonicida should be carried out at 15°C. High yield broth cultures for vaccine production should be incubated at 10°C or lower. Significance and Impact of the Study:,This study is, to the best of our knowledge, the first to identify different optimal temperatures in a bacterial species cultured on physically different types of media. The evidence presented suggests that V. salmonicida and possibly other bacteria destined for vaccine use in poikilothermic organisms should be cultured at temperatures consistent with that at which disease occurs. [source]

    The effect of toluidine blue on the survival, dormancy and outer membrane porin proteins (OmpC and OmpF) of Salmonella typhimurium LT2 in seawater

    JOURNAL OF APPLIED MICROBIOLOGY, Issue 6 2002
    R. Ozkanca
    Aims: To study the relationship between changes in the composition of the outer membrane proteins and the survival of Salmonella typhimurium LT2 in filtered autoclaved seawater containing Toluidine Blue (TB) dye as a photosensitizer. Methods and Results: In samples exposed to TB and excited by artificial visible light, the total viable (TVC) and respiring cell counts (RCC) showed that, although the TVC declined to an undetectable level in 6·5 h, the RCC showed that some cells were still capable of respiration. The porin protein composition changed gradually with OmpC and OmpF becoming undetectable by sodium dodecyl sulphate-polyacrylamide gel electrophoresis after 8 h of incubation. Hydrogen peroxide-pretreated cells survived longer compared with the control. Conclusions: Oxidative pretreatment of Salm. typhimurium protects cells from some of the effects of sunlight in the presence of photosensitizers. The changes in porin proteins may play a role in this protection. Significance and Impact of the Study: The study shows that the survival of bacteria under conditions of stress is the result of a linked series of reactions. [source]

    Purification and characterization of a bacteriocin-like compound (Lichenin) produced anaerobically by Bacillus licheniformis isolated from water buffalo

    JOURNAL OF APPLIED MICROBIOLOGY, Issue 4 2001
    P. Pattnaik
    Aims:,To characterize a bacteriocin-like factor from Bacillus licheniformis 26 L-10/3RA isolated from buffalo rumen. Methods and Results:,The culture supernatant exhibited the antibacterial activity against a number of indicator organisms in a cut-well agar assay under anaerobic conditions. The inhibitory component was purified by following ammonium sulphate precipitation, gel filtration and ion exchange chromatography and confirmed to be a single peptide. A single band on tricine-sodium dodecyl sulphate-polyacrylamide gel electrophoresis confirmed that the peptide was purified to homogeneity and having an estimated molecular mass of approximately 1400 dalton. Complete amino acid sequence of the peptide yielded 12 amino acids from the N-terminal end (ISLEICXIFHDN). No homology with previously reported bacteriocins was observed and has been designated as Lichenin. Lichenin was found to be hydrophobic, sensitive to atmospheric oxygen, retained biological activity even after boiling for 10 min and was active over a pH range of 4·0,9·0. Conclusions:,The Lichenin represents the first anaerobiosis specific expression of bacteriocin-like compound isolated from Bacillus licheniformis 26 L-10/3RA of buffalo rumen origin. Significance and Impact of the Study:,Lichenin could be a potential condidate for manipulating the rumen function at molecular level intended for improving the productivity of the ruminant. [source]

    Purification and characterization of tannin acyl hydrolase from Aspergillus niger MTCC 2425

    JOURNAL OF BASIC MICROBIOLOGY, Issue 6 2003
    Rita Bhardwaj
    The present investigation was carried out for increasing the yield of tannase of Aspergillus niger and the physico-chemical characterization of this enzyme. Homogenization and detergent pretreatments did not have any remarkable effect on the extraction of enzyme protein. However, extraction of fungal pigments and proteins was observed to have high pH dependence, and maximum enzyme extraction was obtained at pH 5.5. The two-step purification protocol gave 51-fold purified enzyme with a yield of 20%. The total tannase activity was made up of nearly equal activity of esterase and depsidase. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of purified tannase protein indicated it to be made up of two polypeptides of molecular weight 102 and 83 kDa. Based on the Michaelis-Menten constant (Km) of tannase for three substrates tested, tannic acid was the best substrate with Km of 2.8 × 10,4M, followed by methyl gallate and propyl gallate. The inhibition was maximum for CaCl2 (58%) whereas EDTA had no modulatory effect on tannase activity. The inhibitor binding constant (KI) of CaCl2 was 5.9 × 10,4M and the inhibition was of noncompetitive type. [source]

