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Protein Bands (protein + bands)
Selected AbstractsBiomarker discovery in rat plasma for estrogen receptor-, actionELECTROPHORESIS, Issue 23 2005Tom G. Holt Dr. Abstract To support in vivo screening efforts for estrogen receptor (ER) subtype selective therapeutic agents, we initiated work to discover surrogate markers (biomarkers) in blood plasma that would change in response to ER subtype-specific action. We used a proteomic approach employing strong anion exchange chromatography (SAX), PAGE, and MS to identify potential plasma markers for selective ER-, action. The methodology was used to compare blood from vehicle-treated rats to blood from rats treated with either 17,-estradiol (an ER-,/ER-, agonist) or compound 1 (17,-ethynyl-[3,2-c]pyrazolo-19-nor-4-androstene-17,-ol, an ER-,-selective agonist). Blood samples were first fractionated by SAX to separate fractions containing dominant common plasma proteins from fractions enriched for less-abundant plasma proteins. 1-D PAGE analysis of fractions depleted of dominant plasma proteins revealed treatment-specific changes in protein profiles. Protein bands that changed reproducibly in response to ER-, action were excised from the gel, separated by capillary LC, and identified by microspray ESI-MS. Using this method, the plasma levels of two proteins, transthyretin and apolipoprotein E, were shown to decrease in response to ER-, agonism. The method lacked the sensitivity to identify the known, 1000-fold less-abundant, estrogenic marker prolactin (PRL). However, using a commercial RIA and immunoblots, we showed that PRL levels increase significantly in response to treatment with the ER-, selective agonist, compound 1. [source] Activity-based mass spectrometric characterization of proteases and inhibitors in human salivaPROTEOMICS - CLINICAL APPLICATIONS, Issue 7 2009Xiuli Sun Abstract Proteases present in oral fluid effectively modulate the structure and function of some salivary proteins and have been implicated in tissue destruction in oral disease. To identify the proteases operating in the oral environment, proteins in pooled whole saliva supernatant were separated by anion-exchange chromatography and individual fractions were analyzed for proteolytic activity by zymography using salivary histatins as the enzyme substrates. Protein bands displaying proteolytic activity were particularly prominent in the 50,75,kDa region. Individual bands were excised, in-gel trypsinized and subjected to LC/ESI-MS/MS. The data obtained were searched against human, oral microbial and protease databases. A total of 13 proteases were identified all of which were of mammalian origin. Proteases detected in multiple fractions with cleavage specificities toward arginine and lysine residues, were lactotransferrin, kallikrein-1, and human airway trypsin-like protease. Unexpectedly, ten protease inhibitors were co-identified suggesting they were associated with the proteases in the same fractions. The inhibitors found most frequently were alpha-2-macroglobulin-like protein 1, alpha-1-antitrypsin, and leukocyte elastase inhibitor. Regulation of oral fluid proteolysis is highly important given that an inbalance in such activities has been correlated to a variety of pathological conditions including oral cancer. [source] Cover Picture: Electrophoresis 5'09ELECTROPHORESIS, Issue 5 2009Article first published online: 3 MAR 200 Issue no. 5 is a special issue on "Fundamentals of Electrophoresis" containing 21 papers including 3 Fast Track papers. The first Fast Track paper deals with imaged CE technology for measuring charge variants of monoclonal antibodies, the second paper is on the dispersion of protein bands in a horseshoe microchannel during IEF while the third Fast Track paper reports a theoretical and experimental study on the irreversible deposition of colloidal particles from electrokinetic microfluidic flow. The remaining 18 papers of this special issue are distributed into seven parts pertaining to various fundamental topics, e.g., electromigration and dielectrophoresis in microchip channels, computer simulation of electromigration, stacking, interaction in electrophoretic systems, thermal effects, electroosmotic flow, etc. [source] Highly sensitive and simple fluorescence staining of proteins in sodium dodecyl sulfate-polyacrylamide-based gels by using hydrophobic tail-mediated enhancement of fluorescein luminescenceELECTROPHORESIS, Issue 19-20 2003Chulhun Kang Abstract Fluorescein has an extremely low luminescence intensity in acidic aqueous media. However, when it was bound to proteins, subsequent increase of luminescence intensity took place. Furthermore, when a hydrophobic tail, such as aliphatic hydrocarbons, was introduced to fluorescein, more dramatic increase of luminescence intensity was observed upon binding to proteins. In the present study, by utilizing this luminescence enhancement, three hydrophobic fluorescein dyes (5-dodecanoyl amino fluorescein, 5-hexadecanoyl amino fluorescein, and 5-octadecanoyl amino fluorescein) were examined as noncovalent fluorescent stains of protein bands in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Effective incorporation of the dyes to proteins in gels was accomplished either simply by adding dyes at the protein fixation step, or by treating gels with a staining solution after the fixation. The sensitivity of this staining method using the fluorescein derivatives