Polyacrylamide Gel Electrophoretic Analysis Research Articles

Component partitioning during microfiltration and diafiltration of whey protein concentrate in the production of whey protein isolate

This study investigated component partitioning during cold microfiltration (MF) and diafiltration (DF) of whey protein concentrate (WPC) feed in producing whey protein isolate (WPI). Significant differences (P < 0.05) were found in the partitioning of components. Specifically, 60.1, 50.2, 6.32, and 75.7% of total solids, protein, fat, and ash, respectively, were partitioned into the permeate stream. Phospholipids comprised 25 and 41.4% of total fat in WPC feed and DF retentate, respectively. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed MFGM-associated proteins were enriched in the MF retentate. The particle size in MF retentate was significantly larger (P < 0.05) than in the MF and DF permeate streams (450, 7.87, and 3.55 nm, respectively), providing evidence of the retention of aggregated whey proteins in the MF retentate. These findings provide an indepth understanding of component partitioning during cold MF of WPC feed, supporting the development of higher value-added ingredients.

Effect of Iron Restriction on the Whole Cell and Outer Membrane Proteins of Riemerella anatipestifer Isolates of Kerala

Globally, riemerellosis is caused by Riemerella anatipestifer, has a major concern, especially around the duck producing countries in variable degrees. A contemplate was led to access the homogeneity/heterogeneity between the Riemerella isolates (RA1 and RA2) by comparing the whole cell and outer membrane protein profiles in iron restricted and repleted condition. On Sodium dodecyl sulphate polyacrylamide gel electrophoretic analysis, both the isolates under iron deprived condition, commonly increased the production of outer membrane proteins, 51 and 75 kDa, among the five increased proteins in each. Also, among the increasingly produced eight and six whole cell proteins of RA1 and RA2, respectively, 38 and 51 kDa were present in both under iron depleted condition. The present study showed heterogeneity in the protein profile of Riemerella isolates, RA1 and RA2. Both the isolates, under iron restricted conditions, not only up and down regulate various whole cell and Omp proteins, but also few novel proteins had been produced in iron depleted environment.

Inducing Systemic Resistance against Cucumber Mosaic Cucumovirus using Streptomyces spp.

Seven Streptomyces spp. were screened for their potential to protect cucumber plants against Cucumber mosaic virus (CMV). Foliage treatment with the Streptomyces culture filtrates resulted in high reduction of the level of disease severity of CMV infection. In contrast, soil treatments were less effective in virus inhibition. Concerning foliage treatment, culture filtrates of S. griseorebens and S. cavourensis showed the highest inhibitory effect (90% and 85%, respectively) when applied 48 hrs prior to virus inoculation. On the other hand, soaking of cucumber seeds for 2hrs in the Streptomyces culture filtrates resulted in the highest viral inhibition. Generally, S. griseorebens recorded the highest percentage of viral inhibition (65%). Direct ELISA was carried-out as a diagnostic tool at the beginning and throughout this study. A number of de-novo synthesized proteins (induced proteins) were detected by polyacrylamide-gel electrophoretic analysis in cucumber leaves treated with culture filtrates of Streptomyces spp. relative to the control treatment. Considerable increase in total phenols as well as peroxidase and polyphenol oxidase activity levels in treated plants before virus inoculation were recorded comparing with the healthy and infected control plants. The highest values were observed in treated cucumber plants with S. griseorebens collected 14 days post virus inoculation.

Mutations in the mitochondrial cysteinyl-tRNA synthase gene, CARS2, lead to a severe epileptic encephalopathy and complex movement disorder

BackgroundMitochondrial disease is often suspected in cases of severe epileptic encephalopathy especially when a complex movement disorder, liver involvement and progressive developmental regression are present. Although mutations in either mitochondrial...

Membrane‐Spanning DNA Nanopores with Cytotoxic Effect

Membrane‐Spanning DNA Nanopores with Cytotoxic Effect

An RMND1 Mutation Causes Encephalopathy Associated with Multiple Oxidative Phosphorylation Complex Deficiencies and a Mitochondrial Translation Defect