    ACTIVITY DISTRIBUTION OF DIGESTIVE PROTEASES FROM NEMIPTERUS VIRGATUS AND THEIR RESPONSES TO pH VALUE AND TEMPERATURE

    JOURNAL OF FOOD PROCESS ENGINEERING, Issue 1 2008
    HONG TAO
    ABSTRACT In the present study, three groups (I,III) of Nemipterus virgatus, with average body weights of 154.36, 250.72 and 329.09 g, respectively, were used to investigate the changes in the activity and distribution of digestive proteases in different organs and sections of the digestive tract. Another group of N. virgatus (average body weight of 188.41 g) was used to analyze the changes in the activity of digestive proteases in response to various pH values and temperatures. The activity of digestive proteases in all analyzed organs increased with the increase of body weight at the range of 154.36,329.09 g. The activities of digestive proteases in the different sections of the digestive tract were compared, and a similar change was found among groups I,III. The activities of digestive proteases from various organs were in a descending order: pylorus ceca > stomach > foregut > midgut > hindgut > hepatopancreas. Through observing the zymograms of substrate,sodium dodecil sulphate-polyacrylamide gel electrophoresis, many kinds of digestive proteases could be found in different organs and the varieties were changed with the change of body weight. Two peaks in the diagram between protease activity and pH value were found at pH 3.0 and 10.0, respectively. The activity under alkaline condition was 60% higher than that under acidic condition. The optimal temperature for protease activity was 50C, while the protease activity at 10C was only 30% of that at 50C. PRACTICAL APPLICATIONS Nemipterus virgatus is one of the most important commercial fishes in the East China Sea and South China Sea. The digestive tract of N. virgatus is rich in digestive proteases and they can be employed as important biotechnological tools. The activities of digestive proteases from various organs and the effects of pH value and temperature on them were investigated in this study. The effect of body weight of N. virgatus was also evaluated. All these information would be helpful to extensively utilize this resource for the fish process industry. [source]

    Rapid identification of pseudomonas avellanae field isolates, causing hazelnut decline in central italy, by repetitive PCR genomic fingerprinting

    JOURNAL OF PHYTOPATHOLOGY, Issue 3 2000
    M. Scortichini
    Pseudomonas avellanae is the main cause of hazelnut (Corylus avellana L.) decline, the so called ,moria', in central Italy where it has already killed more than 30 000 trees. Its current identification is very long requiring biochemical, physiological and nutritional tests as well as pathogenicity tests and takes not less than 6 months for its completion. In the present study the reliability of the repetitive polymerase chain reaction (rep-PCR) technique for a rapid and accurate identification of such a pathogen was compared with the traditional identification method. In order to assess the variability of the pathogen, REP, BOX and ERIC primer sets were used in preliminary work to generate genomic fingerprints of 60 P. avellanae reference strains previously isolated from different areas of hazelnut cultivation. ERIC primers yielded the most discriminative clustering of strains that were grouped according to their geographic origin. Sixty field isolates collected from hazelnut orchards of central Italy, planted with different cultivars, during the years 1996,98 were submitted to either the traditional identification methods or to rep-PCR by using ERIC primers. The latter technique accurately identified all the isolates that were also identified by the traditional methods. Whole-cell protein analysis by means of sodium dodecyl sulphate-polyacrylamide gel electrophoresis confirmed this achievement. Rep-PCR can be successfully adopted for the rapid and accurate identification of P. avellanae in central Italy and it constitutes a very useful tool for the sanitation of the area. Zusammenfassung Eine schnelle Bestimmung von Pseudomonas avellanae -Feldisolaten, dem Erreger einer HaselnuIapoplexie in Zentralitalien, durch rep-PCR genomisches Fingerprinting [source]

    Novel fibrinogen mutation ,314Thr,Pro (fibrinogen AI duPont) associated with hepatic fibrinogen storage disease and hypofibrinogenaemia

    LIVER INTERNATIONAL, Issue 10 2010
    Stephen O. Brennan
    Abstract Mutation in fibrinogen genes may lead to quantitative or qualitative disorders that result in bleeding, thrombosis or hepatic fibrinogen storage disease. Only three mutations in the fibrinogen , gene have been identified that cause hepatic endoplasmic reticulum storage of mutant fibrinogen. To investigate the possibility of hepatic fibrinogen storage disease in a 4-year-old male with persistently elevated serum aminotransferases and preserved synthetic function except for a prolonged INR. After informed consent, liver and blood samples were obtained. Liver sections were examined by light microscopy, anti-fibrinogen immunolabelling and electron microscopy. Purified fibrinogen was analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and reverse phase high performance liquid chromatography; DNA sequencing was performed using a BigDye Terminator (v. 3.1) cycle sequencing kit. Four-year-old male with persistently elevated transaminases with an INR 1.5 but otherwise normal synthetic function. Fibrinogen activity and thrombin clotting time were abnormal at 0.47 g/L and 46 s respectively. Hepatic histological examination revealed portal inflammatory infiltrates with bridging fibrosis. Clumped eosinophilic material was observed in hepatocytes that was immunoreactive to fibrinogen antisera. Ultrastructural examination showed cytoplasmic inclusions arrayed in fingerprint-like patterns. DNA sequence analysis revealed heterozygosity for a novel ,314Thr ,Pro mutation (fibrinogen AI duPont) in the fibrinogen , gene. Protein analyses showed normal patterns of A,, B, and , chains suggesting that the variant , allele was not expressed in plasma fibrinogen. We describe only the fourth mutation to be identified, ,314Thr,Pro (fibrinogen AI duPont), giving rise to hypofibrinogenaemia and hepatic fibrinogen storage disease. [source]

