Distinct Protein Bands Research Articles

Phospho-substrate profiling of Epac-dependent protein kinase C activity.

Exchange protein directly activated by cAMP (Epac) and protein kinase A are effectors for cAMP with distinct actions and regulatory mechanisms. Epac is a Rap guanine nucleotide exchange factor that activates Rap1; protein kinase C (PKC) is a major downstream target of Epac-Rap1 signaling that has been implicated in a variety of pathophysiological processes, including cardiac hypertrophy, cancer, and nociceptor sensitization leading to chronic pain. Despite the implication of both Epac and PKC in these processes, few downstream targets of Epac-PKC signaling have been identified. This study characterized the regulation of PKC activity downstream of Epac activation. Using an antibody that recognizes phospho-serine residues within the consensus sequence phosphorylated by PKC, we analyzed the 1-dimensional banding profile of PKC substrate protein phosphorylation from the Neuro2A mouse neuroblastoma cell line. Activation of Epac either indirectly by prostaglandin PGE2, or directly by 8-pCPT-2-O-Me-cAMP-AM (8pCpt), produced distinct PKC phospho-substrate protein bands that were suppressed by co-administration of the Epac inhibitor ESI09. Different PKC isoforms contributed to the induction of individual phospho-substrate bands, as determined using isoform-selective PKC inhibitors. Moreover, the banding profile after Epac activation was altered by disruption of the cytoskeleton, suggesting that the orchestration of Epac-dependent PKC signaling is regulated in part by interactions with the cytoskeleton. The approach described here provides an effective means to characterize Epac-dependent PKC activity.

Isolation and Characterization of Clostridium perfringens from Suspected Cases of Necrotic Enteritis (NE) in Broiler Chicken

Considering necrotic enteritis (NE) as an economically important problem of broiler industry, a study was undertaken to establish the association of Clostridium perfringens with the disease. Screening of 35 fecal samples/intestinal scrapping of broiler chicken, either clinically affected or died due to the suspected form of NE revealed isolation of C. perfringens from 13 (37.14%) samples. Among the isolates, 10 were identified as toxin type A, while remaining three as toxin type C. One of the toxin type A revealed the presence of additional virulence-associated genes, netB and tpeL. Irrespective of toxin types, C. perfringens isolates showed resistance towards Ciprofloxacin (76.92%), followed by Norfloxacin (53.85%), Tetracycline (46.15%), Colistin (23.07%) and Levofloxacin (15.38%), of which eight could exhibit resistance towards multiple antimicrobial agents. The release of C. perfringens toxins was found to be more, during the 6-8 hrs of incubation with 13 distinct protein bands including of cpa, netB and tpeL protein. The toxin type A with additional virulence-associated genes was found to be more pathogenic in mice pathogenicity test.

Quantity and Functionality of Protein Fractions Isolated from 3 Ecotypes of Indigenous Chicken in Kenya

<p><em>The aim of this study was to evaluate the effect of the cluster ecotype and the part of chicken on nutritional composition, and functionality of sarcoplasmic and myofibrillar proteins that are most relevant to the technological features of chicken meat. Over 50 chickens from each ecotype cluster purchased, slaughtered and the meat stored under refrigeration at -20<sup>o</sup>C and later on transferred in cooler box on ice and flown to South Africa, at the Durban University of Technology. Protein fractions were extracted with a cocktail of Sodium Chloride buffer (50mM NaCl, 50mM Tris HCl; 75mM DTT and 1mM EDTA at pH 7) and quantified by Bradford method. One dimensional Sodium Dodecyl Polyacrylamide Gel Electrophoresis (SDS PAGE) was applied to separate protein fractions. Emulsifying capacity, emulsifying stability, solubility, and in vitro digestibility were determined on the total protein isolates. Significant differences in band expressions were recorded for the myofibrillar and the sarcoplasmic proteins. The three ecotypes had high quality proteins with all the limiting and essential amino acids at concentrations higher than FAO/WHO recommended daily allowance for adults and children. Distinct protein bands at larger molecular weight proteins >100 kDa, corresponding to Myosin Heavy Chain, medium fractions 75 kDa and 45 kDa and even lower molecular weight fraction <25 kDa were present in the chicken breast and the thighs. It concludes that Indigenous chicken protein isolates’ nutritional and functional properties are affected by part of chicken and ecotype clusters. </em></p>