was approximately 1 ng/band for most proteins. For some cases, protein bands containing as low as 0.1 ng were successfully visualized. In addition, the detection sensitivity showed much less protein-to-protein variation than silver staining. This new staining method was also successfully applied to two-dimensional electrophoresis of rat brain proteins. Its overall sensitivity was comparable to that of silver staining. [source] Sodium dodecyl sulfate versus acid-labile surfactant gel electrophoresis: Comparative proteomic studies on rat retina and mouse brainELECTROPHORESIS, Issue 4 2003Simone König Abstract A long-chain derivative of 1,3-dioxolane sodium propyloxy sulfate, with similar denaturing and electrophoretic properties as SDS, and facilitated protein identification following polyacrylamide gel electrophoresis (PAGE) for Coomassie-stained protein bands, has been tested. Comparative acid-labile surfactant/sodium dodecyl sulfate two-dimensional (ALS/SDS 2-D)-PAGE experiments of lower abundant proteins from the proteomes of regenerating rat retina and mouse brain show that peptide recovery for mass spectrometry (MS) mapping is significantly enhanced using ALS leading to more successful database searches. ALS may influence some procedures in proteomic analysis such as the determination of protein content and methods need to be adjusted to that effect. The promising results of the use of ALS in bioanalytics call for detailed physicochemical investigations of surfactant properties. [source] Postendocytic Provitellin Processing in the Growing Oocyte of the Short Horned Grasshopper, Oxyajaponicajaponica (Orthoptera: Acrididae)ENTOMOLOGICAL RESEARCH, Issue 1 2004Sae Youll CHO ABSTRACT Polyclonal antibodies made against 86 kDa (86 k), 80 kDa (80 k) and 54 kDa (54 k) vitellins of Oxya japonica japonica are used for Western blotting. Anti-80k vitellin antibody is cross-reacted with a 95 kDa (95 k) vitellin. While 95 k vitellin is present both in the female hemolymph and in the oocyte, 80 k vitellin is detected only in the oocyte and the laid egg. In the growing oocytes, as 95 k vitellin is faded out gradually, 80 k vitellin is accumulated increasingly, indicating postendocytic processing of 95 k vitellin brings 80 k vitellin. Further conforming the hypothesis, partial digestion of 95 k vitellin with pepsin and ,-chymotrypsin makes several protein bands of molecular weight around 80 kDa. Thus, the 95k vitellin may have a cleavage site (s) to produce 80 k vitellin which forms fairly stable tertiary structure. In the reduced condition (20 mM glutathion), both 95 k and 80 k vitellins were digested throughly by endogenous proteinase at pH 4. Both 86 k and 54 k vitellins, respectively, show no apparent molecular weight changes in the growing oocyte and in the hemolymph. [source] Antioxidative activity of water extracts from the yam (Dioscorea opposita Thunb.) tuber mucilage tororoEUROPEAN JOURNAL OF LIPID SCIENCE AND TECHNOLOGY, Issue 6 2006Takeshi Nagai Abstract A water extract as a viscous solution was obtained from the yam Dioscorea opposita tuber mucilage tororo, and its functional properties were demonstrated. The protein content was about 280,,g/mL extract, and the main protein bands with an MW of ,33,kDa without 2-mercaptoethanol (2-ME) and ,31,kDa with 2-ME were detected by SDS-PAGE. The water extract possessed high antioxidative activity and scavenging activities against superoxide anion and hydroxyl radicals. However, it showed no inhibitory activity against angiotensin,I-converting enzyme. The yam tuber contains relatively high contents of vitamins, different micro- and macroelements, enzymes, and dietary fibers. The yam D.,opposita tuber will be increasingly regarded as a health-promoting food. [source] Biogenesis of Yersinia pestis PsaA in recombinant attenuated Salmonella Typhimurium vaccine (RASV) strainFEMS MICROBIOLOGY LETTERS, Issue 2 2010Ascención Torres-Escobar Abstract Yersinia pestis PsaA is an adhesin important for the establishment of bacterial infection. PsaA synthesis requires the products of the psaEFABC genes. Here, by prediction analysis, we identified a PsaA signal sequence with two signal peptidase (SPase) cleavage sites, type-I and type-II (SPase-I and SPase-II). By Edman degradation and site-directed mutagenesis, the precise site for one of these Spase-I PsaA cleavage sites was located between alanine and serine at positions 31 and 32, respectively. Yersinia pestis psaA expression and the role of the PsaB and PsaC proteins were evaluated in recombinant attenuated Salmonella Typhimurium vaccine strains. PsaA was detected in total extracts as a major 15-kDa (mature) and 18-kDa (unprocessed) protein bands. PsaA synthesis was not altered by a ,A31,,S32 double-deletion mutation. In contrast, the synthesis of PsaA (,A31,,S32) in Y. pestis and delivery to the supernatant was decreased. Otherwise, substitution of the amino acid cysteine at position 26 by valine involved in the SPase-II cleavage site did not show any effect on the secretion of PsaA in Salmonella and Yersinia. These results help clarify the secretion pathway of PsaA for the possible development of vaccines against Y. pestis. [source] Physico-biochemical parameters and protein profiles of sperm from beluga Huso husoJOURNAL OF APPLIED ICHTHYOLOGY, Issue 5 2010P. Li Summary Basic physico-biochemical parameters and protein profiles of sperm from beluga (Huso huso), have been evaluated. The results show a stable