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Protein Extracts (protein + extract)
Kinds of Protein Extracts Selected AbstractsAntifungal Activity Stability of Flaxseed Protein Extract Using Response Surface MethodologyJOURNAL OF FOOD SCIENCE, Issue 1 2008Y. Xu ABSTRACT:, The stability of the antifungal activity of flaxseed (Linum usitatissimum) protein extract was evaluated in this study. Response surface methodology (RSM) using Box,Behnken factorial design was used to evaluate the effects of treatment variables, that is, temperature (50 to 90 °C), time (1 to 29 min), and pH (2 to 8), on the residual antifungal activity (RAA) against Penicillium chrysogenum, Fusarium graminearum, Aspergillus flavus, and a Penicillium sp. isolated from moldy noodles. Regression analyses suggested that the linear terms of the temperature and time had significant (P < 0.05) negative effects on the RAA against all test fungi, whereas that of pH had a significant (P < 0.1) positive role on the RAA of all 3 fungi. In addition, the RAA was significantly (P < 0.05) affected by the quadratic terms of time for all fungi, and the quadratic term of temperature played a significant (P < 0.1) role on RAA against F. graminearum. One interaction term (temperature-pH) was found to significantly (P < 0.1) affect the RAA against both Penicillium strains tested. The results indicated that , 90% antifungal activity was lost after the protein extracts were heated at 90 °C for 8 min except for F. graminearum. At pasteurization condition, , 50% activity was retained except for P. chrysogenum. The results also suggested that neutral and alkaline pH favored the antifungal activity stability of the protein extracts. Thus, flaxseed protein might be promising if used as a preservative in foods with neutral or alkaline pH requiring mild heat treatments. [source] Ubiquitination of TMV Coat Protein Aggregates in Infected Tobacco LeavesJOURNAL OF PHYTOPATHOLOGY, Issue 11-12 2003J. Hamacher Abstract Protein extracts from Tobacco mosaic virus (TMV) infected young tobacco leaves exhibiting strong symptoms contain higher amounts of ubiquitin conjugates in comparison with non-infected control plants presumably due to stress reactions of host cells. Western blots with antibodies against TMV indicate as well an increase in coat protein or in ubiquitinated coat protein subunits. Immunogold labelling of infected leaf material revealed the accumulation of large amounts of coat protein in opaque inclusions which also reacted strongly with ubiquitin antibodies. These inclusions appear in close vicinity to chloroplasts in chlorotic areas of the leaf. It is concluded that most of the ubiquitin conjugates found in Western blots are due to ubiquitinated coat protein. As a consequence, a considerable amount of coat protein cannot be used for correct capsid construction as the stabilizing lysine residues are blocked by ubiquitination. [source] Generation of high-quality protein extracts from formalin-fixed, paraffin-embedded tissuesPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 15 2009Maria Filippa Addis Abstract A wealth of information on proteins involved in many aspects of disease is encased within formalin-fixed paraffin-embedded (FFPE) tissue repositories stored in hospitals worldwide. Recently, access to this "hidden treasure" is being actively pursued by the application of two main extraction strategies: digestion of the entangled protein matrix with generation of tryptic peptides, or decrosslinking and extraction of full-length proteins. Here, we describe an optimised method for extraction of full-length proteins from FFPE tissues. This method builds on the classical "antigen retrieval" technique used for immunohistochemistry, and allows generation of protein extracts with elevated and reproducible yields. In model animal tissues, average yields of 16.3,,g and 86.8,,g of proteins were obtained per 80,mm2 tissue slice of formalin-fixed paraffin-embedded skeletal muscle and liver, respectively. Protein extracts generated with this method can be used for the reproducible investigation of the proteome with a wide array of techniques. The results obtained by SDS-PAGE, western immunoblotting, protein arrays, ELISA, and, most importantly, nanoHPLC-nanoESI-Q-TOF MS of FFPE proteins resolved by SDS-PAGE, are presented and discussed. An evaluation of the extent of modifications introduced on proteins by formalin fixation and crosslink reversal, and their impact on quality of MS results, is also reported. [source] Enhanced resistance to foliar fungal pathogens in carrot by application of elicitorsANNALS OF APPLIED BIOLOGY, Issue 1 2009J. Jayaraj Abstract Treatment of greenhouse-grown carrot plants with salicylic acid (SA) (100 ,m), chitosan (0.02%) and the nutrient-chelate product Alexin (1%) followed 10 h later by inoculation with the necrotrophic fungal pathogens Alternaria radicina and Botrytis cinerea significantly reduced disease development 10 days after inoculation (d.a.i.) compared with control plants sprayed with water. The most effective treatment was chitosan, followed by Alexin and SA. Additional sprays of elicitors resulted in significantly lower disease development 25 d.a.i. Treated plants had elevated transcript levels of pathogenesis-related protein 1 (PR1), chitinase, lipid transfer protein (LTP), chalcone synthase, nonexpressor of PR1 and pathogenesis-related protein 5 (PR5) genes compared with control plants when assayed 10,70 h after treatment. The activity of peroxidase, polyphenoloxidase, phenylalanine ammonia-lyase, chitinase, ,-1,3-glucanase and lipoxygenase was significantly increased in elicitor-treated plants compared with control plants 12,72 h after treatment. Microscopic examination of treated leaves revealed reduced fungal growth and colonisation, 48 h after treatment, accompanied by fewer lesions at 120 h, compared with the control. Protein extracts from elicitor-treated plants reduced spore germination and germ tube elongation of the pathogens in vitro by 30,45%. Elicitor-treated plants accumulated higher amounts of total phenolics, 6-methoxymellin and H2O2 compared with the control. Both chitosan and Alexin induced responses similar to that of SA, suggesting that these