Differentiation of bovine from porcine gelatin capsules using gel electrophoresis method

Gelatin is commonly used in food supplements and in the form of soft or hard capsules. The source of gelatins is usually from porcine and bovine, and less commonly from vegetable and fish. Nevertheless, these different origins of gelatin have much similarity in term of structures, physicochemical properties and amino acid sequences. Due to these reasons, differentiation of the source of gelatins has been very difficult. In our present study, differentiation of sources of gelatin was made possible in a simplified yet economical method. Sample was prepared using ammonium sulfate precipitation and subjected to gel electrophoresis for protein separation. We have found a fraction of proteins which is able to differentiate porcine and bovine gelatins accurately, with distinctive protein bands in SDS-PAGE at 140 kDa and 110 kDa for bovine and porcine samples, respectively. This method was verified by 13 double-blinded gelatin samples, all the 13 samples were accurately identified.

Abolishment of proximal tubule albumin endocytosis does not affect plasma albumin during nephrotic syndrome in mice

The megalin/cubilin receptor complex is required for proximal tubular endocytosis and degradation of filtered albumin. An additional high-capacity retrieval pathway of intact albumin for the recovery of large amounts of filtered albumin has been proposed, possibly involving cooperation between megalin/cubilin and the neonatal Fc receptor. To clarify the potential role of such a pathway, we examined the effects of megalin/cubilin gene inactivation on tubular albumin uptake and plasma albumin levels in nephrotic, podocin knockout mice. Immunofluorescence microscopy of megalin/cubilin/podocin knockout mouse kidneys demonstrated abolishment of proximal tubule albumin uptake, in contrast to the excessive albumin accumulation observed in podocin knockout mice compared to controls. Correspondingly, urinary albumin excretion was increased 1.4 fold in megalin/cubilin/podocin compared to podocin knockout mice (albumin/creatinine: 226 vs. 157 mg/mg). However, no difference in plasma albumin levels was observed between megalin/cubilin/podocin and podocin knockout mice, as both were reduced to approximately 40% of controls. There were no differences in liver albumin synthesis by mRNA levels and protein abundance. Thus, megalin/cubilin knockout efficiently blocks proximal tubular albumin uptake in nephrotic mice but plasma albumin levels did not differ as a result of megalin/cubilin-deficiency, suggesting no significance of the megalin/cubilin-pathway for albumin homeostasis by retrieval of intact albumin.

Organic macromolecules in shells of Arctica islandica: comparison with nacroprismatic bivalve shells

A detailed characterisation of the organic composition of Arctica islandica (Linnaeus, 1767) shells with homogenous microstructure is compared with nacroprismatic shells of Pinctada fucata martensii (Dunker, 1872), Hyriopsis cumingii (Lea, 1852) and Diplodon chilensis patagonicus (d’Orbigny, 1835). Thermogravimetric analysis shows lowest total organic contents of 1.65 wt% for A. islandica shells, while all nacroprismatic shells are higher (3.14–4.13 wt%). Macromolecules extracted from the nacroprismatic shells are dominated by hydrophobic amino acids (~ 54%) in the acid extracts, while EDTA-extracts are moderately rich in aspartate and glutamate (16% in total) and in glycine–alanine (42%). In comparison, A. islandica shells have higher concentrations of proline, glycine and aspartate (ca 40%). Infrared spectroscopy shows some acidic protein bands in A. islandica, which cannot be found in the nacroprismatic shells. Alcian Blue and/or modified silver staining methods reveal many prominent bands. Protein bands at around 10, 14, 17, 21, 26, 31, 40 and 55 kDa are detected in A. islandica shells for the first time, thus may constitute a new set of proteins in mollusc shells. SDS-PAGE exhibits apparent molecular weights from 5 to 63 kDa in nacroprismatic shells. Distinct protein bands at around 17 and 26 kDa in A. islandica shells may correspond to a post-translational modification of proteins; these prominent bands, however, are absent in the nacroprismatic samples. Contrarily, SDS-PAGE of both, homogeneous and nacroprismatic shell microstructures show nonacidic-matrix-proteins.

