SDS-PAGE analysis of whole cell protein and outer memrbane protein patterns of clinical isolates of Burkholderia pseudomallei

Objective:To investigate the banding patterns of whole cell protein(WCP) and outer membrane protein (OMP) of Burkholderia pseudomallei(B.pseudomallei) in clinical isolates from patients with melioidosis. Methods:WCP and OMP of of B.pseudomallei in 50 clinical isolates,from 47 patients with melioidosis were prepared and separated by polyacrylamide gel electrophoresis(SDS-PAGE) using 10%gels and stained with Coomassie brilliant blue.The banding patterns were compared by using a laser densitometer and dendrogram. Results:There were 6 different banding patterns of WCP and 2 types of OMP.Type 1 -5 WCP had 8 common protein bands at 19.0 - 45.0 kDa with identical OMP pattern.The banding patterns of WCP in type 6 were distinct from the others and also its OMP profile.The majority of clinical isolates(37/50,74%) were in type 1 WCP.Of the remaining isolates,8 were in type 2,2 in type 3,and one each was in type 4 to 6.There was no significant association between the WCP typing and the demographic or clinical features of the investigated patients.Conclusion:Despite the wide variation of clinical features of melioidosis,the results of this study show that B.pseudomallei had a few differences in the WCP and OMP profiles.Therefore typing of WCP and OMP,using SDS-PAGE analysis,could be an alternative method for phenotypic differentiation in clinical isolates of B.pseudomallei.

Comparison of Lipopolysaccharide and Protein Immunogens from Pathogenic Yersinia enterocolitica Bio-serotype 1B/O:8 and 2/O:9 using SDS-PAGE

Objective Yersinia enterocolitica is an extracellular pathogen and its related antigens interact with the host immune system. We investigated the difference in immunological characteristics between a highly pathogenic and poorly pathogenic strain of Y. enterocolitico. Methods We used SDS-PAGE and western blotting to characterize lipopolysaccharide （LPS）, Yersinio outer membrane proteins （Yops）, membrane proteins, and whole-cell proteins from poorly pathogenic Y. enterocolitico bio-serotype 2/0：9, isolated from China, and highly pathogenic bio-serotype 1B/O：8, isolated from Japan. Results These two strains of Y. enterocolitica had different LPS immune response patterns. Comparison of their Yops also showed differences that could have accounted for their differences in pathogenicity. The membrane and whole-cell proteins of both strains were similar; immunoblottting showed that the 35 kD and perhaps the 10 kD proteins were immunogens in both strains. Conclusion The major antigens of the two strains e and membrane proteins, as shown by comparing preparations. citing the host immune protein samples with response were the LPS reference and purified

Analysis of Genetic Variation of Seed Proteins in the Genus Vigna and among Its Relatives Cultivated in China

The genetic variation of seed proteins was assayed by SDSPAGE for 24 cultivars belonging to 5 species in Vigna and 7 species in its 7 relative genera cultivated in China. There were 48 polymorphic subunit bands discriminated from electrophoretic profiles. Two dendrograms were constructed by UPGMA cluster analyses using PHYLIP3.6 respectively. Variation among genera or species was larger than that among lower taxonomic categories level. Little variation among cuhivars of yardlong bean （Vigna sesquipedalis ） and small variation of lablab （ Lablab purpureus）, pea （Pisum sativum）, or sword bean （Canavalia gladiata）, but large variation of soybean or rice bean in their origin of China were all revealed. The seed proteins profiles of traditionally regarded as typical species in Vigna such as yardlong bean, rice bean and small bean were more similar than mungbean （Vigna radiata） and black gram （Vigna mungo） were. Mungbean and black gram had distinct seed proteins pattern, they should be of two species.