    Visceral schistosomiasis of domestic animals in India: humoral immune status of infected cattle, sheep and goats against major polypeptide antigens of Schistosoma indicum and S. spindale

    PARASITE IMMUNOLOGY, Issue 4 2004
    A. Singh
    SUMMARY Polypeptide profiles of Schistosoma indicum and S. spindale adult worm homogenates were obtained by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Humoral immune status of infected cattle, sheep and goats against Schistosoma indicum and S. spindale Ags was determined by immunoblot analysis and by indirect ELISA using four major polypeptides of approximate molecular masses 45 kDa, 40 kDa, 28 kDa and 15 kDa electro-eluted from the gel slices. Cattle sera samples had higher levels of antibodies against Si/s40 and Si/s28 than against Si/s45 antigen. Reasons have been discussed for the absence of detectable levels of anti-Si/s28, -Si/s45 and -Si/s40 antibodies in a significant number of sera samples from S. indicum egg-positive sheep. [source]

    Allelic variants of granule-bound starch synthase proteins in European bread wheat varieties

    PLANT BREEDING, Issue 4 2000
    C. Marcoz-Ragot
    Abstract The composition of 324 European wheat cultivars were analysed at the three granule-bound starch synthase (GBSS I) loci. Protein separation was first made by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE. A specific two-dimensional (2D) electrophoresis (immobilized pH gradient × SDS-PAGE) using an Immobiline dry strip in the first dimension was developed to resolve the GBSS I proteins more clearly and to confirm some results. Very low polymorphism was found. Among the 324 cultivars analysed, only one carried a Wx-A1 null allele (Wx-A1b) and none was found to have the Wx-2D null allele. As described in the literature the Wx-B1 locus was more polymorphic and the null allele was encountered in 11 cultivars. The use of 2D electrophoresis allowed us to find another type of variant which presented as having thicker band with same mobility as the Wx-D1 protein in SDS-PAGE. Twelve per cent of the cultivars analysed presented this band and could have been previously mistaken for cultivars carrying the Wx-B1 null allele. Indeed this band probably corresponded to the Wx-B1, or Wx-B1e allele overlapping with the Wx-D1a allele in SDS-PAGE. [source]

    Genetic relationships between South African wheat cultivars as measured by gliadin banding patterns

    PLANT BREEDING, Issue 3 2000
    M. T. Labuschagne
    Abstract The use of gliadins in cultivar identification is a well-known practice that is used in many countries. The aim of this study was to determine the gliadin banding patterns of 35 commercial South African wheat cultivars and to use these data to determine genetic relationships between the cultivars using sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The index of genetic similarity was used to calculate pairwise distance matrices, which were converted to a dendogram. Most of the gliadin bands fell within the nomenclature used. Fourteen cultivars had one novel band and six had two novel bands. All the cultivars could be distinguished from one another. Genetic distances between clusters were small. This suggested that there was not enough unique genetic variability to set any cluster apart from the others. [source]

    Glycoproteomics of N -glycosylation by in-gel deglycosylation and matrix-assisted laser desorption/ionisation-time of flight mass spectrometry mapping: Application to congenital disorders of glycosylation

    PROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 10 2005
    Dijana
    Abstract A general strategy for the structural evaluation of N -glycosylation, a common post-translational protein modification, is presented. The methods for the release of N -linked glycans from the gel-separated proteins, their isolation, purification and matrix-assisted laser desorption/ionisation-mass spectrometry (MALDI-MS) analysis of their mixtures were optimised. Since many glycoproteins are available only at low quantities from sodium dodecyl sulphate-polyacrylamide gel electrophoresis or two-dimensional gels, high attention was paid to obtain N -glycan mixtures representing their actual composition in human plasma by in-gel deglycosylation. The relative sensitivity of solid MALDI matrices for MS analysis of acidic N -glycans was compared. The most favourable results for native acidic N -glycans were obtained with 2,4,6-trihydroxyacetophenone monohydrate/diammoniumcitrate as a matrix. This matrix provided good results for both neutral and acidic mixtures as well as for methylated N -glycans. In the second part of this paper the potential of such an optimised MS strategy alone or in combination with high pH anion-exchange chromatography profiling for the clinical diagnosis of congenital disorders of glycosylation is presented. [source]