Extraction and characterisation of lemon, orange and grapefruit seeds press cake proteins

The aim of this study was to characterise the proteins of lemon (Citrus limon var. Kutdiken), orange (Citrus sinensis var. Dortyol) and grapefruit (Citrus paradisivar. Beyaz) seeds extracted from their defatted press cakes. The press cakes were collected from the cold oil pressing of the seeds which were pre-treated by different techniques. Ultrasound-assisted alkaline (pH=9.0) extraction and isoelectric point (pH=4.0) precipitation procedure yielded around 34.47-80.95% proteins. The extracted seed proteins were in good quality for colour and antioxidant capacity. The extracted-lyophilised proteins were more soluble at pH 11-12 and below 2. The viscosity of the protein dispersions ranged from 3.67 to 25.70 cP. Sixteen amino acids were quantified in the samples, while asparagine, cysteine, hydroxyproline and tryptophan were absent. Thermal denaturation temperature and enthalpy change of denaturation were reported for the first time. The gel electrophoresis indicated four distinct protein bands at around 10...

Effects of microbial transglutaminase on physicochemical properties, electrophoretic patterns and sensory attributes of veggie burger.

The main objective of this study was to investigate the effects of microbial-transglutaminase (MTGase 0-0.75%)/sodium-caseinate (SC 0-2%) as crosslinker agents on proximate analysis, binding properties (expressible moisture and shrinkage), texture analysis, electrophoretic patterns, instrumental color, and sensory properties of veggie burgers. Addition of SC and MTGase positively affected shrinkage and expressible moisture. It also increased hardness, springiness, chewiness, and cutting-force of burgers. Presence of SC had no effects on cohesiveness of burgers. Total protein and ash of samples were increased by treatment with SC. The lightness (L*) of samples was significantly decreased by 0.75% MTGase. No significant influence of SC on samples color parameters was observed. The results indicated that distinct protein bands were not formed on the SDS-PAGE of burger samples and resulted in a smearing pattern on the gel. When soy-protein was incubated with MTGase, a progressive decrease in the intensity of the bands corresponding to the subunits 7S and 11S globulins was observed concomitant with disappearance of A3 and B3 bands. Electrophoresis pattern of gluten was slightly changed after MTGase treatment. There were significant differences in color, taste, appearance, mouth feel, and overall acceptability between treated and control samples. Results suggest that production of veggie burgers using MTGase alone or in combination with SC brings about covalent cross-linking between homologous and heterologous proteins to form high-molecular weight polymers, thereby improving the mechanical properties of veggie burgers and profoundly increases the acceptability of the end product.

Influence of Randomly Inserted Feruloyl Esterase A on β-Glucosidase Activity in Trichoderma reesei

As a well-known industrial fungus for cellulase production, the strain RUT-C30 of Trichoderma reesei was selected to produce the feruloyl esterase A (FAEA) by a random integration protocol. The strong promoter of cellobiohydrolase 1 (cbh1) gene was used to drive the expression of FAEA. Using double-joint PCR protocol, Pcbh1-faeA-TtrpC expression cassette was successfully constructed and co-transformed into RUT C30 strain of T. reesei. One transformant with high feruloyl esterase yield (3.44±0.16IU/mL) was obtained through plate screening and named TrfaeA1. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of fermentation supernatant from transformant TrfaeA1 showed a distinct protein band appearing at the position of about 34kDa, indicating that faeA gene has been successfully expressed in T. reesei. Compared with that in original RUT C30 strain, β-glucosidase production in transformant TrfaeA1 was significantly increased by about 86.4%, reaching 63.2IU/mL due to the random insertion of faeA. Moreover, the total secretion protein and filter paper activities of the transformant TrfaeA1 were also improved by up to 5.5 and 4.3%, respectively. The present results indicated that the random insertion strategy could be an effective and feasible method to improve and optimize the cellulase system of filamentous fungi.

Identification and characterization of Orf virus 050 protein proteolysis.

