Nondenaturing Polyacrylamide Gel Electrophoresis Research Articles

Purification and characterization of laccase from Marasmius species BBKAV79 and effective decolorization of selected textile dyes.

A novel laccase-producing white-rot fungus, Marasmius sp. BBKAV79 (Genbank Accession Number-KP455496, KP455497), was isolated and subjected to purification, characterization and dye decolorization study. The purified enzyme was obtained with a specific activity of 0.226 U mg−1 protein and a final yield of 13.5 %. The enzyme was found to be a monomeric protein with a molecular mass of ~75 kDa as estimated by non-denaturing polyacrylamide gel electrophoresis (PAGE) and further confirmed with zymogram analysis. The optimal pH and temperature of the laccase was recorded to be 5.5 and 40 °C, respectively. The metal ions Hg2+ and Ag+ were found to drastically inhibit the activity of laccase at the rate of 96.6 and 96.5 %, respectively. Nevertheless, Fe3+ was found to inhibit laccase activity at 40 %. Phenylmethanesulfonyl fluoride (PMSF) strongly inhibited the laccase activity, and additives viz, sodium dodecyl sulfate (SDS), hydrogen peroxide (H2O2) and sodium chloride (NaCl) were known to follow the earlier pattern of enzyme inhibition. The values of kinetic parameters K m and V max for purified laccase were noted at 3.03 mM and 5 μmol min−1, respectively, for guaiacol as substrate. The textile dyes were decolorized at a range of 72–76 % and 88–93 % when treated with Marasmius sp. BBKAV79 and purified laccase, respectively. Based on the outcome of the present investigation, it could be, therefore, inferred that laccase isolated from Marasmius sp. BBKAV79 effectively decolorizes the textile dyes; however, the metal ions Hg2+, Ag+ and Fe3+ and agents like PMSF, SDS, H2O2 and NaCl pose an effective inhibitory potential under specified physicochemical conditions.

Effects of jinggangmycin on peroxidase and esterase isozymes and phytotoxin from Rhizoctonia solani AG-1 IA, the causal agent of rice sheath blight. Pirinç kabuğu yanığı nedeni Rhizoctonia solani AG-1 IA tarafından üretilen peroksidaz, esteraz izozimleri ve fitotoksinler üzerinde jinggangmycin etkisi

Abstract Objective: To explore the effects of jinggangmycin on the pathogenicity of rice sheath blight. Methods: The effects of jinggangmycin on peroxidase and esterase isozymes from Rhizoctonia solani Kuhn AG-1 IA were compared using non-denaturing polyacrylamide gel electrophoresis (PAGE). Rs-toxins were obtained from the improved Richard medium amended with jinggangmycin at different concentrations. Subsequently, bioassays of Rs-toxin, including virulence to rice tissues, seedling wilting and inhibition to the seed germination were conducted. Results: The results of PAGE showed that when the jinggangmycin concentration increased to 50 μg/mL, two of peroxidase isozyme bands became apparently weaker compared with the controls, indicating that jinggangmycin could considerably weaken the activity of the peroxidase isozyme. In addition, three extra bands of esterase isozyme were induced by jinggangmycin when the concentration increased to 50 μg/mL, indicating that jinggangmycin could affect the metabolism of esterase isozyme. The bioassays of Rs-toxin showed that jinggangmycin could weaken the virulence of Rs-toxin in rice tissues. Conclusion: It was concluded that the control role of jinggangmycin in rice sheath blight might be due to the effects of jinggangmycin on the peroxidase and esterase isozymes and Rs-toxin virulence.