Mutations in the genes composing the mitochondrial translation apparatus are an important cause of a heterogeneous group of oxidative phosphorylation (OXPHOS) disorders. We studied the index case in a consanguineous family in which two children presented with severe encephalopathy, lactic acidosis, and intractable seizures leading to an early fatal outcome. Blue native polyacrylamide gel electrophoretic (BN-PAGE) analysis showed assembly defects in all of the OXPHOS complexes with mtDNA-encoded structural subunits, and these defects were associated with a severe deficiency in mitochondrial translation. Immunoblot analysis showed reductions in the steady-state levels of several structural subunits of the mitochondrial ribosome. Whole-exome sequencing identified a homozygous missense mutation (c.1250G>A) in an uncharacterized gene, RMND1 (required for meiotic nuclear division 1). RMND1 localizes to mitochondria and behaves as an integral membrane protein. Retroviral expression of the wild-type RMND1 cDNA rescued the biochemical phenotype in subject cells, and siRNA-mediated knockdown of the protein recapitulated the defect. BN-PAGE, gel filtration, and mass spectrometry analyses showed that RMND1 forms a high-molecular-weight and most likely homopolymeric complex (∼240kDa) that does not assemble in subject fibroblasts but that is rescued by expression of RMND1 cDNA. The p.Arg417Gln substitution, predicted to bein a coiled-coil domain, which is juxtaposed to a transmembrane domain at the extreme C terminus of the protein, does not alter thesteady-state level of RMND1 but might prevent protein-protein interactions in this complex. Our results demonstrate that the RMND1 complex is necessary for mitochondrial translation, possibly by coordinating the assembly or maintenance of the mitochondrial ribosome.

Purification of C-Phycocyanin from Spirulina platensis by Single-Step Ion-Exchange Chromatography

C-Phycocyanin is a natural blue pigment with many commercial applications in foods, cosmetics, and medicines. In this paper we describe the extraction and purification of C-phycocyanin from the cyanobacterium Spirulina platensis. The procedure is based on adsorption of impurities with chitosan and activated charcoal then one-step ion-exchange chromatography. The dry algal powder was soaked in potassium phosphate buffer for 2 h to furnish crude phycocyanin extract of purity 0.93. The crude extract was then treated with chitosan and activated charcoal for 5 min, which increased the purity to 2.78. After chromatography on DEAE Sephadex A-25, the purity of phycocyanin was improved to 4.3. The identity of the purified phycocyanin was confirmed by sodium dodecyl sulfate–polyacrylamide gel electrophoretic analysis and by spectroscopic measurements (UV–visible spectrophotometry and spectrofluorimetry). Compared with conventional methods, this method was simple, inexpensive, and time-saving.

Mycoremediation of congo red dye by filamentous fungi.

Azo, anthroquinone and triphenylmethane dyes are the major classes of synthetic colourants, which are difficult to degrade and have received considerable attention. Congo red, a diazo dye, is considered as a xenobiotic compound, and is recalcitrant to biodegradative processes. Nevertheless, during the last few years it has been demonstrated that several fungi, under certain environmental conditions, are able to transfer azo dyes to non toxic products using laccases. The aim of this work was to study the factors influencing mycoremediation of Congo red. Several basidiomycetes and deuteromycetes species were tested for the decolourisation of Congo red (0.05 g/l) in a semi synthetic broth at static and shaking conditions. Poor decolourisation was observed when the dye acted as the sole source of nitrogen, whereas semi synthetic broth supplemented with fertilizer resulted in better decolourisation. Decolourisation of Congo red was checked in the presence of salts of heavy metals such as mercuric chloride, lead acetate and zinc sulphate. Decolourisation parameters such as temperature, pH, and rpm were optimized and the decolourisation obtained at optimized conditions varied between 29.25- 97.28% at static condition and 82.1- 100% at shaking condition. Sodium dodecyl sulphate polyacrylamide gel electrophoretic analysis revealed bands with molecular weights ranging between 66.5 to 71 kDa, a characteristic of the fungal laccases. High efficiency decolourisation of Congo red makes these fungal forms a promising choice in biological treatment of waste water containing Congo red.

English

This study reports the induction of somatic embryos in Mucuna pruriens. Different explants cultured on MS medium supplemented with 11.31 iM 2,4-D produced golden yellow embryogenic callus that induced synchronized embryo development on MS basal liquid medium. Organization of pre-embryonic mass was noticed 15 d after sub culturing the callus, which progressively developed to globular, heart, torpedo and cotyledonary shaped embryos. Attempts to germinate them did not succeed as the cotyledonary embryos turned characteristic red and subsequently brown before finally turning black and unresponsive. The reaction was consistent across different media constituents, hormonal concentrations and pH ranges. Supplementation of anti-oxidants was also ineffective. Polyacrylamide gel electrophoretic analysis of the brown and non-brown embryos revealed quantitative differences in protein contents between the two. More detailed study is necessary to establish the precise cause for embryo browning and ways of its regulation.

A new genetically determined plasma protein polymorphism in the laboratory rabbit.