The Orf virus 050 (ORFV050) gene is located in the core region of the ORFV genome. It is similar to Vaccinia virus (VV) Copenhagen L4R, and encodes the DNA-binding virion core protein VP8, which has structures similar to the VV P25K core protein and may undergo similar proteolytic processing during virus assembly. Three conserved Ala-Gly-X motifs at putative cleavage sites were identified in ORFV050. To investigate the proteolysis of ORFV050 and its participation in viral assembly, full-length and site-directed mutant ORFV050 recombinant proteins were constructed and expressed. Two distinct protein bands of 28.5 and 25kDa were detected in the infected cells using anti-ORFV050 polyclonal antiserum. A potential cleavage site was identified at amino acids 30-32 of ORFV050. Mutation of AG/A to (R) in ORFV050 abolished the process of proteolysis. ORFV050 is a late gene synthesized during viral replication in the host cytoplasm. According to these results, we conclude that ORFV050 undergoes proteolysis and plays an important role in viral assembly.

Composition and Potency Characterization of Mycobacterium avium subsp. paratuberculosis Purified Protein Derivatives.

Mycobacterium avium subsp. paratuberculosis (MAP) purified protein derivatives (PPDs) are immunologic reagents prepared from cultured filtrates of the type strain. Traditional production consists of floating culture incubation at 37°C, organism inactivation by autoclaving, coarse filtration, and protein precipitation. Three traditional production PPDs were used in this study including lot 9801, which served as a reference and has been used in the field for decades. Alternative production PPDs (0902A and 0902B), in which the autoclaving step was removed, were also analyzed in this study. SDS-PAGE analysis revealed protein smearing in traditional PPDs, but distinct bands were observed in the alternative PPD preparations. Antibody bound distinct protein bands in the alternative PPDs by immunoblot analysis, whereas an immunoreactive smear was observed with the traditional PPDs. Mass spectrometry identified 194 proteins among three PPD lots representing the two different production methods, ten of which were present in all PPDs examined. Selected proteins identified by mass spectrometry were recombinantly expressed and purified from E. coli and evaluated by the guinea pig potency test. Seven recombinant proteins showed greater erythema as compared to the reference PPD lot 9801 in paired guinea pigs and were able to stimulate interferon-gamma production in blood from Johne’s positive animals. These results suggest that autoclaving culture suspensions is not a necessary step in PPD production and specific proteins could supplant the PPD antigen for intradermal skin testing procedures and for use as in-vitro assay reagents.

Thymus-Derived PTH (TPTH) Is Increased after Thyroparathyroidectomy in C57BL6/Kalwrij Mice and Modulates Mouse Sensitivity to 5TGM1 Myeloma Cell Line

We have previously reported a significant correlation between serum PTH levels and myeloma responses to proteasome inhibition and have recently demonstrated a crucial role for the parathyroid hormone receptor 1 (PTHR1) in vitro and in vivo. In this study the effect of abrogation of parathyroid-derived PTH was tested in 5TGM1 myeloma transplanted C57BL6/KaLwRij mice. To interrogate PTH activity in vivo, thyroparathyroidectomy (TX-PTX) was performed on the C57BL6/KaLwRij mice before or after the transplantation of 5TGM1 cells. Surgical success was confirmed by the rapid loss of measureable circulating PTH. In TX-PTX mice bearing transplanted 5TGM1 tumors, serum PTH levels was noted to recover 3-4 weeks after surgery and to achieve levels consistently higher than baseline levels pre TX-PTX. These data raised the compelling possibility of an alternative PTH source. To determine the potential source of the recovered PTH levels, multiple organs were examined. Whole tissue lysates were analyzed by Western blot and revealed a faint but specific band from thymic tissue, as expected with no obvious bands detected in 13 other tissues investigated including the liver, lung, kidney, ovary and testis. In addition, murine PTH mRNA transcripts from thymus RNA were measured by Real-Time PCR, using a specifically designed primer set that spans exons 1 and 2, since intron 1 is >1kb. These primers specifically discriminate mRNA transcripts from genomic DNA and improve the assay sensitivity for the detection of PTH transcripts. Using these custom-designed primers, control thymus expressed measureable PTH mRNA transcripts that was significantly elevated in TX-PTX mice. Since the thymus is considerably bigger than the parathyroid glands, PTH was immunoprecipitated before performing Western blot analysis. Following immunoprecipitation, a distinct PTH protein band was observed from the thymus in both TX-PTX and control mice.These data suggest that the thymus is the major source of serum PTH in TX-PTX mice and that the organ can compensate for the loss of serum PTH from the parathyroid gland. We propose that thymus-derived PTH in the TX-PTX mouse may contribute to the control of the 5TGM1 murine myeloma model. DisclosuresZangari:University of Arkansas for Medical Sciences: Employment; Novartis: Research Funding; Millennium: Research Funding; Onyx: Research Funding.