elicitors may activate the salicylate pathway, leading to induction of defence genes, enzymes, phytoalexin and phenolics, which collectively reduced fungal colonisation. [source] CK2,tes gene encodes a testis-specific isoform of the regulatory subunit of casein kinase 2 in Drosophila melanogasterFEBS JOURNAL, Issue 5 2002Alla I. Kalmykova An earlier described CK2,tes gene of Drosophila melanogaster is shown to encode a male germline specific isoform of regulatory ,,subunit of casein kinase 2. Western-analysis using anti-CK2,tes Ig revealed CK2,tes protein in Drosophila testes extract. Expression of a CK2,tes,,-galactosidase fusion protein driven by the CK2,tes promoter was found in transgenic flies at postmitotic stages of spermatogenesis. Examination of biochemical characteristics of a recombinant CK2,tes protein expressed in Escherichia coli revealed properties similar to those of CK2,: (a) CK2,tes protein stimulates CK2, catalytic activity toward synthetic peptide; (b) it inhibits phosphorylation of calmodulin and mediates stimulation of CK2, by polylysine; (c) it is able to form (CK2,tes)2 dimers, as well as (CK2,)2(CK2,tes)2 tetramers. Using the yeast two-hybrid system and coimmunoprecipitation analysis of protein extract from Drosophila testes, we demonstrated an association between CK2,tes and CK2,. Northern-analysis has shown that another regulatory (,,) subunit found recently in D. melanogaster genome is also testis-specific. Thus, we describe the first example of two tissue-specific regulatory subunits of CK2 which might serve to provide CK2 substrate recognition during spermatogenesis. [source] Antifungal Activity Stability of Flaxseed Protein Extract Using Response Surface MethodologyJOURNAL OF FOOD SCIENCE, Issue 1 2008Y. Xu ABSTRACT:, The stability of the antifungal activity of flaxseed (Linum usitatissimum) protein extract was evaluated in this study. Response surface methodology (RSM) using Box,Behnken factorial design was used to evaluate the effects of treatment variables, that is, temperature (50 to 90 °C), time (1 to 29 min), and pH (2 to 8), on the residual antifungal activity (RAA) against Penicillium chrysogenum, Fusarium graminearum, Aspergillus flavus, and a Penicillium sp. isolated from moldy noodles. Regression analyses suggested that the linear terms of the temperature and time had significant (P < 0.05) negative effects on the RAA against all test fungi, whereas that of pH had a significant (P < 0.1) positive role on the RAA of all 3 fungi. In addition, the RAA was significantly (P < 0.05) affected by the quadratic terms of time for all fungi, and the quadratic term of temperature played a significant (P < 0.1) role on RAA against F. graminearum. One interaction term (temperature-pH) was found to significantly (P < 0.1) affect the RAA against both Penicillium strains tested. The results indicated that , 90% antifungal activity was lost after the protein extracts were heated at 90 °C for 8 min except for F. graminearum. At pasteurization condition, , 50% activity was retained except for P. chrysogenum. The results also suggested that neutral and alkaline pH favored the antifungal activity stability of the protein extracts. Thus, flaxseed protein might be promising if used as a preservative in foods with neutral or alkaline pH requiring mild heat treatments. [source] Utilizing a library of synthetic affinity ligands for the enrichment, depletion and one-step purification of leech proteinsJOURNAL OF MOLECULAR RECOGNITION, Issue 3 2008Dexian Dong Abstract Although the concept of affinity purification using synthetic ligands had been utilized for many years, there are few articles related to this research area, and they focus only on the affinity purification of specific protein by a defined library of synthetic ligands. This study presents the design and construction of a 700-member library of synthetic ligands in detail. We selected 297 ligand columns from a 700-member library of synthetic ligands to screen leech protein extract. Of the 297, 154 columns had an enrichment effect, 83 columns had a depletion effect, 36 columns had a one-step purification effect, and 58 columns had a one-step purification via flowthrough effect. The experimental results achieved by this large library of affinity ligands provide solid convincing data for the theory that affinity chromatography could be used for the enrichment of proteins that are present in low abundance, the depletion of high abundance proteins, and one-step purification of special proteins. Copyright © 2008 John Wiley & Sons, Ltd. [source] CpKLP1: A CALMODULIN-BINDING KINESIN-LIKE PROTEIN FROM CYANOPHORA PARADOXA (GLAUCOPHYTA)JOURNAL OF PHYCOLOGY, Issue 4 2000Salah E. Abdel-Ghany KCBP (kinesin-like calmodulin [CaM]-binding proteins), a member of the carboxy-terminal kinesin-like proteins (KLPs), is unique among KLPs in having a CaM-binding domain (CBD). CaM-binding KLPs have been identified from flowering plants and the sea urchin. To determine if CaM-binding KLP is present in phylogenetically divergent protists, we probed Cyanophora paradoxa protein extract with affinity-purified KCBP antibody. The KCBP antibody detected a polypeptide with a molecular mass of about 133 kDa in the crude extract. In a CaM,Sepharose column-purified fraction, the same band was detected with both KCBP antibody and biotinylated CaM. In a PCR reaction using degenerate primers corresponding to two conserved regions in the motor domain of kinesin, a 500-bp fragment (CpKLP1) was amplified from a cDNA library. The predicted amino acid sequence of CpKLP1 showed significant sequence similarity with KCBPs. In phylogenetic analysis, CpKLP1 fell into the KCBP group within the carboxy-terminal subfamily. These biochemical data, sequence, and phylogenetic analysis strongly suggest the presence of a calmodulin-binding KLP in C. paradoxa and that it is related to Ca2+/calmodulin regulated KLPs from plants. This is the first report on identification of any motor protein in C. paradoxa. Furthermore, our data suggest that CaM-binding KLPs may have evolved long before the divergence of plants and animals. [source] The detection, correlation, and comparison of peptide precursor and product ions from data independent