    Cross-protection elicited in channel catfish (Ictalurus punctatus Rafinesque) immunized with a low dose of virulent Edwardsiella ictaluri strains

    AQUACULTURE RESEARCH, Issue 8 2009
    Victor S Panangala
    Abstract Cross-protection of channel catfish (Ictalurus punctatus Rafinesque) immunized with a low dose of virulent Edwardsiella ictaluri and challenged with six E. ictaluri strains was examined in four trials. The relative per cent survival among low-dose immunized and then challenged fish ranged from 27.7% to 100%. Significant protection (P<0.05), with the exception of strain ATCC-33202, was conferred by immunization with a given E. ictaluri strain challenged either with a homologous or a heterologous strain. Antibody titres of pooled serum collected on day 22 from surviving fish examined by enzyme-linked immunosorbent assay (ELISA) ranged from 1:40 to 1:320, but no differences were apparent among different vaccinated groups. The protein profiles of six E. ictaluri strains examined by Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) showed a relatively homogeneous pattern. Immuno-blots probed with pooled serum from immunized and challenged fish showed a pattern similar to LPS-reaction patterns observed with E. ictaluri in other studies. Since the present studies further corroborate that E. ictaluri is a clonal bacterial species with no apparent antigenic differences, it is possible that immunization with a single E. ictaluri field strain should confer protection against any other strain. [source]

    Digestive peptidases and proteinases in the midgut gland of the pink shrimp Farfantepenaeus paulensis (Crustacea, Decapoda, Penaeidae)

    AQUACULTURE RESEARCH, Issue 7 2009
    Diego Souza Buarque
    Abstract Proteases from the midgut gland of the Farfantepenaeus paulensis juveniles were assessed. Enzyme activity was determined using protease substrates and inhibitors. The effect of pH, temperature and calcium on proteolytic activity was assayed. Caseinolytic activity was analysed in substrate-sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Trypsin, chymotrypsin and leucine aminopeptidase activity was detected. Proteolytic activity was strongly inhibited by the specific trypsin inhibitors. Tosyl-phenylalanine chloromethyl ketone inhibited 59.3% of chymotrypsin activity. The greatest trypsin-like activity occurred at pH 8.0 and 45 °C. Chymotrypsin-like activity reached maximal values at alkaline pH (7.2,9.0) and 55 °C. CaCl2 did not increase trypsin-like activity, but rather inhibited it at concentrations of 30 (20%), 50 (30%) and 100 mM (50%). The substrate-SDS-PAGE zymogram revealed eight proteinase bands. Two possibly thermal-resistant (85 °C, 30 min) chymotrypsin isoforms were found, which were inhibited by phenyl-methyl-sulphonyl-fluoride. Aminopeptidase activity of enzyme extracts (Arg, Leu, Lys, Phe and Val) and the recommended concentrations of these essential amino acids in penaeid shrimp diets were positively correlated (P<0.05). Beause protein digestion involves the combined action of different enzymes, adequate knowledge of shrimp digestion and enzyme characteristics is required for the assessment of the digestive potential of different feed sources and development of in vitro digestibility protocols. [source]

    Shift of C3 deposition from localization in the glomerulus into the tubulo-interstitial compartment in the absence of secreted IgM in immune complex glomerulonephritis

    CLINICAL & EXPERIMENTAL IMMUNOLOGY, Issue 1 2008
    C. Vaculik
    Summary The role of secretory IgM in protecting kidney tissue from immune complex glomerulonephritis induced by 4 mg horse spleen apoferritin and 0·05 mg lipopolysaccharide has been investigated in mutant mice in which B cells do not secrete IgM, but are capable of expressing surface IgM and IgD and secreting other Ig isotypes. Glomerular size, number of glomeruli per cross-section, glomerular cellularity and urine content of protein and creatinine was comparable in treated secreted IgM (sIgM)-deficient and wild-type mice. Assessment of urinary proteins by sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed a 30 kDa low molecular weight protein in treated sIgM-deficient animals only, reflecting dysfunction of proximal tubules. A shift of bound C3 from glomeruli to the tubulo-interstitial compartment in sIgM-deficient mice also suggests tubulo-interstitial damage. In contrast, local C3 synthesis within the kidney tissue did not differ between the two treated groups. Apoptosis physiologically present to maintain kidney cell homeostasis was increased slightly in treated wild-type mice. These results indicate that secretory IgM can protect the tubulo-interstitial compartment from immune complex-induced damage without having an effect on the glomerulus. [source]