Long Noncoding RNA Ceruloplasmin Promotes Cancer Growth by Altering Glycolysis.

Long noncoding RNAs (lncRNAs) significantly influence the development and regulation of genome expression in cells. Here, we demonstrate the role of lncRNA ceruloplasmin (NRCP) in cancer metabolism and elucidate functional effects leading to increased tumor progression. NRCP was highly upregulated in ovarian tumors, and knockdown of NRCP resulted in significantly increased apoptosis, decreased cell proliferation, and decreased glycolysis compared with control cancer cells. In an orthotopic mouse model of ovarian cancer, siNRCP delivered via a liposomal carrier significantly reduced tumor growth compared with control treatment. We identified NRCP as an intermediate binding partner between STAT1 and RNA polymerase II, leading to increased expression of downstream target genes such as glucose-6-phosphate isomerase. Collectively, we report a previously unrecognized role of the lncRNA NRCP in modulating cancer metabolism. As demonstrated, DOPC nanoparticle-incorporated siRNA-mediated silencing of this lncRNA in vivo provides therapeutic avenue toward modulating lncRNAs in cancer.

Monoclonal Antibodies That Recognize the Alkylation Signature of Antimalarial Ozonides OZ277 (Arterolane) and OZ439 (Artefenomel).

The singular structure of artemisinin, with its embedded 1,2,4-trioxane heterocycle, has inspired the discovery of numerous semisynthetic artemisinin and structurally diverse synthetic peroxide antimalarials, including ozonides OZ277 (arterolane) and OZ439 (artefenomel). Despite the critical importance of artemisinin combination therapies (ACTs), the precise mode of action of peroxidic antimalarials is not fully understood. However, it has long been proposed that the peroxide bond in artemisinin and other antimalarial peroxides undergoes reductive activation by ferrous heme released during hemoglobin digestion to produce carbon-centered radicals that alkylate heme and parasite proteins. To probe the mode of action of OZ277 and OZ439, this paper now describes initial studies with monoclonal antibodies that recognize the alkylation signature (sum of heme and protein alkylation) of these synthetic peroxides. Immunofluorescence experiments conducted with ozonide-treated parasite cultures showed that ozonide alkylation is restricted to the parasite, as no signal was found in the erythrocyte or its membrane. In Western blot experiments with ozonide-treated Plasmodium falciparum malaria parasites, distinct protein bands were observed. Significantly, no protein bands were detected in parallel Western blot experiments performed with lysates from ozonide-treated Babesia divergens, parasites that also proliferate inside erythrocytes but, in contrast to P. falciparum, do not catabolize hemoglobin. However, subsequent immunoprecipitation experiments with these antibodies failed to identify the P. falciparum proteins alkylated by OZ277 and OZ439. To the best of the authors’ knowledge, this shows for the first time that antimalarial ozonides, such as the artemisinins, alkylate proteins in P. falciparum.