Two-dimensional Electrophoresis Analysis of Proteins Extracted from Alexandrium sp. LC3

Two-dimensional electrophoresis（2-DE） of protein extracted and purified from Alexandrium sp. LC3 was conducted. In the SDS-PAGE study, the relative molecular weights of the proteins were mainly in the range of 14 kDa-31 kDa and 43 kDa-66 kDa, and more proteins were detected between 14kDa and 31 kDa. With the improved protein preparation, the two-dimensional electrophoresis patterns indicated that the relative molecular weights of the proteins were between 14kDa and 100kDa, and most of them ranged from 14 kDa to 31 kDa. This was consistent with the result of the SDS-PAGE analysis. The isoelectric points were found to lie between 3.0 and 8.0, and most of them were in the range of 3.0-6.0. Better separation effect was acquired with pre-prepared immobilized gradient （IPG） strip （pH 3-5.6）, and about 320 protein spots could be visualized on the 2-DE map by staining. Within pH 3-l0 and pH 3-5.6 strips, the protein samples of Alexandrium sp. LC3 could be separated well.

Analysis of Protein Content and Genetic Diversity in Pea Germplasm in Tibet

To grasp protein content and composition of pea resource in Tibet Plateau,54 pea materials from different eco-geographical environments of Tibet were collected and arranged in this paper.Based on SDS-PAGE,electrophoresis and genetic diversity analysis of water-solubility and salt-solubility proteins from 54 pea materials were conducted,and the relationship between geographical ecological factors(longitude,latitude and altitude) and total protein content was studied.The research results showed that total protein contents of 54 pea materials were between17.58% and 28.67%,in which water-solubility protein accounted for 86.12%-91.40%,while salt-solubility protein accounted for 4.76-8.29%.Total protein content of Tibet pea showed significantly positive correlation with longitude,certain positive correlation with latitude and certain negative correlation with altitude.SDS-PAGE of water-solubility and salt-solubility proteins from 54 pea materials respectively detected 1588 and 699 protein bands.Based on different mobility ratios,there were 43 kinds of water-solubility protein bands,and diversity index was between 0 and 0.5.Its relative molecular weight was between 24.87 and 149.54 ku,showing the low molecular weight region of24.71-50.41 ku and high molecular weight region of 56.34-88.08 ku.There were 24 salt-solubility protein bands based on different mobility ratios,with the diversity index of 0-0.5,and relative molecular weight was between 24.85 and 91.24 ku.According to the altitude,54 pea resources were divided into 4 geographical groups.Gene diversity indexes of each group were respectively 0.23,0.18,0.35 and 0.31,and Shannon information indexes were respectively 0.33,0.41,0.52 and 0.46.It showed that the variation of pea protein was related to altitude.In clustering analysis,the tested resources were divided into seven classes,showing that water-solubility and salt-solubility proteins could reflect genetic relationship among germplasm resources at certain degree.The research could provide theoretical basis for the development of Tibet pea resources and selection of good parents.

Expression of 87 kD protein in the broth culture filtrate of Helicobacter pylori and its association with the vacuolating effect

Objective:To studythevacuolatingeffectof Helicobacter pylori(H.pylori).Method:Thevacuolatingef-fectanditsrelationshipwithvacuolatingcytotoxinantigen(an87kDprotein)wereinvestigatedby themethodof cy-totoxictest,SDS-PAGEandscanning.Result:Of the62clinicalisolates,43strainswereH.pylori(Toxin+)with vacuolatingeffect,whiletheotherswereH.pylori(Toxin-)withoutvacuolatingeffect.Altogether78.26%(36/46)patientswithpepticulcerwereinfectedwithH.pylori(Toxin+)strains,andonly42.86%(6/14)who hadgastritiswere infectedwithH.pylori(Toxin+)strains,withsignificantdifferencebetweenthem(χ 2 =4.83,P<0.05).A proteinwith relativemolecularmassof87kD was identifiedin thebrothculturefilter(BCF)of30.23%H.pylori(Toxin+)strains(13/43)butinnoneof thatof H.pylori(Toxin-)strains,andthedifferencewas statisticallysignificant(P<0.05).There was a significantandconcordantrelationshipbetweentheOD valueof theproteinbandandthetiterof vacuolating activityof H.pylori(Toxin+)(r=0.67andP<0.05by linearregressionanalysis).Conclusion:H.pylori(Toxin+)were moreoftenassociatedwithpepticulcerousdiseasesthanwithgastritisdiseases.Thevacuolatingeffectof H.pylori(Toxin+)maybe causedby the87kDprotein.