EVALUATION OF GENETIC DIVERSITY IN CACAO COLLECTED FROM KOLAKA, SOUTHEAST SULAWESI, USING SSR MARKERS

Kolaka, which is located in Southeast Sulawesi, has long been known as one of cacao production centers in Indonesia. Therefore, many different cacao germplasms can be found in this region. The study aimed to evaluate genetic diversity and relationships of 12 cacao genotypes collected from Kolaka. Genomic DNA was extracted by using a modified CTAB method. Meanwhile, genetic diversity was analyzed based on 16 SSR markers, which then separated by 6% non-denaturing polyacryl-amide gel electrophoresis. The result showed that all of those markers, 14 markers exhibited polymorphism and subsequently used for data analysis using NTSYS and PowerMarker program. About 70 different alleles were generated from 12 cacao genotypes analyzed with an average of 5 alleles per locus. Average value of polymorphism information content (PIC) resulted in this study was 0.59. The cluster analysis using UPGMA method based on the genetic similarity coefficient revealed that all cacao genotypes were separated into three major groups. The first group consisted of five cacao genotypes, the second one held four cacao genotypes, whereas the third group contained three genotypes. This result indicates that three genotypes that clustered separately from the others could be used as a good clonal candidate for cacao breeding program. The information resulted from this present study would be useful for future cacao breeding program, especially in efforts to release a new variety.

EVALUATION OF GENETIC DIVERSITY IN CACAO COLLECTED FROM KOLAKA, SOUTHEAST SULAWESI, USING SSR MARKERS

Kolaka, which is located in Southeast Sulawesi, has long been known as one of cacao production centers in Indonesia. Therefore, many different cacao germplasms can be found in this region. The study aimed to evaluate genetic diversity and relationships of 12 cacao genotypes collected from Kolaka. Genomic DNA was extracted by using a modified CTAB method. Meanwhile, genetic diversity was analyzed based on 16 SSR markers, which then separated by 6% non-denaturing polyacryl-amide gel electrophoresis. The result showed that all of those markers, 14 markers exhibited polymorphism and subsequently used for data analysis using NTSYS and PowerMarker program. About 70 different alleles were generated from 12 cacao genotypes analyzed with an average of 5 alleles per locus. Average value of polymorphism information content (PIC) resulted in this study was 0.59. The cluster analysis using UPGMA method based on the genetic similarity coefficient revealed that all cacao genotypes were separated into three major groups. The first group consisted of five cacao genotypes, the second one held four cacao genotypes, whereas the third group contained three genotypes. This result indicates that three genotypes that clustered separately from the others could be used as a good clonal candidate for cacao breeding program. The information resulted from this present study would be useful for future cacao breeding program, especially in efforts to release a new variety.

Chitosan-myristate nanogel as an artificial chaperone protects neuroserpin from misfolding.

Background:Molecular chaperon-like activity for protein refolding was studied using nanogel chitosan-myristic acid (CMA) and the protein neuroserpin (NS), a member of the serine proteinase inhibitor superfamily (serpin).Materials and Methods:Recombinant his-tag fusion NS was expressed in Escherichia coli. For confirmation of refolding of the purified NS, structural analysis was performed by circular dichroism and spectrofluorometric along with its inhibitory activity, which was assayed by single-chain tissue plasminogen activator. For evaluating NS aggregation during preparation, the samples were separated on a 7.5% (w/v) nondenaturing polyacrylamide gel electrophoresis. MA and chitosan covalently join together by the formation of amide linkages through the 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide-mediated reaction. The morphology and size of the prepared CM nanogel were characterized by transmission electron microscopy and scanning electron microscopy.Results:Heating at different temperatures (25°C, 37°C, 45°C, 65°C, 80°C) results in a further rise in β-structures accompanied by a fall of helices and no significant change in random coils. Structural changes in NS in the presence of CMA nanogel were less than that in the absence of CMA nanogel. Mater nanogel effectively prevented aggregation of NS during temperature induced protein refolding by the addition of cyclodextrins. The nanogel activity resembled the host-guest chaperon activity.Conclusion:These conditions, called conformational disorders, include Alzheimer's, Parkinson's, Huntington's disease, the transmissible spongiform encephalopathies, prion diseases, and dementia. Nanogels can be useful in recovery of the structural normality of proteins in these diseases.