Evaluation of antibacterial activity and innate immune components in skin mucus of Indian major carp,Cirrhinus mrigala

The present work has been undertaken to analyse the antibacterial activity and innate immune components in the skin mucus of Indian major carp, Cirrhinus mrigala. Skin mucus was extracted separately in triple-distilled water (TDW), 3% acetic acid (3% AA) or 0.1% trifluoroacetic acid (1% TFA). All mucus extracts exhibited different spectrum of the antibacterial activity against different groups of pathogenic bacteria. Protein profiling by polyacrylamide gel electrophoresis revealed a series of protein bands in the TDW extract, four major protein bands in the AA extract and two protein bands in the TFA extract. Tandem mass spectrometry analysis of distinct protein bands identified potential innate immune factors – histone H2A, histone H3, histone H4, haemoglobin, cofilin and nucleoside diphosphate kinase in the TDW extract, and ubiquitin and histone H2B isoforms in acidic extracts of skin mucus of C. mrigala. The presence of these innate immune molecules suggests that skin mucus play an important role in the protection of the fish against microbial invasion.

Copper induction and differential expression of laccase in Aspergillus flavus.

Aspergillus flavus was isolated from soil and exhibited laccase activity under both constitutive and copper induced conditions. Spiking the medium with 1 mM copper sulfate resulted in an increase in the activity which reached 51.84 U/mL, a distinctive protein band was detected at 60 kDa. The extracellular enzyme was purified 81 fold using gel filtration chromatography and resulted in two different laccase fractions L1 and L2, the latter had a higher enzymatic activity which reached 79.57 U/mL and specific activity of 64.17 U/μg protein. The analysis of the spectrum of the L2 fraction showed a shoulder at 330 nm which is characteristic for T2/T3 copper centers; both copper and zinc were detected suggesting that this is an unconventional white laccase. Primers of laccase gene were designed and synthesized to recover specific gene from A. flavus . Sequence analysis indicated putative laccase (Genbank ID: JF683612) at the amino acid level suggesting a close identity to laccases from other genera containing the copper binding site. Decolorization of textile waste water under different conditions showed possible application in bioremediation within a short period of time. The effect of copper on A. flavus was concentration dependent.

Detecting monoclonal immunoglobulins in human serum using mass spectrometry

Established guidelines from the International Myeloma Working Group recommend diagnostic screening for patients suspected of plasma cell proliferative disease using protein electrophoresis (PEL), free light chain measurements and immunofixation electrophoresis (IFE) of serum and urine in certain cases. Plasma cell proliferative disorders are generally classified as monoclonal gammopathies given most are associated with the excess secretion of a monoclonal immunoglobulin or M-protein. In clinical practice, the M-protein is detected in a patients’ serum by the appearance of a distinct protein band migrating within regions typically occupied by immunoglobulins. Given each M-protein is comprised by a sequence of amino acids pre-defined by somatic recombination unique to each clonal plasma cell, the molecular mass of the M-protein can act as a surrogate marker. We established a mass spectrometry based method to assign molecular mass to the immunoglobulin light chain of the M-protein and used this to detect the presence of M-proteins. Our method first enriches serum for immunoglobulins, followed by reduction to separate light chains from heavy chains, followed by microflow LC-ESI-Q-TOF MS. The multiply charged light chain ions are converted to their molecular mass and reconstructed peak area calculations are used for quantification. Using this method, we term “monoclonal immunoglobulin Rapid Accurate Molecular Mass” or miRAMM, the presence of M-proteins can be reliably detected with superior sensitivity compared to current gel-based PEL and IFE techniques.

Evaluation of Indirect ELISA and Western Blotting for the Diagnosis of Amphistomes Infection in Cattle and Buffaloes