LC-MS with data dependant LC-MS/MSPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 6 2009Scott J. Geromanos Abstract The detection, correlation, and comparison of peptide and product ions from a data independent LC-MS acquisition strategy with data dependent LC-MS/MS is described. The data independent mode of acquisition differs from an LC-MS/MS data acquisition since no ion transmission window is applied with the first mass analyzer prior to collision induced disassociation. Alternating the energy applied to the collision cell, between low and elevated energy, on a scan-to-scan basis, provides accurate mass precursor and associated product ion spectra from every ion above the LOD of the mass spectrometer. The method therefore provides a near 100% duty cycle, with an inherent increase in signal intensity due to the fact that both precursor and product ion data are collected on all isotopes of every charge-state across the entire chromatographic peak width. The correlation of product to precursor ions, after deconvolution, is achieved by using reconstructed retention time apices and chromatographic peak shapes. Presented are the results from the comparison of a simple four protein mixture, in the presence and absence of an enzymatically digested protein extract from Escherichia coli. The samples were run in triplicate by both data dependant analysis (DDA) LC-MS/MS and data-independent, alternate scanning LC-MS. The detection and identification of precursor and product ions from the combined DDA search results of the four protein mixture were used for comparison to all other data. Each individual set of data-independent LC-MS data provides a more comprehensive set of detected ions than the combined peptide identifications from the DDA LC-MS/MS experiments. In the presence of the complex E. coli background, over 90% of the monoisotopic masses from the combined LC-MS/MS identifications were detected at the appropriate retention time. Moreover, the fragmentation pattern and number of associated elevated energy product ions in each replicate experiment was found to be very similar to the DDA identifications. In the case of the corresponding individual DDA LC-MS/MS experiment, 43% of the possible detectable peptides of interest were identified. The presented data illustrates that the time-aligned data from data-independent alternate scanning LC-MS experiments is highly comparable to the data obtained via DDA. The obtained information can therefore be effectively and correctly deconvolved to correlate product ions with parent precursor ions. The ability to generate precursor-product ion tables from this information and subsequently identify the correct parent precursor peptide will be illustrated in a companion manuscript. [source] A comparative proteomic evaluation of culture grown vs nodule isolated Bradyrhizobium japonicumPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 10 2006Annamraju D. Sarma Abstract Total protein extract of Bradyrhizobium japonicum cultivated in HM media were resolved by 2-D PAGE using narrow range IPG strips. More than 1200,proteins were detected, of which nearly 500,proteins were analysed by MALDI-TOF and 310,spots were tentatively identified. The present study describes at the proteome level a significant number of metabolic pathways related to important cellular events in free-living B.,japonicum. A comparative analysis of proteomes of free-living and nodule residing bacteria revealed major differences and similarities between the two states. Proteins related to fatty acid, nucleic acid and cell surface synthesis were significantly higher in cultured cells. Nitrogen metabolism was more pronounced in bacteroids whereas carbon metabolism was similar in both states. Relative percentage of proteins related to global functions like protein synthesis, maturation & degradation and membrane transporters were similar in both forms, however, different proteins provided these functions in the two states. [source] Quantitative nuclear proteomics reveals new phenotypes altered in lymphoblastoid cellsPROTEOMICS - CLINICAL APPLICATIONS, Issue 3 2009Paul Brennan Dr. Abstract B-lymphocytes are essential for the production of antibodies to fight pathogens and are the cells of origin in 95% of human lymphomas. During their activation, and immortalisation by Epstein,Barr virus (EBV) which contributes to human cancers, B-lymphocytes undergo dramatic changes in cell size and protein content. This study was initiated to compare the proteome of two B-cell lines, from the same individual, that reflect different patterns of activation, one is EBV negative and the other is EBV positive. Using isobaric tags, LC-MALDI TOF-TOF and subcellular fractionation, we quantified 499 proteins from B-cells. From a detergent lysed protein extract, we identified 34 proteins that were differentially expressed in EBV-immortalised B-cells. By analysing a nuclear extract, we identified a further 29 differentially expressed proteins with only four proteins shared between the two extracts, illustrating the benefit of subcellular fractionation. This analysis has identified proteins involved in the cytoskeletal phenotype of activated B-cells and the increased antigen recognition in EBV-immortalised cells. Importantly, we have also identified new regulators of transcription and changes in ribonuclear proteins that may contribute to the increased cell size and immortalisation of lymphoblastoid cells. [source] Identification of endo- and exo-polygalacturonase activity in Lygus hesperus (Knight) salivary glands,ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 2 2009Maria de la Paz Celorio-Mancera Abstract Polygalacturonase (PG) activity found in the salivary gland apparatus of the western tarnished plant bug (WTPB, Lygus hesperus Knight) has been thought to be the main chemical cause of the damage inflicted by this mirid when feeding on its plant hosts. Early viscosity and thermal stability studies of the PG activity in L. hesperus protein extracts were difficult to interpret. Thus, it has been suggested that one or more PG protein(s) with different hydrolytic modes of action are produced by this mirid. In order to understand the quantitative complexity of