Effect of Low Temperature Stress on Protein Content of Toona Sinensis Seedlings and Expression of Specific Protein

[Objective] The paper was to study the changes of protein content and component in leaves of Toona Sinensis under low-temperature stress.[Method] Potted experiment was adopted,and SDS-PAGE electrophoresis was also used to analyze the dynamic changes of protein in T.Sinensis leaves.[Result] Low temperature stress could change the content and component in leaves of T.Sinensis,but different provenances had different performance under different temperatures,so did the same provenance under different stress periods.SDS-PAGE electrophoresis results indicated that the change law of provenances of Xixia of Henan and Nanjing of Jiangsu with stronger cold resistance was similar,showing the change trend of "increase-decrease-increase".Protein was greatly expressed after stress for 1 d,the color of band became darker;the content gradually decreased in the following second and third day,and the color of bands was lighter;the content began to increase at the forth day.Two provenances induced the specific proteins with molecular weights of 27.6 and 22.5 kD,respectively.The soluble protein content of provenance of Xiapu of Fujian with relatively weak cold resistance was gradually increased,but no new protein bands were induced.The changes of protein band color of various provenances in SDS-PAGE electrophoresis was basically consistent with the changes of protein content,the provenance with stronger cold resistance could induce the production of specific proteins.[Conclusion] The study provided theoretical basis for under the molecular mechanism of plant cold resistance,which had great significance in theory and practice.

Comparative studies on seed protein characteristics in eight lines of two Gossypium species

Background:In order to achieve the targets aiming at the improvement of protein quality,knowledge regarding seed protein fractions and polypeptides constituting them in different crops is essential.Besides having high nutritional value as animal feed and human food,the protein isolates from cottonseed meal have also been proven promising as industrial raw materials for a number of applications.As far as Indian work on the characterization of cotton seed proteins is concerned,relatively meagre reports are available.Keeping in mind the importance of cotton seed proteins,lines belonging to Gossypium arboreum L.(Indian cotton)and G.hirsutum L.(American cotton)which are grown in all the major cotton growing states in India were selected for analysing their seed protein characteristics.Results:Whereas G.arboreum(A-genome)lines revealed a lower range of seed protein content i.e.19.5-24.3%,an upper range(21.8~29.5%)could be observed in lines of G.hirsutum(AD-genome).Globulins represented dominating fraction in both species followed by albumins,glutelins and prolamins.A significant positive correlation between albumins/globulins and seed protein content in G.arboreum/G.hirsutum,respectively,was observed.Intraspecific electrophoretic variation in seed protein extracts was observed in the region of molecular weight 22 kDa-27 kDa in lines of both the species;however some lines with A-genome showed similarity in banding pattern with AD-genome.Four polypeptides with disulphide-linkages were also reported for the first time.Albumins were observed to reveal more variations in their electrophoretic pat-tern between the lines of two species followed by globulins.Conclusion:On the basis of present and previous studies,screening the lines with low or high protein content will lead the selection of lines with superior polypeptide fraction important for nutritional and industrial purposes.On comparing the composition and behaviour of four 2-S linked polypeptides with other plant groups,these were suggested to be legumin-like in nature.The similarity in banding patterns between the lines of A-genome and AD-genome species marked towards the close evolutionary relationship between these two.Albumin fractions on the basis of our results could be taken for cultivar differentiation in cotton crop.

Protein Kinase C Enhances the Swelling-Induced Chloride Current in Human Atrial Myocytes