Secretory component mediates Candida albicans binding to epithelial cells.

Candida albicans attaches to oral surfaces via a number of mechanisms including adherence mediated by salivary components adsorbed to the C. albicans cell surface. Our goal was to identify the salivary molecules involved. Biotinylated salivary polypeptides that were bound by C. albicans were detected in extracts from washed, saliva-treated yeast cells by polyacrylamide gel electrophoresis and electroblot or immunoblot transfer analysis and purified by electroelution. Purified material was tested for the ability to promote the adherence of radiolabelled C. albicans yeast cells to cultured epithelial monolayers. Three of the polypeptides bound by C. albicans cells were identified as components of secretory IgA, including secretory component. Using non-denaturing polyacrylamide gel electrophoresis, we demonstrated that secretory component could be detected in its free form in saliva, and was bound by yeast cells. Secretory component which was purified by electroelution from non-denaturing PAGE-separated saliva, without detectable complete IgA, promoted adherence of yeast cells to cultured epithelial monolayers in a dose-dependent fashion. These results indicate that despite the inhibitory effect on adherence of IgA specific to C. albicans, IgA components, in particular secretory component, also promote binding to cultured epithelial monolayers.

Nondenaturing polyacrylamide gel electrophoresis to study the dissociation of the p53·MDM2/X complex by potentially anticancer compounds

A new analytical method to study the dissociation of the complexes between the oncosuppressor p53 and its negative modulators murine double-minute protein 2 (MDM2) or MDMX, is proposed. This technique is reliable to determine the dissociative power exerted by small molecules on the complex taking advantage of the appearance of migrating MDM2 or MDMX in a native polyacrylamide gel, when inhibitors are added to the complex mixture. Therefore, we propose this new approach to easily screen library of compounds, with potential pharmacological anticancer activity.

Heme Stabilization of α-Synuclein Oligomers during Amyloid Fibril Formation.

α-Synuclein (αSyn), which forms amyloid fibrils, is linked to the neuronal pathology of Parkinson's disease, as it is the major fibrillar component of Lewy bodies, the inclusions that are characteristic of the disease. Oligomeric structures, common to many neurodegenerative disease-related proteins, may in fact be the primary toxic species, while the amyloid fibrils exist either as a less toxic dead-end species or even as a beneficial mechanism for clearing damaged proteins. To alter the progression of the aggregation and gain insights into the prefibrillar structures, we determined the effect of heme on αSyn oligomerization by several different techniques, including native (nondenaturing) polyacrylamide gel electrophoresis, thioflavin T fluorescence, transmission electron microscopy, atomic force microscopy, circular dichroism, and membrane permeation using a calcein release assay. During aggregation, heme is able to bind the αSyn in a specific fashion, stabilizing distinct oligomeric conformations and promoting the formation of αSyn into annular structures, thereby delaying and/or inhibiting the fibrillation process. These results indicate that heme may play a regulatory role in the progression of Parkinson's disease; in addition, they provide insights into how the aggregation process may be altered, which may be applicable to the understanding of many neurodegenerative diseases.

Association of 14-bp insertion/deletion polymorphism of HLA-G gene with idiopathic recurrent miscarriages in infertility center patients in Yazd, Iran