the WTPB salivary PG activity, PG purification from a protein extract from salivary glands excised from L. hesperus insects was performed using affinity and ion exchange chromatography. To elucidate the qualitative complexity of the purified PGs, the digestion products generated by the PGs were separated using high performance anion exchange chromatography with pulsed amperometric detection. At least five PG proteins were detected; these differing in terms of their glycosylation, mass-to-charge ratios, and/or molecular mass. The characterization of the products generated by these PGs showed that endo- and exo-acting PGs are produced by WTPB. Although none of the PGs was purified to homogeneity, the present work provides biochemical evidence of a multiplicity of PGs that degrade the pectin component of the plant tissue in different fashions. The implications of these findings affect the understanding of WTPB feeding damage and, potentially, help identify ways to control this important crop pest. Arch. Insect Biochem. Physiol. 2008. © 2008 Wiley-Liss, Inc. [source] Biochemical mechanisms of insecticide resistance in the diamondback moth (DBM), Plutella xylostella L. (Lepidopterata: Yponomeutidae), in the Sydney region, AustraliaAUSTRALIAN JOURNAL OF ENTOMOLOGY, Issue 4 2009Vincent Y Eziah Abstract Following the detection of resistant diamondback moth (DBM) populations to synthetic pyrethroid, organophosphorus and indoxacarb insecticides in the Sydney Basin, a study of the major biochemical mechanisms was conducted to determine the type of resistance in these populations. The activity of cytochrome P450 monooxygenases increased two- to sixfold when compared with the susceptible strain. Up to a 1.9-fold increase in esterase activity in resistant strains compared with the susceptible strain was observed. In vitro inhibition studies showed that profenofos, methamidophos and chlorpyrifos strongly inhibited the esterases while permethrin and esfenvalerate resulted in less than 30% inhibition. Qualitative analysis of the esterases using native polyacrylamide gel electrophoresis showed four bands in both the susceptible and resistant individuals with more intense staining in the resistant individuals. The development of these bands was inhibited by methamidophos and chlorpyrifos pretreatment of the protein extract while permethrin and esfenvalerate did not exhibit this effect. Glutathione S-transferase (GST) activity was significantly higher in two field populations compared with the remaining populations. Overall, the study showed that the mechanisms of insecticide resistance in the DBM populations in the area studied were due to cytochrome P450 monooxygenases, esterase and GSTs, and possibly other non-metabolic mechanisms that were not investigated in the present study. [source] Purification, identification and preliminary crystallographic studies of a 2S albumin seed protein from Lens culinarisACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 8 2008Pankaj Gupta Lens culinaris (lentil) is a widely consumed high-protein-content leguminous crop. A 2S albumin protein (26.5,kDa) has been identified using NH2 -terminal sequencing from a 90% ammonium sulfate saturation fraction of total L. culinaris seed protein extract. The NH2 -terminal sequence shows very high homology to PA2, an allergy-related protein from Pisum sativum. The 2S albumin protein was purified using a combination of size-exclusion and ion-exchange chromatography. Crystals of the 2S seed albumin obtained using the hanging-drop vapour-diffusion method diffracted to 2.5,Å resolution and were indexed in space group P41 (or P43), with unit-cell parameters a = b = 78.6, c = 135.2,Å. [source] Rapid and selective isolation of ,-xylosidase through an activity-based chemical approachBIOTECHNOLOGY JOURNAL, Issue 2 2006Lee-Chiang Lo Dr. Abstract ,-Xylosidase is a key enzyme in the xylanolytic system with a great potential in many biotechnological applications, especially in the food as well as the pulp and paper industries. We have developed a chemical approach for the rapid screening and isolation of ,-xylosidase. Activity probe LCL-6X targeting ,-xylosidase was utilized in this study. It carries a ,-xylopyranosyl recognition head, a latent trapping device consisting of a 2-fluoromethylphenoxyl group, and a biotin reporter group. The biotin reporter group serves both as a readout device and as a tool for enriching the labeled proteins. LCL-6X could selectively label a model ,-xylosidase from Trichoderma koningii. All other bystander proteins used in this study, including phosphorylase b, BSA, ovalbumin, carbonic anhydrase, and trypsin inhibitor, gave negligible cross-labeling effect. With the assistance of streptavidin agarose beads and mass spectrophotometry for the recovery and identification of the biotinylated proteins, we demonstrated that LCL-6X could be successfully applied to identify a bi-functional enzyme with ,- L -arabinofuranosidase/,-xylosidase activity from the total protein extract of a Pichia expressing system and a prospective ,-xylosidase in the culture medium of Aspergillus fumigatus. The ,-xylosidase activities from numerous microbes were also screened using the LCL-6X probe. Preliminary results showed significant differences among these microbial sources and some distinct protein bands were observed. Thus, we have successfully developed a novel chemical probe that has potential applications in xylan-related research. [source] Kiwifruit allergy: actinidin is not a major allergen in the United KingdomCLINICAL & EXPERIMENTAL ALLERGY, Issue 9 2007J. S. A. Lucas Summary Background Actinidin has previously been reported as the major allergen in kiwifruit. Objectives To investigate the relevance of actinidin in a well-characterized population of UK patients with kiwifruit allergy. Methods To identify the allergens in kiwifruit, using Western blots, we examined the IgE-binding patterns of 76 patients with a history of kiwifruit allergy, 23 of who had had a positive double-blind, placebo-controlled food challenge. In addition, IgE binding to purified native actinidin was studied in 30 patients, and to acidic and basic isoforms of recombinant actinidin in five patients. Inhibition of IgE