Swelling-activated chloride currents（ICl.swell） are thought to play a role in several physiologic and pathophysiologic processes and thus represent a target for therapeutic approaches. However, the mechanism of ICl.swell regulation remains unclear. In this study, we used the whole-cell patch-clamp technique to examine the role of protein kinase C（PKC） in the regulation of ICl.swell in human atrial myocytes. Atrial myocytes were isolated from the right atrial appendages of patients undergoing coronary artery bypass and enzymatically dissociated. ICl.swell was evoked in hypotonic solution and recorded using the whole-cell patch-clamp technique. The PKC agonist phorbol dibutyrate（PDBu） enhanced ICl.swellin a concentration-dependent manner, which was reversed in isotonic solution and by a chloride current inhibitor, 9-anthracenecarboxylicacid. Furthermore, the PKC inhibitor bis-indolylmaleimide attenuated the effect and 4α-PDBu, an inactive PDBu analog, had no effect on ICl.swell. These results, obtained using the whole-cell patch-clamp technique, demonstrate the ability of PKC to activate ICl,swell in human atrial myocytes. This observation was consistent with a previous study using a single-channel patch-clamp technique, but differed from some findings in other species.

A Simple Procedure for Extraction of Surface Protein of Salmonella Serotypes and Escherichia coli Strains Isolated from Poultry and Pigs

Salmonella and E.coli possess different surface protein structures that can induce protective immune responses.Identification of these proteins capacitates development of diverse applications in prevention and diagnosis that contribute to effectively control disease-causing enterobacteria pathogens such as Salmonella and E.coli.A simple procedure for obtaining protein complexes of Salmonella serotypes and E.coli is performed in this study.A sonication process with heat treatment of whole bacteria induced the release of protein complexes.Concentration of the protein extract was quantified using protein quantification Kits-Rapid,and protein complex profile was obtained by SDS-PAGE(Sodium dodecyl sulfate polyacrylamide gel electrophoresis)and silver staining.The concentrations of protein ranged from 29.45 to 45.35μg/mL in the Salmonella protein extracts,and from 25.35 to 36.72μg/mL in the E.coli protein extracts.Six major groups of proteins from E.coli(YfiO,NipB,OmpF,YfgL,Talc,YaeT)and four major groups of proteins from Salmonella(Flagellin,OmpA,Porin,SEF21)were preliminarily determined by a simple procedure of extraction based on the molecular weight.

Coconut Press Cake Alkaline Extract—Protein Solubility and Emulsification Properties

The solubility and the emulsification properties of a crude freeze dried alkaline protein extract (APE), 30% protein, obtained from coconut milk press cake by one step extraction at pH 11, were characterized at pH 2 to 11, and the cream and subnatant fractions of the emulsion studied by SDS-PAGE electrophoresis. The protein solubility followed U profile, showing a minimum at pH 3 to 4, close to but not identical to reported iso-electric points of 4 - 5 for many coconut protein fractions. The extract showed good capacity to form oil-in-water emulsion outside the low solubility pH range. The bands that appeared to play a role in the emulsification were found at 32 and 42 kDa in SDS-PAGE electrophoresis, but the most predominant absorbed band was at 23 kDa.

幽门螺杆菌外膜蛋白基因omp22克隆、表达和鉴定

目的:克隆幽门螺杆菌外膜蛋白基因omp22,构建幽门螺杆菌omp22基因与麦芽糖结合蛋白基因融合表达载体,并进行诱导表达,鉴定融合蛋白免疫原性,为幽门螺杆菌疫苗研究提供依据。方法:利用PCR技术从Helicobacter pylori郑州分离株MEL-Hp27染色体DNA上扩增omp22基因,并将其克隆到表达载体pMAL-c2X中,转化大肠杆菌(E.coli TB1),用IPTG诱导目的基因表达,SDS-PAGE方法对表达产物进行分析,Western blot鉴定其免疫原性。结果:PCR方法扩增的omp22基因长度约为540 bp。经酶切鉴定,插入到表达载体的基因片段与预期目的DNA片段相一致;SDS-PAGE结果显示表达产物分子量约为22 ku,融合蛋白的表达量约占全菌总蛋白的26%。结论:成功克隆并构建了omp22基因高效原核表达系统,为幽门螺杆菌基因工程组分疫苗的研究奠定基础。