spermatozoan velocity (ranging from 136.23 ± 5.63 to 105.78 ± 5.27 ,ms,1) and motility during 2 min post activation, as well as a long duration of the overall spermatozoa motility period (up to 5 min). Mean values were determined for seminal plasma protein concentration (0.29 ± 0.16 mg ml,1), spermatozoa concentration (0.28 ± 0.27 × 109 spz ml,1), seminal plasma osmolality (51.33 ± 4.91 mOsmol kg,1), the pH (8.49 ± 0.01) and Na+ (18.97 ± 3.65 mm), K+ (2.83 ± 1.36 mm), Ca2+ (0.19 ± 0.06 mm), Mg2+ (0.49 ± 0.23 mm) and Cl, (6.33 ± 0.58 mm) concentrations. Moreover, in seminal plasma, five protein bands with molecular weights (MW) of 71, 49, 46, 34, 29 kDa were identified. In spermatozoa, about 100 spots with molecular weights varying from 26.5 to 107 kDa and iso-electric points ranging from 5 to 9.5 were found. The observed physiological and biochemical properties, together with protein patterns, should be considered for the development of methods for controlled reproduction and sperm cryopreservation for the highly endangered beluga sturgeon. [source] Ethanol-induced alterations of the antioxidant defense system in rat kidneyJOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 6 2006Diana Dinu Abstract We report here the effects of chronic ethanol consumption on the antioxidant defense system in rat kidney. Thirty-two male Wistar rats were randomly divided in two identical groups and were treated as follows: control group (water for fluid) and the ethanol-fed group (2 g/kg body weight/24 h). The animals were sacrificed after 10 weeks, and respectively 30 weeks of ethanol consumption, and the renal tissue was isolated and analyzed. Results revealed that kidney alcohol dehydrogenase activities increased significantly after ethanol administration, but the electrophoretic pattern of alcohol dehydrogenase isoforms was unmodified. The SDS polyacrylamidegel electrophoretic study of kidney proteins has revealed the appearance of two new protein bands after long-term ethanol consumption. The kidney reduced glutathione/oxidized glutathione ratio decreased, indicating an oxidative stress response due to ethanol ingestion. The malondialdehyde contents and xanthine oxidase activities were unchanged. The antioxidant enzymatic defense system showed a different response during the two periods of ethanol administration. After 10 weeks, catalase, glutathione peroxidase, glutathione reductase, and glucose-6-phosphate dehydrogenase were activated, while superoxide dismutase, glutathione transferase, and ,-glutamyltranspeptidase levels were stationary. After 30 weeks, superoxide dismutase and glutathione peroxidase activities were unmodified, but catalase, glutathione transferase, ,-glutamyltranspeptidase, glutathione reductase, and glucose-6-phosphate dehydrogenase activities were significantly increased. Remarkable changes have been registered after 30 weeks of ethanol administration for glutathione reductase and glucose-6-phosphate dehydrogenase activities, including an increase by 106 and 216' of control values, respectively. These results showed specific changes in rat kidney antioxidant system and glutathione status as a consequence of long-term ethanol administration. © 2005 Wiley Periodicals, Inc. J Biochem Mol Toxicol 19:386-395, 2005; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20101 [source] Effects of nickel poisoning on expression pattern of the 72/73 and 94 kDa stress proteins in rat organs and in the COS-7, HepG2, and A549 cell linesJOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 1 2005N. Hfaiedh Abstract The present study deals with the effects of Ni on the expression level of three stress proteins, namely, the cytosolic HSP72 and HSP73, and the reticulum-associated GRP94. Experiments were carried out on "Wistar" female rats daily injected with 4 mg NiCl2 per kg body weight for 1, 3, 5, and 10 days. Another set of experiments were carried out using cell lines, derived from the monkey kidney (COS-7), and from human tumors of the lung (A549) and liver (HepG2). Cells were cultured for 4 days in the permanent presence of 100, 200, or 400 µM NiCl2. In control rats, stress proteins pattern was found to be tissue specific: two protein bands of 96 and 94 kDa were immunodetected with the anti-GRP94 antibody in kidney and liver extracts, whereas only the 96 kDa band was present in ovary extracts. HSP73 was present in kidney, liver, and ovary whereas HSP72 was only found in kidney. In kidney of nickel-treated animals, HSP73 and the 96 kDa proteins were overexpressed whereas HSP72 was strongly down regulated. No such effect was observed in liver or ovary. Similarly, in nickel-treated cell lines, HSP72 was downregulated and GRP94 (96 kDa protein) was overexpressed. HSP73 expression appeared moderately increased in A549 cells but decreased in COS-7 cells. Because long-term caloric restriction was reported to reduce free radical generation in cells, the effect of 1 month food restriction (50%) was tested in rats as a possible way to lower oxidative damages induced by Ni. No significant effect on HSP expression was observed. © 2005 Wiley Periodicals, Inc. J Biochem Mol Toxicol 19:12,18, 2005; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20056 [source] FILM FORMING MECHANISM AND MECHANICAL AND THERMAL PROPERTIES OF WHEY PROTEIN ISOLATE-BASED EDIBLE FILMS AS AFFECTED BY PROTEIN CONCENTRATION, GLYCEROL RATIO AND PULLULAN CONTENTJOURNAL OF FOOD BIOCHEMISTRY, Issue 3 2010MAHAMADOU ELHADJI GOUNGA