binding to kiwifruit protein extract by purified native actinidin was investigated by both inhibition immunoblots and inhibition ELISAs using pooled sera. Results Twelve protein bands in kiwifruit protein extract were bound by IgE. A protein band with a molecular weight of 38 kDa was the major allergen recognized by 59% of the population. IgE did not bind to actinidin in the kiwifruit protein extract, or to purified native or recombinant forms of actinidin during Western blotting. Pooled sera bound to kiwifruit protein extract but not purified actinidin on ELISA, and pre-incubating sera with actinidin did not inhibit IgE binding to kiwifruit protein extract on immunoblot or ELISA. Conclusion A novel 38 kDa protein, not actinidin, is the major allergen in this large study population. Identification of major allergens in one patient group is therefore not necessarily reproducible in another; therefore, major allergens should not be defined until there is a sufficient body of data from diverse geographical and cultural populations. [source] Mass spectrometrical analysis of the mitochondrial carrier Aralar1 from mouse hippocampusELECTROPHORESIS, Issue 11 2010Seok Heo Abstract Aralar1 is a mitochondrial aspartate/glutamate carrier and a key component of the malate,aspartate NADH shuttle system. An analytical approach to obtain high sequence coverage is important to predict conformation, identify splice variants and binding partners or generate specific antibodies. Moreover, a method allowing determination of Aralar1 from brain samples is a prerequisite for evaluating a biological role. Sucrose gradient ultracentrifugation was applied to enrich native membrane protein fractions and these were run on blue-native PAGE, followed by multidimensional gel electrophoresis. Spots from the third-dimensional gel electrophoresis were in-gel digested with trypsin, chymotrypsin and subtilisin. Subsequently, peptides were analyzed by nano-ESI-LC-MS/MS using collision-induced dissociation and electron transfer dissociation modes. ModiroÔ v1.1 along with Mascot v2.2 software was used for data handling. Aralar1 could be clearly separated, unambiguously identified and characterized from protein extracts of mouse hippocampus by the use of the multidimensional gel electrophoretic steps. The combined sequence coverage of Aralar1 from trypsin, chymotrypsin and subtilisin digestions was 99.85%. The results provide the basis for future studies of Aralar1 at the protein chemical rather than at the immunochemical level in the brain and thus challenge and enable determination of Aralar1 levels required for understanding biological functions in health and disease. [source] Detection of carbonyl-modified proteins in interfibrillar rat mitochondria using N, -aminooxymethylcarbonylhydrazino- D -biotin as an aldehyde/keto-reactive probe in combination with Western blot analysis and tandem mass spectrometryELECTROPHORESIS, Issue 6 2008Woon-Gye Chung Abstract There is now a large body of supporting data available that links oxidative modifications of proteins to a large number of diseases, degenerative disorders and aging. However, the detailed analysis of oxidative protein modifications remains challenging. Here, we report a new efficient method for identification of oxidatively modified proteins in complex biological samples which is based on the use of an aldehyde-reactive probe, N,-aminooxymethylcarbonylhydrazino- D -biotin (ARP), in combination with Western-type analyses and MS. The biotinylated hydroxylamine derivative forms a chemically stable oxime derivative with the aldehyde/keto group found in carbonyl-modified proteins. The biotin tag is detected by avidin affinity staining. ARP-positive proteins are subsequently subjected to in-gel trypsinization and MS/MS for protein identification. We demonstrate the usefulness of the method for the analysis of protein extracts obtained from interfibrillar heart mitochondria (IFM) from young and old rats. In this study, we identified as putative major protein targets of oxidative modifications the mitochondrial matrix protein, aconitase, the inner mitochondrial membrane protein, ADP/ATP translocase, and constituents of the electron transport chain complexes IV and V. An age-related increase of carbonyl levels was found for aconitase and ATP synthase. [source] Apparent growth phase-dependent phosphorylation of malonyl coenzyme A:acyl carrier protein transacylase (MCAT), a major fatty acid synthase II component in Mycobacterium bovis BCGFEMS MICROBIOLOGY LETTERS, Issue 1 2003Indrajit Sinha Abstract Probing protein extracts from exponentially growing and stationary phase cultures of Mycobacterium bovis BCG with anti-phospho amino acid antibodies revealed a 31-kDa anti-phospho threonine antibody-reactive protein specific to growing culture. The corresponding protein was purified via two-dimensional gel electrophoresis and identified via mass spectrometry to be malonyl coenzyme A:acyl carrier protein transacylase (MCAT), a component of the fatty acid biosynthetic pathway. MCAT tagged with histidine reacted with anti-phospho threonine antibody and was positive in an in-gel chemical assay for phospho proteins. Analysis of the growth phase dependence of MCAT-His phosphorylation and protein levels showed that phosphorylated MCAT-His can be detected only in growing culture. In contrast, MCAT-His protein level was growth phase-independent. These results suggest that MCAT may be a substrate of a protein kinase and phosphatase, and that aspects of fatty acid synthesis in tubercle bacilli are regulated by protein phosphorylation. [source] Enhanced Activation of Cyclooxygenase-2 Downregulates Th1 Signaling Pathway in Helicobacter pylori -infected Human Gastric MucosaHELICOBACTER, Issue 3 2007Antonia Pellicanò Abstract Background:, Evidence suggests that an impaired T-cell response against Helicobacter pylori plays a role in the pathogenesis of H. pylori -related diseases. Cyclooxygenase (COX) 2 has been shown to inhibit the production of T-helper (Th) 1 cytokines. This study aimed to ascertain whether COX-2 downregulates Th1 signaling pathway in human gastric mucosa colonized by H. pylori. Methods:, COX-2 expression and prostaglandin E2 (PGE2) production