Two-dimensional agarose (pH 5.4)-polyacrylamide (pH 9.0) gel electrophoretic analysis of rabbit plasma proteins, followed by protein staining with Coomassie Brilliant Blue G250, revealed genetic polymorphism of a new, unidentified protein in the post-albumin region. This protein was tentatively designated as post-albumin with locus symbol Pa. Family data supported the hypothesis that the three different Pa phenotypes observed were controlled by two codominant autosomal alleles at a single locus. Each of the Pa homozygous types showed one fraction while the Pa heterozygous type had a pair of fractions. The Pa protein did not show activity to be ceruloplasmin, esterase, hemopexin, haptoglobin or protease (trypsin and chymotrypsin) inhibitor and it was also not likely to be the vitamin D-binding globulin (Gc protein). Pa polymorphism was observed in both of the breeds (New Zealand White and White of Dendermonde) studied.

An OmpA Family Protein, a Target of the GinI/GinR Quorum-Sensing System in Gluconacetobacter intermedius , Controls Acetic Acid Fermentation

Via N-acylhomoserine lactones, the GinI/GinR quorum-sensing system in Gluconacetobacter intermedius NCI1051, a gram-negative acetic acid bacterium, represses acetic acid and gluconic acid fermentation. Two-dimensional polyacrylamide gel electrophoretic analysis of protein profiles of strain NCI1051 and ginI and ginR mutants identified a protein that was produced in response to the GinI/GinR regulatory system. Cloning and nucleotide sequencing of the gene encoding this protein revealed that it encoded an OmpA family protein, named GmpA. gmpA was a member of the gene cluster containing three adjacent homologous genes, gmpA to gmpC, the organization of which appeared to be unique to vinegar producers, including "Gluconacetobacter polyoxogenes." In addition, GmpA was unique among the OmpA family proteins in that its N-terminal membrane domain forming eight antiparallel transmembrane beta-strands contained an extra sequence in one of the surface-exposed loops. Transcriptional analysis showed that only gmpA of the three adjacent gmp genes was activated by the GinI/GinR quorum-sensing system. However, gmpA was not controlled directly by GinR but was controlled by an 89-amino-acid protein, GinA, a target of this quorum-sensing system. A gmpA mutant grew more rapidly in the presence of 2% (vol/vol) ethanol and accumulated acetic acid and gluconic acid in greater final yields than strain NCI1051. Thus, GmpA plays a role in repressing oxidative fermentation, including acetic acid fermentation, which is unique to acetic acid bacteria and allows ATP synthesis via ethanol oxidation. Consistent with the involvement of gmpA in oxidative fermentation, its transcription was also enhanced by ethanol and acetic acid.

Different expression patterns of intact forms of squamous cell carcinoma antigens between normal and malignant cervical squamous epithelial tissues: Nondenaturing polyacrylamide gel electrophoretic analysis

Squamous cell carcinoma antigen (SCCA), a 45-kDa tumor-associated serpin, mainly consists of two highly homologous molecules, SCCA1 and SCCA2, which possess unique proteinase inhibitory properties. Importantly, our previous study demonstrated that an intact structure of SCCAs, and not a cleaved form yielded by interacting with target proteinase, is essential for their function as a serpin. The aim of this study is therefore, to develop a simple method of analyzing expression patterns of intact forms of SCCAs (functional SCCAs) in cervical squamous epithelial tissues and to investigate whether there are any differences in the expression of intact forms of SCCAs between normal and malignant cervical squamous epithelial tissues. We used nondenaturing polyacrylamide gel electrophoresis (PAGE) with immunoblotting. The newly generated antibody, Pab Y2, recognizes only intact form of SCCAs, while the conventional antibody, Mab 27, reacts with the cleaved form of SCCA1 as well as intact forms of SCCAs. Nondenaturing PAGE using Pab Y2 showed that an intact form of SCCAs in the heat-treated tissue extract at 60 degrees C for 2 h was separated into at least five bands, termed as bands A-E from cathode to anode. By comparison with two-dimensional electrophoresis patterns of SCCAs, it was found that the first three bands, i.e. bands A-C, are derived from the intact form of SCCA1, while the other two bands, i.e. band D and E are from the intact form of SCCA2. Specifically, band E, but not band D, of SCCA2 is apparently increased in squamous cell carcinomas compared with normal squamous epithelium. In conclusion, this novel analytical approach will be useful for investigating the different expression patterns of functional SCCAs between normal and malignant cervical squamous epithelial tissues.