ABSTRACT Tensile strength (TS), elongation at break (EAB) and elastic modulus (EM) of edible films prepared from 5, 7 and 9% whey protein isolate (WPI) plasticized with different levels of glycerol (Gly) (WPI : Gly = 3.6:1, 3:1 and 2:1) were investigated in order to completely characterize WPI-Gly films. On increasing protein concentration an increase in TS and EAB was observed. On the other hand, increasing Gly led to a decrease in TS and EM, while EAB increased. The addition of pullulan (Pul) into the film forming solution (FFS) increased EAB while TS, EM and thermal properties were reduced. This suggested that Pul had a similar effect as plasticizers. Films with higher Pul content showed lighter protein bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Fourier transform infrared spectroscopy showed that hydrogen bonding was high in WPI : Pul films as compared with the control. This is attributed to the protein-polysaccharide interactions brought about by the dominance of Pul in the FFS. PRACTICAL APPLICATIONS This work describes some physical properties of films based on blends of whey protein isolate (WPI) and pullulan (Pul), made after a previous study on some characteristics of films based on pure WPI plasticized by glycerol. The most studied proteins in the edible films technology being gluten and WPI, the use of Pul in mixture with WPI is considered as a new investigation to explore the utilization of WPI-Pul in edible film and coating materials applied to food products. Furthermore, the use of WPI-Pul films and coatings could potentially extend the shelf life and improve the stability of the coated products as shown by the resultant properties in this investigation and previous works. [source] Effect of Chemically Modified Soy Proteins and Ficin-tenderized Meat on the Quality Attributes of SausageJOURNAL OF FOOD SCIENCE, Issue 1 2003R. Ramezani ABSTRACT: The purpose of this investigation was to use ficin-tenderized meat and cysteine-modified soy proteins in the production of bologna and to evaluate the effect of these modifications on water-holding capacity (WHC), emulsion stability (ES), texture, and protein solubility. The effect of ficin on meat protein was also evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Results indicated that both ficin-tenderized meat and modified soy proteins substantially improved WHC, ES, and other quality factors. SDS-PAGE results showed the disappearance of several protein bands in ficin-treated meat. Solubility of meat proteins increased when ficin was used for meat tenderization. The results of this study indicated that some quality attributes of meat products can be improved by enzymatic and chemical modification of protein sources in the manufacture of meat products. [source] Hepatic covalent adduct formation with zomepirac in the CD26-deficient mouseJOURNAL OF GASTROENTEROLOGY AND HEPATOLOGY, Issue 1 2002MIN WANG Abstract Background and Aims: Zomepirac (ZP), a non-steroidal anti-inflammatory drug (NSAID), has been reported to cause immune-mediated liver injury. In vivo, ZP is metabolized to a chemically reactive acyl glucuronide conjugate (ZAG) which can undergo covalent adduct formation with proteins. Such acyl glucuronide-derived drug-protein adducts may be important in the development of immune and toxic responses caused by NSAID. We have shown using immunoabsorptions that the 110 kDa CD26 (dipeptidyl peptidase IV) is one of the hepatic target proteins for covalent modification by ZAG. In the present study, a CD26-deficient mouse strain was used to examine protein targets for covalent modification by ZP/metabolites in the liver. Methods and Results: The CD26-deficient phenotype was confirmed by immunohistochemistry, flow cytometry analysis, RT-PCR, enzyme assay and immunoblotting. Moreover, by using monoclonal antibody immunoblots, CD26 was not detected in the livers of ZP-treated CD26-deficient mice. Immunoblots using a polyclonal antiserum to ZP on liver from ZP-treated mice showed three major sizes of protein bands, in the 70, 110 and 140 kDa regions. Most, but not all, of the anti-ZP immunoreactivity in the 110 kDa region was absent from ZP-treated CD26-deficient mice. Conclusion: These data definitively showed that CD26 was a component of ZP-modified proteins in vivo. In addition, the data suggested that at least one other protein of approximately 110 kDa was modified by covalent adduct formation with ZAG. [source] Potential of ,flat' fibre evanescent wave spectroscopy to discriminate between normal and malignant cells in vitroJOURNAL OF MICROSCOPY, Issue 2 2007Z. HAMMODY Summary The present study focuses on evaluating the potential of flattened AgClBr fibre-optic evanescent wave spectroscopy (FTIR-FEWS) technique for detection and identification of cancer cells in vitro using cell culture as a model system. The FTIR-FEWS results are compared to those from FTIR-microspectroscopy (FTIR-MSP) method extensively used to identify spectral properties of intact cells. Ten different samples of control and malignant cells were measured in parallel by the above two methods. Our results show a significant similarity between the results obtained by the two methodologies. The absorbance level of Amide I/Amide II, phosphates and carbohydrates were significantly altered in malignant compared to the normal cells using both systems. Thus, common biomarkers such as Amide I/Amide II, phosphate and carbohydrate levels can be derived to discern between