were determined in total proteins extracted from freshly obtained gastric biopsies of H. pylori -infected and uninfected patients by Western blotting and enzyme-linked immunosorbent assay (ELISA). Phosphorylated (p)STAT4, pSTAT1, T-bet, and pSTAT6 expression and interleukin (IL)-12, interferon (IFN)-,, and IL-4 production were also determined by Western blotting and ELISA, respectively, in total protein extracts from gastric biopsy cultures of H. pylori -infected patients treated without and with COX-2 inhibitor NS-398. Results:, Enhanced expression of COX-2 and production of PGE2 was found in H. pylori -infected compared to uninfected patients. COX-2 inhibition significantly increased expression of Th1 transcription factors along with production of IL-12 and IFN-,. By contrast, no changes in the expression of STAT6 and production of IL-4 were found. Conclusion:, This study provides a mechanism by which H. pylori may actually interfere with normal T-cell activation in human gastric mucosa, possibly enhancing its pathogenicity. The use of COX-2 selective inhibitors as immunomodulators in the course of H. pylori infection deserves investigations. [source] Expression of Fas and Fas ligand in human testicular germ cell tumoursINTERNATIONAL JOURNAL OF ANDROLOGY, Issue 2 2009E. Baldini Summary In the present study, we analysed the expression of Fas ligand (FasL) and its cognate receptor Fas in 14 seminomatous testicular germ cell tumours (TGCT) and six normal testicular tissues obtained following orchiectomy. Tissue samples have been processed to prepare either total RNA or protein extracts or fixed and embedded in paraffin for immunohistochemistry (IHC) experiments. Quantitative RT-PCR experiments demonstrated in TGCT a significant (p < 0.01) increase of the FasL mRNA expression of 21.1 ± 5.4 fold, with respect to normal tissues. On the contrary, in the same cancer tissues, the levels of Fas mRNA were significantly (p < 0.01) reduced to 0.27 ± 0.06 fold. These observations were confirmed in western blot experiments showing a significant increase of FasL and a concomitant decrease of Fas proteins in testicular cancer tissues, with respect to normal testis. Moreover, IHC experiments showed a strong FasL immuno-reactivity in six out of eight TGCT samples analysed, while Fas immuno-positivity was found in cancer cells of only two TGCT tissues. In addition, in all tumour samples, infiltrating lymphocytes were Fas positive. However, no correlation could be observed between Fas or FasL mRNA variations and clinical parameters such as patient's age, TNM stage or tumour size. We also compared the serum levels of soluble FasL (sFasL) of 15 patients affected by seminomatous TGCT, of four patients with non-seminomatous TGCT and six age-matched healthy males. No significant differences in sFasL serum level could be identified. In conclusion, our data demonstrated that the majority of seminomas are characterized by an increased expression of FasL and a concomitant reduction of Fas, with respect to human normal testis, and that sFasL serum level is not a tumour marker for patients affected by TGCT. [source] Decreased pyruvate kinase M2 activity linked to cisplatin resistance in human gastric carcinoma cell linesINTERNATIONAL JOURNAL OF CANCER, Issue 4 2004Byong Chul Yoo Abstract Resistance to anticancer drugs is a major obstacle preventing effective treatment of disseminated cancers. Understanding the molecular basis to chemoresistance is likely to provide better treatment. Cell lines resistant to cisplatin or 5-fluorouracil (5-FU) were established from human gastric carcinoma cell lines SNU-638 and SNU-620. Comparative proteomics involving 2-dimensional gel electrophoresis (2-DE) and matrix-associated laser desorption ionization-mass spectroscopy (MALDI-MS) was performed on protein extracts from these parental and drug-resistant derivative lines to screen drug resistance-related proteins. Pyruvate kinase M2 (PK-M2) was identified as a protein showing lower expression in cisplatin-resistant cells compared to parental cells. Consistent with this finding, PK-M2 activity was also lower in cisplatin-resistant cells. Suppression of PK-M2 expression by antisense oligonucleotide resulted in acquired cisplatin resistance in SNU-638 cells. Furthermore, PK-M2 activity in 11 individual human gastric carcinoma cell lines positively correlated with cisplatin sensitivity. Taken together, PK-M2 protein and activity levels were lower in cisplatin-resistant human gastric carcinoma cell lines compared to their parental cell lines. Furthermore, suppression of PK-M2 expression using antisense oligonucleotides increased cisplatin resistance. These data clearly link PK-M2 and cisplatin resistance mechanisms. © 2003 Wiley-Liss, Inc. [source] Decorin transfection in human mesangial cells downregulates genes playing a role in the progression of fibrosisJOURNAL OF CLINICAL LABORATORY ANALYSIS, Issue 4 2002Antonia Costacurta Abstract The proteoglycan decorin inhibits TGF-,; therefore, it could antagonize progression of fibrotic diseases associated with activation of TGF-,1. The effect of decorin transfection in human mesangial cells (HMCs) on the expression of genes related to kidney fibrosis was investigated. HMCs, isolated from glomeruli of healthy portions of human kidneys removed due to carcinoma, were histochemically typed. Decorin cDNA cloned in a eukaryotic expression vector was transfected into HMCs. Gene expression of fibrogenetic cytokines and fibrotic proteins TGF-,1, PDGF-,, ,1 collagen type IV, ,1 collagen type I, fibronectin, and tenascin was analyzed, by reverse transcription polymerase chain reaction (RT-PCR), 24 hr after transfection. Immunoblotting analysis of protein extracts using anti-decorin IgG, revealed a positive signal of about 52 MDa, corresponding to the molecular weight of decorin, in cultures transfected with the decorin gene. Decorin mRNA increased about 12 times in cultures transfected with the construct pCR3.1-Deco. Cells with increased decorin synthesis showed a 61% decrease of TGF-,1 mRNA, a 71% reduction of ,1 collagen type IV mRNA, and a 29% reduction of fibronectin mRNA. This study is the first to investigate decorin transfection into human mesangial