Characterization of heat-induced aggregates of globulin from common buckwheat ( Fagopyrum esculentum Moench)

Some physicochemical properties and the microstructure of heat-induced aggregates of globulin from common buckwheat ( Fagopyrum esculentum Moench) (BWG) formed at 100 °C in 0.01 M phosphate buffer containing 1.0 M NaCl, pH 7.4 were studied. Differential scanning calorimetric (DSC) analysis shows a re-distribution of native and extensively denatured proteins in the heat-induced aggregates of BWG, particularly in the ISA fraction. Sodium dodecyl sulfate polyacrylamide gel electrophoretic (SDS-PAGE) analysis suggests the occurrence of both dissociation and association of molecules and the involvement of intermolecular disulfide linkages during thermal aggregation. Transmission electron microscopy (TEM) reveals that native BWG appeared as uniform compact globules with diameters ranging between 11.7 and 12.5 nm. TEM examination of the buffer-soluble aggregates, fractionated by sucrose density gradient ultracentrifugation, demonstrates the formation of strand-like small aggregates and large compact globular soluble macroaggregates.

Chromosome 6U from Aegilops geniculata Roth Carrying Powdery Mildew Resistance in Bread Wheat

The production and isolation of a wheat—Aegilops geniculata disomic addition line and of monosomic substitution line resistant to powdery mildew at the seedling and adult plant stages are described. Cytological analysis revealed the presence of an acrocentric chromosome in the resistant derivatives. Polyacrylamide gel electrophoretic analysis indicated differences in the β-zone of the reduced by mercaptoethanol treatment seed protein pattern between the wheat parent cv. Trakya and the resistant derivatives. A component with a high staining intensity in the β-zone was assumed to be a marker for the chromosome 6 of Ae. geniculata. The isoenzyme activity at the Got-1 and Est-4 loci of the lines was lower than that of wheat. By analyzing the isoenzymes of the Amp-1 locus of the addition plants, a quantitative marker of Ae. geniculata 6U chromosome was identified. The progeny of the plants with the 2n = 43 and 2n = 42 chromosomes carrying the chromosome 6U added or mono-substituted for the wheat 6D chromosome segregated for powdery mildew resistance. The mono-and/or double conditions for this chromosome could be maintained by selecting resistant plants in the segregated populations.

Water-Soluble Complexes Formed by Natural Polyphenols and Bovine Serum Albumin: Evidence from Gel Electrophoresis

The formation of water-soluble complexes from combinations of various polyphenols and bovine serum albumin (BSA) was analyzed by gel electrophoresis. Sodium dodecylsulfate (SDS) and native polyacrylamide gel electrophoretic (PAGE) analyses clearly showed complexes formed from BSA with oenothein B, corilagin, (+)-catechin, procyanidin B3, and gallic acid derivatives; however, it was difficult to distinguish between the complexes containing a single BSA molecule and the BSA molecule itself by using size-exclusion chromatography (SEC). Combining SDS and native PAGE analyses showed instability in the macromolecular complexes formed from pentagalloylglucose (PGG) and BSA. Research also revealed that the oxidation of (-)-epigallocatechin gallate (EGCG) contributed to the formation of more stable macromolecular complexes from EGCG and BSA.

Biochemical characterization of resistance against Alternaria helianthi in cultivated and wild sunflowers

The biochemical basis of resistance to the leaf spot/blight pathogen A. helianthi was compared in six wild Helianthus species, possessing three ploidy levels (dlploid, telrapiold and hexaplotd) and different degrees of resistance to helianthi, and cultivated sunflower (H. annuus cv. CO-4 susceptible check) in terms of sugar, phenols and isozymes of peroxidase. polyhenol oxidase and chitinase. The resistant species of wild sunflowers (H. tuberosus. H. occidentalis) possessed higher levels of constitutive as well as induced total phenols and total sugars as compare with cultivated sunflower and susceptible wild sunflowers. Polyacrylamide gel electrophoretic (PAGE) isozyme analysis of defence-related enzymes showed a positive correlation of resistance with chitinase and polyphenol oxidase and a negative correlation with peroxidase.