HLA-G is supposed to play a pivotal role in tolerance of the semi-allogeneic graft in pregnancy by inhibiting the cytotoxic functions of T and NK cells. A 14-bp insertion and/or deletion polymorphism in exon-8 has a possible role in HLA-G expression. The present study analyzed the 14-bp insertion/deletion polymorphism in normal pregnancy and recurrent miscarriage patients in order to discover a possible correlation between the 14-bp polymorphism and recurrent miscarriage (RM). In this study, genomic DNA from 200 RM patients and 200 normal fertile control individuals using the routine salting out method were isolated. Exon-8 of HLA-G gene of the two groups were amplified using polymerase chain reaction and analyzed by electrophoresis on 10% non-denaturing polyacrylamide gel electrophoresis containing ethidium bromide and visualized under ultraviolet light. HLA-G allele frequencies and genotypes in RM women and the fertile control group were compared using a Chi-square test. The results showed that there was a difference in allelic frequencies of 14-bp insertion polymorphism between fertile controls and RM patients; the frequency of +14 bp/−14 bp heterozygotes was significantly higher in RM patients as compared with fertile controls. Furthermore, the frequency of +14-bp insertion allele was significantly higher in those with RM as compared with normal fertile controls. From the findings here, it was concluded that a 14-bp insertion/deletion polymorphism in exon 8 could play a possible role in recurrent miscarriages. These results might ultimately be of significance for clinicians and those involved in understanding infertility and RM.

Performance of Bell Pepper Seeds in Response to Drum Priming with Addition of 24-Epibrassinolide

Drum priming systems are among the most common methods of seed priming for commercial treatment. The supply of water to the seeds is controlled by physical means, and seeds are able to reach a desired moisture content without the use of osmotic solutions for hydration control. The brassinosteroid (BR), 24-epibrassinolide (24-EpiBL), has been shown previously to have a significant effect on seed performance. Therefore, the aim of this study was to verify the feasibility of drum priming associated with added 24-EpiBL on enhancement of bell pepper seed performance. Two bell pepper cultivars (AF-6 and AF-7) were tested, and each cultivar represented, respectively, by three and four seed lots with different initial physiological potentials. Seed performance was determined by evaluating standard germination, initial seedling growth, and changes on the antioxidant activity of superoxide dismutase (SOD), catalase (CAT), and peroxidase (POX). Seed Vigor Imaging System (SVIS®) was used to assess the initial seedling growth. The nondenaturing polyacrylamide gel electrophoresis (PAGE) technique was used to evaluate possible changes in the enzymatic antioxidant system. Several advantages were verified in the drum priming technique with added 24-EpiBL compared with the traditional procedure (water alone). Germination time was reduced followed by a seedling growth increase. Concomitantly, seed enzymatic activity was improved. However, results showed different response for each enzyme. Drum priming with 24-EpiBL demonstrates viability for commercial treatment and enhancement of bell pepper seeds.

Chromogenic assessment of the three molybdo-selenoprotein formate dehydrogenases in Escherichia coli

Escherichia coli synthesizes three selenocysteine-dependent formate dehydrogenases (Fdh) that also have a molybdenum cofactor. Fdh-H couples formate oxidation with proton reduction in the formate hydrogenlyase (FHL) complex. The activity of Fdh-H in solution can be measured with artificial redox dyes but, unlike Fdh-O and Fdh-N, it has never been observed by chromogenic activity staining after non-denaturing polyacrylamide gel electrophoresis (PAGE). Here, we demonstrate that Fdh-H activity is present in extracts of cells from stationary phase cultures and forms a single, fast-migrating species. The activity is oxygen labile during electrophoresis explaining why it has not been previously observed as a discreet activity band. The appearance of Fdh-H activity was dependent on an active selenocysteine incorporation system, but was independent of the [NiFe]-hydrogenases (Hyd), 1, 2 or 3. We also identified new active complexes of Fdh-N and Fdh-O during fermentative growth. The findings of this study indicate that Fdh-H does not form a strong complex with other Fdh or Hyd enzymes, which is in line with it being able to deliver electrons to more than one redox-active enzyme complex.