normal and cancer cells. However, marked differences are also noted between the two methodologies in the protein bands due to CH3 bending vibration (1480,1350 cm,1). The spectral differences may be attributed to the variation in the penetration depth of the two methodologies. The use of flattened fibre rather than the standard cylindrical fibre has several practical advantages and is considered as an important step towards in vivo measurements in real time, such as that of skin nevi and melanoma using special designs of fibre-optic,based sensors. [source] Reduced expression of MAb6B4 epitopes on chondroitin sulfate proteoglycan aggrecan in perineuronal nets from cerebral cortices of SAMP10 mice: A model for age-dependent neurodegenerationJOURNAL OF NEUROSCIENCE RESEARCH, Issue 6 2008Yuko Saitoh Abstract The accelerated senescence-prone SAMP10 mouse strain is a model for age-dependent neurodegeneration and is characterized by brain atrophy and deficits in learning and memory. Because perineuronal nets play an important role in the synaptic plasticity of adult brains, we examined the distributions of molecules that constitute perineuronal nets in SAMP10 mouse brain samples and compared them with those in control SAMR1 mouse samples. Proteoglycan-related monoclonal antibody 6B4 (MAb6B4) clearly immunostained perineuronal nets in SAMR1 mice cortices, but the corresponding immunostaining in SAMP10 mice was very faint. MAb6B4 recognizes phosphacan/PTP, in immature brains. However, this antibody recognized several protein bands, including a 400-kDa core glycoprotein from chondroitin sulfate proteoglycan in homogenates of mature cortices from SAMR1 mice. The 400-kDa band was also recognized by antiaggrecan antibodies. The aggrecan core glycoprotein band was also detectable in samples from SAMP10 mice, but this glycoprotein was faintly immunostained by MAb6B4. Because MAb6B4 recognized the same set of protein bands that the monoclonal antibody Cat-315 recognized in mature cerebral cortices of SAMR1 mice, the MAb6B4 epitope appears to be closely related to that of Cat-315 and presumably represents a novel type of oligosaccharide that attaches to aggrecans. The Cat-315 epitope colocalized with aggrecan in perineuronal nets from SAMR1 mouse brain samples, whereas its expression was prominently reduced in SAMP10 mouse brain samples. The biological significance of the MAb6B4/Cat-315 epitope in brain function and its relationship to the neurodegeneration and learning disabilities observed in SAMP10 mice remain to be elucidated. © 2007 Wiley-Liss, Inc. [source] Inhibition of neuronal migration by JONES antibody is independent of 9-O-acetyl GD3 in GD3-synthase knockout miceJOURNAL OF NEUROSCIENCE RESEARCH, Issue 7 2007Chia-Ron Yang Abstract It has been shown previously that the migration of granule neurons in neonatal cerebellum can be inhibited by a monoclonal antibody (Mab) JONES. Because the inhibition is presumed to be mediated through binding of the JONES antibody to 9-O-acetyl GD3, we used GD3-synthase knockout (GD3S,/,) mice that do not express 9-O-acetyl GD3 and also have no detectable defect in brain development, to examine the mechanism of the inhibitory effect. We found no difference between the migration of granule neurons in the neonatal cerebellar explant culture in GD3S,/, mice and in wild-type mice. Addition of the Mab JONES, but not Mab R24 or A2B5, in the culture medium blocked the neuronal migration in the explant culture of the wild-type mice. The inhibitory effect of Mab JONES was also observed, however, in the explant culture of GD3S,/, mice. Immuno-HPTLC analysis showed at least two JONES-positive glycolipids bands in the lipid extract of GD3S+/+ mice, and none was detected in that of GD3S,/, mice. Western blot analysis of the cerebellum homogenate of wild-type and GD3S,/, mice identified at least 3 JONES-positive protein bands, one of which is ,1-integrin. Because the JONES antibody also blocked neuronal migration in the cerebellar explant culture of GD3S,/, mice that do not express 9-O-acetyl-GD3, it suggested an alternative mechanism for the inhibitory effect of the antibody, at least in the GD3S knockout mice, and the inhibitory effect of the JONES antibody on neuronal migration could be mediated through its binding to ,1-integrin. © 2007 Wiley-Liss, Inc. [source] Development of a Sensitive Serological Method for Specific Detection of Latent Infection of Macrophomina phaseolina in CowpeaJOURNAL OF PHYTOPATHOLOGY, Issue 1 2009Leonard Afouda Abstract A double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was developed for the specific detection and quantification of Macrophomina phaseolina in plant tissue. Both polyclonal antisera produced against immunogens from mycelium and culture filtrate of M. phaseolina detected the fungus in mycelial and plant extracts, although the antibodies raised against mycelium were more sensitive. No cross-reaction occurred with Rhizopus stolonifer, Pythium ultimum, Mucor hiemalis, Fusarium oxysporum, Septoria nodorum, Rhizoctonia solani, Sclerotinia sclerotiorum, Phytophthora infestans and Aspergillus niger. In enzyme assays, activity of the endo-acting hydrolytic enzymes 1,3-,-glucanase and, less, cellulase, but not xylanase was detected in infected plants. DAS-ELISA was more sensitive than the 1,3-,-glucanase assay. In polyacrylamide gel electrophoresis (PAGE) up to 18 protein bands were observed, with four bands occurring in the 12 tested