Monoamine oxidase inhibitory activities of the scorpion Mesobuthus gibbosus (Buthidae) venom peptides

In the present study, crude venom of Mesobuthus gibbosus (Buthidae), a scorpion distributed all over Anatolia was isolated and purified by the Sephadex G-50 gel filtration and high pressure liquid chromatographic (HPLC) separation. Two of the five fractions (fractions 4 and 5) obtained from the Sephadex G-50 filtration and detected as lethal on mice and Musca domestica larvae in in vivo toxicity tests, were independently subjected to the HPLC separation. Only one of seven fractions (fraction 5.5*) obtained from the HPLC separation of the fraction 5 was found to be extremely lethal. Sodium dodecylsulfate polyacrylamide gel electrophoretic (SDS-PAGE) analysis of the crude venom and its chromatographic fractions demonstrated that crude venom consisted of peptides with molecular weights of 6500–210,000 Da. The neurotoxic fraction 5.5* appeared as a single band of 28,000 Da and two bands of 6200 and 22,000 Da in SDS-PAGE under non-reducing and reducing conditions, respectively, suggesting that it might consist of two chains attached by a disulfide bridge. Fractions 5 and 5.5* inhibited monoamine oxidase A (MAO-A) of rat liver reversibly and non-competitively, in a concentration-dependent manner. Fraction 5.5* appeared as a potent and specific MAO-A inhibitor with a K i value of 0.12 mg venom protein ml −1. The inhibitory effect of venom peptide 5.5* on MAO-A was found to be dependent on the preincubation time suggesting that the peptide binds to some site other than the substrate-binding site. Results of the present study demonstrated that M. gibbosus venom contains a peptide with specific MAO-A inhibitory activity which may be responsible for the anxiogenic effects of the scorpion venoms on animals and humans.

Purification of recombinant green fluorescent protein by three-phase partitioning

The technique of three-phase partitioning (TPP) was used to purify the green fluorescent protein (GFP) in a single step. TPP uses a combination of ammonium sulphate and tert-butanol to precipitate proteins from their crude extracts. In the first round of TPP with 20% ammonium sulphate saturation at the ratio of crude to tert-butanol 1:1 (v/v), most of the GFP remains in the lower aqueous phase. When subjected to a second round of TPP with 60% ammonium sulphate saturation at the ratio of crude to tert-butanol 1:2 (v/v) gives 78% recovery of GFP with a 20-fold purification. The sodium dodecyl sulphate–polyacrylamide gel electrophoretic (SDS–PAGE) analysis of purified preparation shows single band. The fluorescence excitation and emission spectra agreed with values reported in literature.

Electrophoretic analysis of genetic variability within Cryptosporidium parvum from imported and autochthonous cases of human cryptosporidiosis in the United Kingdom.

Cryptosporidium parvum oocyst DNA samples (n = 184) from humans with cryptosporidiosis contracted during foreign travel or during outbreaks in the United Kingdom were characterized genetically and categorized by single-strand conformation polymorphism (SSCP)-based analysis of the small-subunit gene (pSSU) (approximately 300 bp) and second internal transcribed spacer (pITS-2) (approximately 230 bp) of nuclear ribosomal DNA. The two recognized genotypes (types 1 and 2) of C. parvum could be readily differentiated by a distinct electrophoretic shift in the pSSU SSCP profile, associated with a nucleotide difference of approximately 1.3 to 1.7%. Of the 102 samples from cases contracted during foreign travel, 88 (86.3%) were identified as C. parvum type 1 and 14 (13.7%) were identified as type 2. For outbreak samples, unequivocal differentiation between type 1 (n = 20; one child nursery outbreak) and type 2 (n = 62; two waterborne outbreaks) was also achieved. Nucleotide variation in pITS-2 (both within and among samples representing each genotype) was substantially greater (10 to 13 different profiles for each genotype, relating to sequence differences of approximately 1 to 42%) than that in pSSU. SSCP analysis of pITS-2 for all samples revealed that some profiles had a broad geographical distribution whereas others were restricted to particular locations, suggesting a link between some subgenotypes and the geographical origin or source. Comparative denaturing polyacrylamide gel electrophoretic analysis revealed the same genotypic identification and a similar subgenotypic classification of samples as SSCP analysis. The findings of this study, particularly the detection of intragenotypic variation by SSCP, should have significant diagnostic implications for investigating transmission patterns and the monitoring of outbreaks.

The determination of flesh productivity and protein components of some fish species after hot smoking

AbstractChemical changes in pike perch (Sander lucioperca L Kottelat 1997), rainbow trout (Oncorhynchus mykiss WALBAUM 1792) and eel (Anguilla anguilla L 1766) after hot smoking were investigated. Some differences in water, protein, lipid, ash and carbohydrate contents of the fish were established as significant (P &lt; 0.01). The flesh productivity of fresh eel and pike perch was found to be very high. Differences in group averages of total microprotein content of fresh, smoked and stored eel, rainbow trout and pike perch were found to be significant (P &lt; 0.01). Using polyacrylamide gel electrophoretic analysis, 18 protein bands in rainbow trout, 12 bands in pike perch and 14 bands in eel were determined. Most of the bands disappeared after smoking and storage owing to denaturation of the fish proteins.© 2001 Society of Chemical Industry