Enzyme Activities of Muscle Tissue Lysates can be Unexpectedly Affected by Protease Inhibitor Cocktails

Previous studies in our lab have shown that the protective use of certain protease inhibitor cocktails with skeletal muscle tissue lysates can decrease protein expression on PAGE (polyacrylamide gel electrophoresis). The purpose of the present study was to determine whether inclusion of cocktails in two different rat skeletal muscle (biceps femoris and rectus abdominis) tissue lysates could affect enzyme activities of endogenous non-proteases. After lysates were incubated with cocktails at 37°C for one hour, they were run on non-denaturing polyacrylamide gel electrophoresis (native-PAGE) and assayed for enzyme activities. Results indicate that acetyl-cholinesterase (AChE) activities of lysates incubated with cocktails were significantly diminished; in contrast, malate dehydrogenase (MDH) and lactate dehydrogenase (LDH) activities of lysates incubated with cocktails were unaffected. When muscle lysates were challenged with high trypsin concentrations (0.14%), enzyme activities of MDH (mitochondrial isozyme) and LDH were inhibited; however, the inclusion of certain cocktails rescued activities. Enzyme activities in cardiac and small intestinal muscle tissue lysates treated with cocktails appear to show a similar pattern. In conclusion, it is suggested that the inclusion of cocktails with tissue lysates should be carefully evaluated when measuring tissue enzyme activity. (Support from MSMC Research Funding and Pace Scholarly Research)

Label-free detection of kanamycin based on a G-quadruplex DNA aptamer-based fluorescent intercalator displacement assay.

This work was the first to report that the kanamycin-binding DNA aptamer (5′-TGG GGG TTG AGG CTA AGC CGA-3′) can form stable parallel G-quadruplex DNA (G4-DNA) structures by themselves and that this phenomenon can be verified by nondenaturing polyacrylamide gel electrophoresis and circular dichroism spectroscopy. Based on these findings, we developed a novel label-free strategy for kanamycin detection based on the G4-DNA aptamer-based fluorescent intercalator displacement assay with thiazole orange (TO) as the fluorescence probe. In the proposed strategy, TO became strongly fluorescent upon binding to kanamycin-binding G4-DNA. However, the addition of kanamycin caused the displacement of TO from the G4-DNA–TO conjugate, thereby resulting in decreased fluorescent signal, which was inversely related to the kanamycin concentration. The detection limit of the proposed assay decreased to 59 nM with a linear working range of 0.1 μM to 20 μM for kanamycin. The cross-reactivity against six other antibiotics was negligible compared with the response to kanamycin. A satisfactory recovery of kanamycin in milk samples ranged from 80.1% to 98.0%, confirming the potential of this bioassay in the measurement of kanamycin in various applications. Our results also served as a good reference for developing similar fluorescent G4-DNA-based bioassays in the future.

Effect of iron stress on Withania somnifera L.: antioxidant enzyme response and nutrient elemental uptake of in vitro grown plants.

In the present study the response of antioxidant enzyme activities and the level of expression of their corresponding genes on bioaccumulation of iron (Fe) were investigated. In vitro germinated Withania somnifera L. were grown in Murashige and Skoog's liquid medium with increasing concentrations (0, 25, 50, 100 and 200µM) of FeSO4 for 7 and 14days. Root and leaf tissues analyzed for catalase (CAT, EC 1.11.1.6), superoxide dismutase (SOD, EC 1.15.1.1) and guaiacol peroxidase (GPX, EC 1.11.1.7), have shown an increase in content with respect to exposure time. Isoforms of CAT, SOD and GPX were separated using non-denaturing polyacrylamide gel electrophoresis and observed that the isoenzymes were greatly affected by higher concentrations of Fe. Reverse transcriptase polymerase chain reaction analysis performed by taking three pairs of genes of CAT (RsCat, Catalase1, Cat1) and SOD (SodCp, TaSOD1.2, MnSOD) to find out the differential expression of antioxidant genes under Fe excess. RsCat from CAT and MnSOD from SOD have exhibited high levels of gene expression under Fe stress, which was consistent with the changes of the activity assayed in solution after 7days of treatment. Analysis by proton induced X-ray emission exhibited an increasing uptake of Fe in plants by suppressing and expressing of other nutrient elements. The results of the present study suggest that higher concentration of Fe causes disturbance in nutrient balance and induces oxidative stress in plant.