isolates deriving from various geographical origin in Niger and Nigeria. The enzyme assays and protein patterns were considered not suitable for specific M. phaseolina detection. Macrophomina phaseolina was essentially located in the roots and hypocotyls, and less in epicotyls and leaves of infected plants. The antibodies were also useful to detect latent infection and the infection of cowpea seeds. [source] Electrophoretic patterns of microwaved and ,-irradiated beef liver proteinsJOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 10 2001S Farag Abstract The effects of ,-irradiation treatments (2.5, 5 and 10,kGy) and microwaves generated from an oven at low and defrost power settings for 0.5, 1 and 2,min on the total proteins and protein patterns of beef liver immediately after treatment and during frozen storage (,18,°C) for different periods were studied. Chemical analyses indicated that the protein content of beef liver was reduced after exposure to ,-radiation or microwaves and also during frozen storage. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was used to illustrate the changes in protein bands of different molecular weights and their percentages before and after exposure to gamma and microwave radiation. The main effect of ,-radiation on the protein patterns of beef liver was the disappearance of some high-molecular-weight protein bands and the development of other bands characterised by moderate and low molecular weights. This finding indicates the degradation of beef liver proteins by ,-irradiation. In contrast, microwave treatment caused an increase in the levels of high-molecular-weight protein bands with a concomitant decrease in low-molecular-weight protein bands. This phenomenon demonstrates the polymerisation of low-molecular-weight proteins under the influence of microwaves. © 2001 Society of Chemical Industry [source] Acetaminophen UDP-glucuronosyltransferase in ferrets: species and gender differences, and sequence analysis of ferret UGT1A6JOURNAL OF VETERINARY PHARMACOLOGY & THERAPEUTICS, Issue 6 2001M. H. Court The principal objective of this study was to determine whether ferrets glucuronidate acetaminophen more slowly compared with other species, and if so investigate the molecular basis for the difference. Acetaminophen-UDP-glucuronosyltransferase (UGT) activities were measured using hepatic microsomes from eight ferrets, four humans, four cats, four dogs, rat, mouse, cow, horse, monkey, pig and rabbit. Gender differences between male and female ferret livers were explored using enzyme kinetic analysis. Immunoblotting of microsomal proteins was also performed using UGT-specific antibodies. Finally, the exon 1 region of UGT1A6, a major acetaminophen-UGT, was sequenced. Glucuronidation of acetaminophen was relatively slow in ferret livers compared with livers from all other species except cat. Gender differences were also apparent, with intrinsic clearance (Vmax/Km) values significantly higher in male compared with female ferret livers. Furthermore, Vmax values correlated with densitometric measurements of two protein bands identified with a UGT1A subfamily-specific antibody. No deleterious mutations were identified in the exon 1 or flanking regions of the ferret UGT1A6 gene. In conclusion, like cats, ferret livers glucuronidate acetaminophen relatively slowly. However, unlike cats, in which UGT1A6 is encoded by a pseudogene and dysfunctional, there are no defects in the ferret UGT1A6 gene which could account for the low activity. [source] IgE-binding components of cultured human keratinocytes in atopic eczema/dermatitis syndrome and their crossreactivity with Malassezia furfurALLERGY, Issue 2 2004O. Kortekangas-Savolainen Background:, Atopic eczema/dermatitis syndrome (AEDS) patients display immunoglobulin E (IgE) reactivity to several antigens, e.g. saprophytic yeasts as Malassezia furfur. AEDS patients also show IgE autoreactivity towards cells of their own tissue including epidermis. Purpose of the study:, The aim of this study was to investigate the IgE autoreactivity of AEDS patients to cultured keratinocytes and to reveal potential crossreacting epitopes in cultured keratinocytes and M. furfur. Material and methods:, Serum samples of 27 AEDS patients were analyzed, of these 13 were M. furfur radioallergosorbent test (RAST) positive and 14 negative. Four urticaria, three psoriasis, and seven nonatopic patients were included as controls. The studies were performed by using IgE immunoblotting and immunoblotting inhibition methods. Results:, Ten IgE-binding protein bands were detected in cultured human keratinocytes by IgE immunoblotting using sera from adult AEDS patients. Anti-keratinocyte IgE antibodies were more associated with elevated S-IgE level than M. furfur RAST. Clear crossreactivity with M. furfur could not be shown. Conclusions:, The possible pathomechanism of anti-keratinocyte IgE antibodies is not due to IgE epitope mimicry of saprophytic yeast and local tissue in AEDS skin. [source] Immediate hypersensitivity to Malassezia furfur in patients with atopic dermatitisMYCOSES, Issue 4 2007A. R. Khosravi Summary Atopic dermatitis (AD) is a chronic pruritic dermatitis that has unknown aetiology. It seems that Malassezia furfur has a role in pathogenesis of AD. The purpose of this study was to evaluate skin responses to M. furfur antigens in AD patients. Malassezia furfur was grown and the yeasts were broken. Cells were centrifuged and supernatants