cells, and supports the use of the decorin gene to control the progression of glomerular and interstitial fibrosis in kidney diseases. © 2002 Wiley-Liss, Inc. [source] Antifungal Activity Stability of Flaxseed Protein Extract Using Response Surface MethodologyJOURNAL OF FOOD SCIENCE, Issue 1 2008Y. Xu ABSTRACT:, The stability of the antifungal activity of flaxseed (Linum usitatissimum) protein extract was evaluated in this study. Response surface methodology (RSM) using Box,Behnken factorial design was used to evaluate the effects of treatment variables, that is, temperature (50 to 90 °C), time (1 to 29 min), and pH (2 to 8), on the residual antifungal activity (RAA) against Penicillium chrysogenum, Fusarium graminearum, Aspergillus flavus, and a Penicillium sp. isolated from moldy noodles. Regression analyses suggested that the linear terms of the temperature and time had significant (P < 0.05) negative effects on the RAA against all test fungi, whereas that of pH had a significant (P < 0.1) positive role on the RAA of all 3 fungi. In addition, the RAA was significantly (P < 0.05) affected by the quadratic terms of time for all fungi, and the quadratic term of temperature played a significant (P < 0.1) role on RAA against F. graminearum. One interaction term (temperature-pH) was found to significantly (P < 0.1) affect the RAA against both Penicillium strains tested. The results indicated that , 90% antifungal activity was lost after the protein extracts were heated at 90 °C for 8 min except for F. graminearum. At pasteurization condition, , 50% activity was retained except for P. chrysogenum. The results also suggested that neutral and alkaline pH favored the antifungal activity stability of the protein extracts. Thus, flaxseed protein might be promising if used as a preservative in foods with neutral or alkaline pH requiring mild heat treatments. [source] Development of a Monoclonal Antibody for Grouper (Epinephelus marginatus) and Wreck Fish (Polyprion americanus) Authentication Using an Indirect ELISAJOURNAL OF FOOD SCIENCE, Issue 6 2003L. Asensio ABSTRACT: A monoclonal antibody generated against soluble muscle proteins from grouper (Epinephelus marginatus) has been used in 2 indirect ELISA formats (microtiter plates and immunostick tubes) for the rapid authentication of grouper and wreck fish (Polyprion americanus). This monoclonal antibody (1A4-MAb) was tested against native and heat-treated (cooked and sterilized) soluble muscle protein extracts from several commonly marketed fish species and only reacted with grouper and wreck fish samples. [source] Regulation and Expression of Progesterone Receptor mRNA Isoforms A and B in the Male and Female Rat Hypothalamus and Pituitary Following Oestrogen TreatmentJOURNAL OF NEUROENDOCRINOLOGY, Issue 3 2002R. E. M. Scott Abstract Progesterone receptors play a central role in neuroendocrine and behavioural regulation. To gain insight into the sex- and tissue-specific regulation of progesterone receptors, protein binding on a progesterone receptor-oestrogen response element and mRNA levels for progesterone receptor (PR)-A and PR-B were compared between female and male rats following oestradiol benzoate replacement treatment in hypothalamic and pituitary tissue. Both male and female pituitary protein extracts demonstrated an increase in nuclear protein binding activity to a progesterone receptor-oestrogen response element following oestradiol benzoate treatment. However, there was a greater difference in total binding activity seen in the female pituitary extracts compared to male pituitary protein extracts. In both cases, reflecting the binding data, oestradiol benzoate pretreatment led to an increase in pituitary PR-B messenger RNA, although this increase was significantly larger in females than in males. Oestradiol benzoate treatment also led to a significant increase in specific binding of hypothalamic nuclear proteins to the progesterone receptor oestrogen response element from both females and male hypothalamic extracts. In addition, PR-B messenger RNA was induced by oestradiol benzoate treatment in the female rat hypothalamus, under circumstances where no PR-A could be detected. The male also demonstrated an increase in PR-B messenger RNA following oestradiol benzoate treatment, with undetectable levels of PR-A, although to a lesser degree than that seen in the female. The predominance of PR-B over PR-A messenger RNA in rat hypothalamus and pituitary, and the quantitative differences between female and male rats, could both contribute to the greater responsiveness of female rats to progesterone with respect to control over luteinizing hormone release from the pituitary, and lordosis behaviour regulated by hypothalamic neurones. [source] Soil-Borne Wheat Mosaic Virus (SBWMV) 37 kDa Protein Rescues Cell-to-Cell and Long-Distance Movement of an Immobile Tobacco Mosaic Virus Mutant in Nicotiana benthamiana, a Non-Host of SBWMVJOURNAL OF PHYTOPATHOLOGY, Issue 1 2005C. Zhang Abstract To verify the role and examine the functional range of the 37 kDa putative movement protein (MP) of soil-borne wheat mosaic virus (SBWMV), the 37 kDa gene was inserted into an infectious tobacco mosaic virus (TMV)-based expression vector (p30B), to generate p30BMP. The 30 kDa cell-to-cell MP gene of TMV was then inactivated (in p30BMP to give p30B,MP) by a frameshift mutation which removed 80 amino acids from its C-terminus. Systemic infection of Nicotiana benthamiana plants occurred following inoculation with in vitro transcripts of p30BMP or p30B,MP. Progeny viral RNAs from inoculated and systemically infected leaves were analysed by reverse transcriptase polymerase chain reaction and ApaI digestion, and by sequencing. The 30 kDa TMV MP or its truncated form were detected, respectively, in Western blots of cell wall protein extracts from p30BMP-transcript or p30B,MP-transcript inoculated or systemically infected N. benthamiana leaves. High levels of SBWMV 37 kDa MP were detected in all cases. The results suggest that the 37 kDa protein of SBWMV, a monocotyledonous-infecting furovirus, can complement both cell-to-cell and long-distance movement functions in a defective heterologous virus (TMV) in N. benthamiana, a non-host