Cloning and characterization of a differentially expressed mitochondrial manganese superoxide dismutase gene from Pleurotus ostreatus

Manganese-containing superoxide dismutase (Mn-SOD) is one of the most important superoxide dismutases found in many eukaryotes and bacteria. In this study, the full-length cDNA of mitochondrial manganese superoxide dismutase from Pleurotus ostreatus (PoMn-SOD) was obtained. It contained 776 nucleotides with an open reading frame (ORF) of 663 bp, encoding 220 amino acid residues. The deduced amino acid sequence showed high identity with the sequences of other basidiomycetous Mn-SODs from Trametes versicolor (71 %) to Laccaria bicolor (77 %). Quantitative real-time PCR (RT-PCR) analysis revealed that PoMn-SOD gene transcripts were abundant in the stage of young fruit bodies and mature fruit bodies. The up-regulation of the PoMn-SOD gene suggested that it was developmental regulated and could play an important role in antagonizing environmental stresses. In addition, another isoform of Mn-SOD was detected by a non-denaturing polyacrylamide gel electrophoresis (PAGE) approach and the highest total Mn-SOD activity (203.9 U/mg) was observed in the stage of mature fruit bodies. These data could provide important reference for functional study of the PoMn-SOD gene and may benefit biotechnological production of Mn-SOD in the future.

Unique molecular signatures of Alzheimer's disease amyloid β peptide mutations and deletion during aggregate/oligomer/fibril formation.

The formation of amyloid β (Aβ) peptide aggregates, oligomers, and fibrils is a dynamic process; however, the kinetics of their formation is not well understood. This study compares the time course of aggregate/fibril formation by transmission electron microscopy (TEM) analyses with that of oligomer/fibril formation by Western blot analysis under native and denaturing conditions. Efforts to deaggregate/defibrillate these peptides by using hexafluoroisopropanol, ammonium hydroxide, or dimethylsulfoxide did not change the nondenaturing polyacrylamide gel electrophoresis (PAGE) footprints or drive the peptides to a monomeric species. Regardless of the pretreatment protocol, TEM analyses reveal that all Aβ peptides (Aβ40, Aβ42, Aβ39E22Δ [Osaka], Aβ40E22G [Arctic], Aβ40E22Q [Dutch], and Aβ40A2T [Icelandic]) immediately formed nonfibrillar, amorphous aggregates when first placed into solution with the Osaka mutation, quickly forming early-stage fibrils. The extent of fibril formation for other Aβ peptides is time dependent, with the Arctic mutation forming fibrils at 1 hr, the Dutch and Icelandic at 4 hr, Aβ42 at 8 hr, and Aβ40 at 24 hr. In contrast, nondenaturing PAGE revealed unique footprints for the different Aβ species. The rapidity of aggregate formation and the rapid transition to fibrils, particularly for the Osaka deletion, suggest an important role for aggregates/fibrils of Aβ in the development of neuronal degeneration.

DNA logic gate based on metallo-toehold strand displacement.

DNA is increasingly being used as an ideal material for the construction of nanoscale structures, circuits, and machines. Toehold-mediated DNA strand displacement reactions play a very important role in these enzyme-free constructions. In this study, the concept of metallo-toehold was utilized to further develop a mechanism for strand displacement driven by Ag+ ions, in which the intercalation of cytosine–cytosine mismatched base pairs on the toeholds provides additional control by varying of the concentration of Ag+ ions. The characteristics of displacement reaction in response to different concentration of Ag+ ions are investigated by fluorescence spectral and non-denaturing polyacrylamide gel electrophoresis. The reaction can successfully occur when the concentration of Ag+ ions is suitabe; excess Ag+ ions block the reaction. Furthermore, the displacement reaction can be tuned and controlled most efficiently under the condition of two C:C mismatched base pairs placed on the six-nt toehold. Based on our research, a mechanism was developed to construct Boolean logic gate AND and OR by employing strand displacement reaction as a tool, Ag+ and Hg2+ as input.