were used as crude extracts (CE). Protein components of CE were separated by sodium dodecylsulphate,polyacrylamide gel electrophoresis (SDS,PAGE). In addition, to fractionate CE antigens, gel filtration chromatography was performed. One hundred and fifteen AD patients were selected for skin-prick test (SPT). In SDS,PAGE, CE showed a total of 19 different protein bands (10,100 kDa). Chromatographic gel filtration with M. furfur proteins showed four major fractions (F). The protein pattern of F1 (tube no. 40) was between 22 and 100 kDa and it was selected for SPT. In SPT, 49.6% and 42.6% patients showed positive reactions with CE and F1 antigens respectively. The most positive results were obtained in 20,29 aged group (P < 0.001). The allergens of M. furfur may have a role in AD signs; it is suggested to use F1 antigens in allergy tests. [source] Airborne viable fungi in Riyadh and allergenic response of their extractsMYCOSES, Issue 9-10 2001A. S. Al-Suwaini Luftbürtige Pilze; Allergenität; Antigenität; Prick-Test; Saudi-Arabien. Summary. The allergenicity and antigenicity of various airborne fungi isolated from the atmosphere of Riyadh were studied. Protein nitrogen contents were estimated and found to range from 0.9 mg ml,1 for Cladosporium to 2.1 mg ml,1 for Aspergillus extracts. Sodium dodecyl sulphate,polyacrylamide gel electrophoresis analysis for those extracts exhibited a number of protein bands of higher molecular weight between 13 and 80 kDa for Alternaria, Ulocladium, Penicillium, Aspergillus and Cladosporium. Extracts in both aqueous and lyophilized forms were sterilized and tested for diagnostic skin prick test in 100 consecutive patients having bronchial asthma and allergic rhinitis. Overall, 13% of patients reacted positively to fungal extracts, revealing allergic sensitization to these fungi. These findings necessitate further investigation as regards the purification and characterization of these local extracts for better diagnostic use in patients in Saudi Arabia. Zusammenfassung. Es wurde die Allergenität und Antigenität mehrerer luftbürtiger Pilze untersucht, die aus der Luft von Riad isoliert worden waren. Hierzu wurden Pilzextrakte hergestellt, deren Proteinstickstoffgehalt zwischen 0.9 mg ml,1 bei Cladosporium und 2.1 mg ml,1 bei Aspergillus lag. Die SDS,PAGE-Analyse zeigte eine Anzahl von Proteinbanden höheren Molekulargewichts zwischen 13 und 80 kDa für Alternaria, Ulocladium, Penicillium, Aspergillus und Cladosporium. Die sowohl wässrigen wie lyophilisierten Extrakte wurden sterilisiert und an 100 unausgewählten Patienten mit Bronchialasthma und allergischer Rhinitis im Pricktest getestet. Ingesamt 13% der Patienten reagierten auf die Extrakte positiv, was für eine allergische Sensibilisierung gegen diese Pilze spricht. [source] Anti-inflammatory responses and oxidative stress in Nippostrongylus brasiliensis -induced pulmonary inflammationPARASITE IMMUNOLOGY, Issue 1 2002Kathryn S. McNeil summary Migration of L3 larvae of Nippostrongylus brasiliensis through the lungs of the rat, during primary infection, was studied at 24 h, 72 h and 8 days. At 24 h p.i., there was evidence of damage to lung epithelial cells and microvasculature, with increased protein and ,-glutamyl transpeptidase in the bronchoalveolar lavage (BAL) fluid. However, there was little evidence of inflammatory cell recruitment. At 24 h p.i., there was a significant reduction in the inflammatory cytokine tumour necrosis factor ,. Superoxide (O2,·) production was also reduced, accompanied by an increase in superoxide dismutase activity. Lipid peroxidation was reduced at 24 h p.i. and L3 larvae were shown to possess high levels of glutathione compared to host lung tissue. Nitric oxide, detected as nitrite, was produced in BAL fluid, and inducible nitric oxide synthase protein was increased by 72 h p.i. There was evidence of peroxynitrite production throughout the infection period with specific protein bands nitrosylated at 75, 30 and 25 kDa. It appears that despite early evidence of lung damage, the inflammation was reduced in response to L3 larvae of N. brasiliensis. [source] Mass spectrometric genomic data mining: Novel insights into bioenergetic pathways in Chlamydomonas reinhardtiiPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 23 2006Jens Allmer Abstract A new high-throughput computational strategy was established that improves genomic data mining from MS experiments. The MS/MS data were analyzed by the SEQUEST search algorithm and a combination of de novo amino acid sequencing in conjunction with an error-tolerant database search tool, operating on a 256 processor computer cluster. The error-tolerant search tool, previously established as GenomicPeptideFinder (GPF), enables detection of intron-split and/or alternatively spliced peptides from MS/MS data when deduced from genomic DNA. Isolated thylakoid membranes from the eukaryotic green alga Chlamydomonas reinhardtii were separated by 1-D SDS gel electrophoresis, protein bands were excised from the gel, digested in-gel with trypsin and analyzed by coupling nano-flow LC with MS/MS. The concerted action of SEQUEST and GPF allowed identification of 2622 distinct peptides. In total 448 peptides were identified by GPF analysis alone, including 98 intron-split peptides, resulting in the identification of novel proteins, improved annotation of gene models, and evidence of alternative splicing. [source] Isolation, Partial Purification, and Immunogenicity