Construction of WCB-11:A novel phiYFP arsenic-resistant whole-cell biosensor

The prediction and assessment of environmental pollution by arsenic are important preconditions of advocating environmental protection and human health risk assessment. A yellow fluorescent protein-based whole-cell biosensor for the detection of arsenite and arsenate was constructed and tested. An arsenic-resistant promoter and the regulatory gene arsR were obtained by PCR from the genome ofEscherichia coli DH5ct, andphiYFP was introduced into E. coli DH5ct as a reporter gene to construct an arsenic-resistant whole-cell biosensor （WCB-11） in which phiYFP was expressed well for the first time. Experimental results demonstrated that the biosensor has a good response to arsenic and the expression ofphiYFP. When strain WCB-11 was exposed to As^3＋ and As^5＋, the expression of yellow fluorescence was time-dependent and dose-dependent. This engineered construct is expected to become established as an inexpensive and convenient method for the detection of arsenic in the field.

Antimicrobial activities of the tissue extracts of Babylonia spirata Linnaeus,1758 (Mollusca:Gastropoda) from Thazhanguda,southeast coast of India

Objective:To investigate the antimicrobial activity of the tissue extracts of Babylonia spirata(B.spirala)against nine bacterial and three fungal patliogeiis.Methods:Crude extract of gastropod was tested for inhibition of bacterial and fungal growth.Antibacterial assay was carried out by disc diffusion method and in vitro antifungal activity was determined against Czapex Dox agar.The antimicrobial activity was measured accordingly based on the inhibition zone around the disc impregnated with gastropod extract.Molecular size of muscle protein was determined using sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE).And fourier transform infrared spectroscopy(FT1R)spectro photometry analysis was also studied.Results:The maximum inhibition zone(12 mm)was observed against Pseudomonas aeruginosa in the crude ethanol extract of B.spirata and the minimum inhibition zone(2 mm)was noticed against Staphylococcus aureus in the crude methanol extract of B.spirala.Water extract of B.spirala showed the highest activity against Vibrio parahaemolyticus,Staphylococcus aureus and Candida albicans.Ethanol,acetone,methanol,chloroform and water extracts showed antimicrobial activity against almost all the bacteria and fungus.Compared with water extracts,ethanol and methanol extracts showed higher activity against all pathogens.The molecular weight of protein of the gastropod sample ranged from 2-110 kDa on SDS-PAGE.FTIR analysis revealed the presence of bioactive compounds signals at different ranges.Conclusions:The research shows that the great medicinal value of the gastropod muscle of B.spirata may be due to high quality of antimicrobial compounds.

Root Extract of Polygonum Hydropiper Alters the Expression of Rat Uterine Protein Profile in Presence and Absence of Ovary in-situ during Periimplantation Period:An Evidence on SDS-PAGE

Objective To study its effects on reproductive performance in albino rats.Methods The chromatographic fraction （CF） of crude methanolic extract of the herb has been subcutaneously administered to female albino rats. Experiments were carried out in adult cyclic females and oavriectomised （OVX） females during early gestation period. Uterine horns were collected following the respective treatment regimen to stud）, the protein profile in 15% gel SDS-PAGE.Results The CF induced changes in the expression of protein in rat uterus. New proteins have been expressed in uterus of adult cyclic females having ovary in-situ. The OVX females treated with CF showed altered uterine protein profile compared with that of OVX control and OVX estradiol-17β （E2） treated rats. The CF exerted its effect on expression of uterine protein during early gestation period in rats. While uterine proteins of CF treated females were similar to that of controls during preimplantation period; many of the proteins on day 6 of gestation have been found either missing or expressed in lesser intensity.Conclusion The root of Polygonum hydropiper contains potential compound（s） which can alter the reproductive performance of female rats modulating uterine protein expression.

SDS-PAGE PROTEIN PATTERN AND ITS ANTIGENICITY ANALYSIS OF DIFFERENT ISOLATES OF SCHISTOSOMA JAPONICUM IN CHINA

Homogenates prepared from S. japonicum adult worms ofdifferent isolates from Anhui, Hubei, Guangxi, Yunnan andSichuan Provinces were analyzed by SDS-PAGE and enzymelinked immunoelectrotransfer blot (EITB) tested with rabbitanti-snails antibody. The results of SDS-PAGE indicated thatwith silver staining both male and female worms of Guangxiisolate showed some definite differences in their protein profile,namely, absence of one band between 50-75 kDa in maleworms and marked reduction in quantity of > 110 and 30 kDabands in female worms. There was no obvious differenceamong other isolates both in male and female worms. TheEITB patterns were similar in S. japonicum of Anhui andHubei, and it was also the case with isolates from Yunnan andSichuau, except that Yuunan female worms had a distinct band