RNA-RNA SELEX.

Systematic evolution of ligands by exponential enrichment (SELEX) protocol is a valuable technique to identify RNA aptamers interacting with RNA structural motifs. RNA aptamers are mainly resolved with affinity column chromatography and electrophoretic mobility shift assay (EMSA). Here, we describe the separation of the RNA aptamers binding to an RNA stem-loop target with affinity chromatography using the column attached the target RNA and nondenaturing polyacrylamide gel electrophoresis to obtain a single predominant RNA aptamer family.

Burkholderia sp. SCMS54 Triggers a Global Stress Defense in Tomato Enhancing Cadmium Tolerance

Cadmium is a toxic element for living organisms. This metal causes different damages to the cell, generating oxidative stress. In order to elucidate cadmium tolerance mechanism and increase tomato plant tolerance by inoculating a Cd-tolerant Burkholderia strain, we analyzed malondialdehyde, hydrogen peroxide content, and the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), and glutathione S-transferase of two strains, one isolated from a soil contaminated with Cd (strain SCMS54) and the other from a soil without Cd (strain SNMS32). Strains SNMS32 and SCMS54 exhibited different SOD, CAT, and GR isoenzyme profiles in non-denaturing polyacrylamide gel electrophoresis analysis, with strain SCMS54 exhibiting an extra isoenzyme for all enzymes (Mn-SOD, CAT I, and GR IV, respectively). Despite accumulating more Cd, strain SCMS54 did not increase peroxide hydrogen and presented a fast antioxidant response (increasing SOD and CAT after 5 h of Cd exposure). In this way, strain SCMS54 exhibited a higher metabolic diversity and plasticity when compared to strain SNMS32, so it was selected for Cd–Burkholderia–tomato interaction studies. Inoculated tomato plants in the presence of Cd grew more than non-inoculated plants with Cd indicating that the SCMS54 increased tomato Cd tolerance. It appears that the strain isolated from Cd-contaminated soil (SCMS54) triggers a global stress response in tomato increasing plant tolerance, which may enable plants to be cultivated in Cd-contaminated soils.

Partial characterization of superoxide dismutase activity in the Barber pole worm-Haemonchus contortus infecting Capra hircus and abomasal tissue extracts

ABSTRACT Objective To determine the activity of superoxide dismutase (SOD) in the male and female haematophagous caprine worms, Haemonchus contortus infecting Capra hircus, and their E/S products and also to analyse the effect of Haemonchus infection on the level of host SOD. Methods The SOD activity was analysed by using the pyrogallol autoxidation assay and non-denaturing polyacrylamide gel electrophoresis followed by specific enzyme staining by riboflavin-nitroblue tetrazolium method. Results The adult females were found to have higher enzyme activity than the male worms. Appreciable amount of SOD activity was also detected in the worm culture medium and female worms secreted more SOD in comparison to the male parasites. The SOD activity was negatively correlated to the worm burden. Statistically significant decrease in SOD activity ( P 0.05) was observed in the heavily infected host tissue in comparison to the control non-infected host tissue. SOD profile of the crude extracts of both the sexes revealed polymorphism and a fast migrating activity band being characteristic of E/S products. The SOD activities were found highly sensitive to potassium cyanide indicating the Cu/Zn form of SOD. Conclusions Haemonchus contortus is a key model parasite for drug and vaccine discovery. The presences of SOD activity in appreciable amount in the parasite as well as its E/S products indicate that it has a well-developed active antioxidant system to protect itself from the host immune attack. SOD could be the target for vaccine development which is the need of the hour as mass drug administration for parasite control has resulted in anthelmintic resistance across the globe and threatens the viability of sheep and goat industry in many regions of the world. The infection with Haemonchus causes a drastic reduction in SOD activity of the host tissue thus effecting its protective potential. One characteristic SOD band was found in the females which was not present in any other preparations and thus could be exploited for further studies on diagnostic/control measures.