of Flagella from Tritrichomonas foetusTHE JOURNAL OF EUKARYOTIC MICROBIOLOGY, Issue 3 2005PHILIP F. JEMILOHUN Abstract. Tritrichomonas foetus, the agent of bovine trichomoniasis, is a flagellate protozoan responsible for substantial economic losses to the dairy and calf industries worldwide. As yet, there is no approved treatment nor is there a sensitive diagnostic method. All these problems suggest that immunization is the best control strategy. In view of this, we isolated and partially purified flagella of the parasite by vortex homogenization followed by low-speed differential centrifugation. The resulting enriched flagellar preparation termed "crude flagellar prep" was purified further by sucrose and percoll gradients. Microscopic analysis showed that the flagellar membrane was intact. Analysis by sodium dodecyl-sulfate polyacrylamide gel electrophoresis revealed three prominent protein bands of 42, 49, and >250 kDa, and several minor bands. Immunoblotting of flagellar and whole-cell extracts revealed many flagellar antigens. [source] Reissner's Fibre Proteins and p73 Variations in the Cerebrospinal Fluid and Subcommissural Organ of Hydrocephalic RatANATOMIA, HISTOLOGIA, EMBRYOLOGIA, Issue 4 2009E. M. Carmona-Calero Summary Reissner's fibre (RF) is formed by the polymerization of the glycoprotein secreted by the subcommissural organ (SCO). The SCO also secretes soluble glycoprotein into the cerebrospinal fluid (CSF); variations in RF and SCO have been reported in hydrocephalus. On the other hand, hydrocephalus and other brain alterations have been described in p73 mutant mice. The p73 belongs to the tumour suppressor p53 protein family and has two isoforms: the TAp73 with apoptotic activity and ,Np73 with anti-apoptotic function. Moreover, the TAp73 isoform is glycosylated and secreted into the CSF. In the present work, we analysed the variations in RF and p73 proteins in the CSF and SCO of spontaneously hydrocephalic rats. Brains from control rats and spontaneously hydrocephalic rats of 12 months of age were used. The SCO sections were immunohistochemically processed with anti-TAp73 and anti-Reissner fibre (AFRU). The spontaneous hydrocephalus presents a decrease in the AFRU immunoreactive material in the SCO and an absence of RF. The anti-TAp73 was also present, slightly decreased, in the hydrocephalic SCO. AFRU and p73 bands were also detected in the CSF by western blot and six AFRU and p73 protein bands of a similar molecular weight were found in the CSF of the control rats. The number of AFRU and p73 bands was lower in the hydrocephalic rats than in the control rats. In conclusion, hydrocephalus produces a decrease in the secretions of the SCO and an absence of RF and a decrease in p73 and RF proteins in the CSF. [source] A simple method for purifying the White Spot Syndrome Virus using ultrafiltrationAQUACULTURE RESEARCH, Issue 6 2009Martina Hilda Gracia-Valenzuela Abstract A very simple and efficient method was developed for isolating intact White Spot Syndrome Virus (WSSV) particles from infected Litopenaeus vannamei tissue. No density gradient centrifugation, ultracentrifugation or protease inhibitors were required for the purification of intact WSSV virions using microfilters (100 kDa cut-off) combined with several steps of conventional centrifugation procedures. A mortality assay was run using healthy shrimp to prove that the virions obtained were infective. The concentrated viral preparations were further studied using polyacrylamide gel electrophoresis (PAGE). At least five distinct protein bands were detected when intact purified WSSV virions were found by sodium dodecyl sulphate-PAGE, followed by Coomassie Brilliant R-250 staining. The estimated molecular weights of these proteins were 23, 24, 29, 32 and 42-kDa, which could correspond to viral protein. Using this method, the virus does not lose its ability to infect healthy shrimp. [source] Effect of immunostimulants on the haemolymph haemagglutinins of tiger shrimp Penaeus monodonAQUACULTURE RESEARCH, Issue 12 2008Roshan Pais Abstract The levels of haemagglutinins in Penaeus monodon, following administration of immunostimulants, ,-glucans and/or vibrio bacterin either orally or by immersion, were studied. The freshly drawn haemolymph was incubated with microbial materials like ,-glucans/vibrio bacterin and serum obtained after coagulation was administered to naïve animals. The immunostimulant treatments either via immersion, feeding or injection were found to cause an increase (P<0.006) in haemagglutination activity (HA) of the haemolymph against mouse erythrocytes. Injection of saline or heterologous haemolymph caused an increase in the HA, but injection of haemolymph serum obtained by clotting haemolymph in the presence of vibrio bacterin or glucan did not bring about an increase in HA. There was no change in the haemolymph protein profile of the groups receiving immunostimulants through immersion or feed. Two protein bands (27 and 30 kDa), which were present in the uninjected group, were found to be overexpressed in the haemolymph-injected groups. Three bands of 17, 21 and 23 kDa, which were absent in control or saline-injected groups, were present in all the haemolymph serum-injected groups. The study indicates that modulation of HA may partly account for the immunomodulatory activity of immunostimulants like ,-glucan or vibrio bacterins. [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] | |||||||||||||||||||