Analysis of Two-Dimensional Gel Electrophoresis Map of Methicillin-Resistant <i>Staphylococcus aureus</i>Treated with Acetone Extract from <i>Canarium odontophyllum</i>Miq. Leaves

Methicillin-resistant Staphylococcus aureus (MRSA) infection is a global health concern that has caused severe health threats over the past decade. Leaves extract of C. odontophyllum has been proven previously as an anti MRSA agent. Proteomics provide a technique that used to analyze the differential of protein expression profile between untreated and treated MRSA with subinhibitory concentrations of acetone extract from C. odontophyllum leaves. This study aims to determine the optimum parameter for analysis of protein expression profile using two-dimension gels electrophoresis (2-DE) for MRSA protein after treatment with acetone extract from C. odontophyllum leaves. Comparison of the Protein Expression Profile (PEP) between the untreated and treated MRSA was analyzed using PDQuest software. The optimum condition for MRSA protein treated with acetone extract from C. odontophyllum leaves to produce the best resolution with greater spot distribution was as follows: 100 μg volume of MRSA protein that loaded after passive rehydration then was run until reaching 25 kVrhs during IEF using 17 cm IPG strip within ranges of pH 4 - 7. Analysis of protein expression from the 2-DE gel map shows that 9 protein spots up-regulated and 41 protein spots were down-regulated with more than 2-fold differences (p < 0.05). This preliminary study on the PEP of MRSA treated with acetone extract of C. odontophyllum leave may provide an insight into the antimicrobial mechanism, which could lead to the identification of target protein for future novel therapeutic development against MRSA infections.

Effects of extrusion conditions on the physicochemical properties of soy protein/gluten composite

Soybean protein-gluten blend was extruded using a co-rotating twin-screw extruder.Effects of different extrusion conditions on the textural properties of extrudates were analyzed by central composite design through the evaluation of texturization index,water holding capacity,and hardness of extruded protein products.Extrusion process variables including feed moisture content(40%-60%),screw speed(12-20 Hz),extrusion temperature(120℃-180℃),and gluten content(16%-32%)were studied by the nonlinear regression equations analysis.The extrusion process was also optimized using response surface methodology(RSM).The influence of moisture content on the hardness of extrudate was the most significant.The optimized results,including feed moisture content,extrusion temperature,screw speed and gluten content were 49.18%-50.15%,155.24℃-159.86℃,16.41-16.73 Hz,20.00%-20.03%,respectively.The microstructures of TISP products were analyzed by scanning electron microscopy(SEM)and the changes of protein molecules before and after extrusion were also analyzed by SDS-PAGE electrophoresis.

Extraction and Characterization of Myofibrillar Proteins from Different Meat Sources:A Comparative Study

In the present study,myofibrillar proteins were extracted from the meat proteins of beef,lamb,chicken,tuna and emperor fish using non-denaturation method,and their physico-chemical and rheological properties were assessed.The myofibrillar proteins of beef,emperor and lamb samples had higher percentage of protein extractability than tuna and chicken samples.The tuna sample showed significantly higher bound bromophenol blue(BPB)value while lamb samples showed lower value(P<0.05).The myofibrillar protein of chicken sample was found to have more ionic and hydrogen bonds than all other myofibrillar samples.The disulphide bonds in tuna and lamb myofibrillar protein samples were significantly higher than other three samples(P<0.05).The myofibrillar protein samples showed major bands myosin heavy chain,α-actinin,desimin,actin,troponin,tropomyosin and myosin light chain with wider molecular weight distribution in the range of 20-200 ku.The myofibrillar proteins exhibited Newtonian and shear thickening nature behaviour at lower protein concentration(1 mg/mL)as revealed by flow profile and visco-elastic analysis using rheometer.