of SBWMV. [source] EFFECT OF LACTIC ACID AND LACTIC ACID BACTERIA TREATMENT ON MYOFIBRILLAR PROTEIN DEGRADATION AND DYNAMIC RHEOLOGY OF BEEFJOURNAL OF TEXTURE STUDIES, Issue 3 2007M. SIGNORINI ABSTRACT Lactic acid has been used as an efficient decontaminant in meats aimed for direct consumption or product fabrication. However, reports on the functionality of proteins extracted from lactic acid-treated meat are scattered. The objective of this work was to study the degradation and gelling ability of myofibrillar protein extracts obtained from beef treated with lactic acid of chemical and microbial origins, stored at 4 and 20C. The gelling ability was considerably reduced by lactic acid treatment as a result of protein denaturation in acid conditions at both storage temperatures. Scanning electron microscopy showed loose structures resulting in low penetration resistance and storage modulus. Treatments with lactic acid or lactic acid bacteria (LAB) had similar effect on tan,, affecting gel rigidity but not elasticity. Penetration in gels obtained from LAB-treated meat was highly correlated with myosin degradation. Lactobacillus carnis -treated meat produced compact gels with high penetration resistance and storage modulus, although the structure became looser with storage time. LAB treatment, although not as efficient as lactic acid as a meat preservative, is a milder process causing less severe changes in meat structure rheology. PRACTICAL APPLICATIONS The potential of lactic fermentation by selected strains is somewhat limited as compared to lactic acid preservation of meat substrates, regarding pH reduction and its consequence on pathogens and spoilage microorganism population reduction. However, lactic acid bacteria (LAB) treatments are milder; therefore, changes in protein structure and rheology are less severe. Lactic acid in its chemical form promotes protein changes, whereas LAB does not. As myofibrillar protein configuration is responsible for most meat functional properties, such as gel and emulsion formation, it is important that protein structure remains unchanged as much as possible. Using nonproteolytic strains, protein degradation can only be altered by endogenous or bacteria-produced enzymes, which can be inhibited by several means. Meat preservation by lactic fermentation with selected strains can be an alternative when keeping meat protein functional properties unaltered. [source] The Neurospora circadian clock regulates a transcription factor that controls rhythmic expression of the output eas(ccg-2) geneMOLECULAR MICROBIOLOGY, Issue 4 2001Deborah Bell-Pedersen The circadian clock provides a link between an organism's environment and its behaviour, temporally phasing the expression of genes in anticipation of daily environmental changes. Input pathways sense environmental information and interact with the clock to synchronize it to external cycles, and output pathways read out from the clock to impart temporal control on downstream targets. Very little is known about the regulation of outputs from the clock. In Neurospora crassa, the circadian clock transcriptionally regulates expression of the clock-controlled genes, including the well-characterized eas(ccg-2) gene. Dissection of the eas(ccg-2) gene promoter previously localized a 68 bp sequence containing an activating clock element (ACE) that is both necessary and sufficient for rhythmic activation of transcription by the circadian clock. Using electrophoretic mobility shift assays (EMSAs), we have identified light-regulated nuclear protein factors that bind specifically to the ACE in a time-of-day-dependent fashion, consistent with their role in circadian regulation of expression of eas(ccg-2). Nucleotides in the ACE that interact with the protein factors were determined using interference binding assays, and deletion of the core interacting sequences affected, but did not completely eliminate, rhythmic accumulation of eas(ccg-2) mRNA in vivo, whereas deletion of the entire ACE abolished the rhythm. These data indicate that redundant binding sites for the protein factors that promote eas(ccg-2) rhythms exist within the 68 bp ACE. The ACE binding complexes formed using protein extracts from cells with lesions in central components of the Neurospora circadian clock were identical to those formed with extracts from wild-type cells, indicating that other proteins directly control eas(ccg-2) rhythmic expression. These data suggest that the Neurospora crassa circadian clock regulates an unknown transcription factor, which in turn activates the expression of eas(ccg-2) at specific times of the day. [source] Expression of hck-tr, a truncated form of the src-related tyrosine kinase hck, in bovine spermatozoa and testisMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 5 2008Louis-Jean Bordeleau Abstract In bull testicular haploid germ cells, an mRNA encoding for hck was detected in addition to another one encoding for hck-tr, a truncated form of the tyrosine kinase hck. As the transcripts were expressed in spermatids, we tried to determine whether hck-tr is present in mature bovine spermatozoa. Two polyclonal antibodies were produced against peptides specific to the N- and C-terminal portions of the truncated protein. Western blot analyses confirmed the presence of hck-tr in total protein extracts of ejaculated bull spermatozoa, and sub-cellular fractionation experiments suggest its presence in both head and flagellum. The truncated protein appears tightly associated with cytoskeletal elements as it could be extracted only with SDS under reducing conditions. When assessed by indirect immunofluorescence, hck-tr was mostly localized at the acrosomal area of the sperm cell and a similar localization was observed on demembranated spermatozoa. Immunohistochemical studies on testis sections revealed protein expression in spermatocytes as well as in round and elongating spermatids. The results presented in this study clearly show the presence of mRNAs encoding for hck and hck-tr in testicular germ cells; hck-tr being translated during spermatogenesis and expressed on mature ejaculated bull spermatozoa. Mol. Reprod. Dev. 75: 828,837, 2008. © 2007 Wiley-Liss, Inc. [source] | ||||||||||||||||