Amphistomes are snail-borne trematodes infect rumens and reticulums causing acute and chronic diseases in cattle and buffaloes. The economical losses caused by Amphistomes resulted from drop in milk and meat yield in addition to mortalities. Eighty four serum samples (50 from cattle and 34 from buffaloes, 30 from amphistomes infected and 54 from amphistomes free cattle and buffaloes) collected from Giza and Garbia governourates, Egypt. The collected Amphistomes were morphologically and histologically classified. The identified worms of Paramphistomum and Carmyerius were used for the preparation of somatic antigens separately. The collected 84 serum sample were tested twice by indirect ELISA, firstly with Paramphistomum somatic antigen and secondly with Carmyerius somatic antigen. The antigenic profiles of adult Paramphistomum spp somatic antigen and Carmyerius gergaerius somatic antigen were analysed by (SDS-PAGE). Four male Rabbit were used for the preparation of hyper-immune serum against Paramphistomum and Carmyerius somatic antigens. Nine serum samples (two rabbit hyper immune sera; one for Paramphistomum and the another for Carmyerius , 3 serum sample from Paramphistomum infected cattle and 3 serum samples from Carmyerius infected cattle and one serum sample from non infected cattle as negative control) were blotted and tested twice at the same time on the nitrocellulose membrane by Western blotting techniques., firstly by using Paramphistomum somatic antigen and secondly with Carmyerius antigen. The results of both ELISA and Western blotting were statistically analysed. The sensitivity, specificity and accuracy for ELISA and Western blotting were (74% and 100%),(82.4% and 33.3%) and (79.76% and77.78%) respectively . The antigenic profile of adult Paramphistomum somatic antigen showed 14 distinct protein bands of protein molecular weights ranging from 11.5 to 174 KDa. While Carmyerius somatic antigen showed 13 distinct protein bands ranging from 11.5 to 166KDa. One distinct immunogenic band at 63 KDa was found to react with all sera from infected cattle and buffaloes with Paramphistomum somatic antigen while the same serum samples gave one distinct immunogenic band at 71 KDa with Carmyerius somatic antigen. It is concluded that ELISA is more reliable test for early diagnosis of amphistomiasis. There is a strong cross immune reaction between Paramphistomum and Carmyerius.

Evaluation of cellulolytic activity in insect digestive fluids

Efficient and low-cost cellulolytic enzymes are urgently needed to degrade recalcitrant plant biomass during the industrial production of lignocellulosic biofuels. Here, the cellulolytic activities in the gut fluids of 54 insect species that belong to 7 different taxonomic orders were determined using 2 different substrates, carboxymethyl cellulose (CMC) (approximating endo-β-1,4-glucanase) and filter paper (FP) (total cellulolytic activities). The use of CMC as the substrate in the zymogram analysis resulted in the detection of distinct cellulolytic protein bands. The cellulolytic activities in the digestive system of all the collected samples were detected using cellulolytic activity analysis. The highest CMC gut fluid activities were found in Coleoptera and Orthoptera, while FP analysis indicated that higher gut fluid activities were found in several species of Coleoptera and Lepidoptera. In most cases, gut fluid activities were higher with CMC than with FP substrate, except for individual Lepidoptera species. Our data indicate that the origin of cellulolytic enzymes probably reflects the phylogenetic relationship and feeding strategies of different insects.

Variability in Indian isolates of Sclerotium rolfsii by SDS-PAGE

The variability among 14 different Indian isolates of Sclerotium was studied on the basis of protein profile generated by Polyacrylamide Gel–Electrophoresis (SDS-PAGE). Different characteristics taken into account while studying the variability were sclerotia formation, non-formation, basidial stage formation and non-formation and type of disease symptom, viz. spotted leaf rot and collar rot. A comparison was done between mycelium of the different isolates as well as the sclerotia of the same isolate. All the 14 isolates showed a significant variation among themselves with respect to number and intensity of major and minor bands. It was intriguing to note that isolate L-Brl showed quiet different pattern from the rest of the isolates. A strong induction of a protein band of ∼15 kDa, probably responsible for the sclerotial development, was observed on comparing the protein profile of mycelium with the protein profile of sclerotia of all the isolates taken into consideration. A 66 kDa protein was prominently present in the isolate (L-Sph) which invariably forms basidiospores, but this protein was not present in the mycelial protein profile. A protein band with molecular weight greater than 98 kDa was prominently present in the total protein profile of isolates which does not form sclerotia in its life cycle. Furthermore, a protein band of ∼12 kDa was consistently present in the isolate which causes collar rot and this protein was absent in isolates which cause spotted leaf rot. Two closely placed distinct protein bands of 43 kDa and 46 kDa were invariably present in all the isolates causing spotted leaf rot. The dendrogram constructed with the help of protein profile so obtained was found to be divided into two prominent clusters. The representative isolate (L-Sph) was also investigated for pectinase, cellulase and xylanase production. The presence of hydrolytic enzymes is significant for contributing pathogenicity to the fungus.