Electrophoretic Protein Profiles Research Articles

Changes of Electrophoretic Protein Profiles of Smoked and Marinated Rainbow Trout (Oncorhynchus mykiss) During Refrigerated Storage

In this study, we aimed to determine the changes of electrophoretic protein profiles of smoked and marinated rainbow trout (Oncorhynchus mykiss) during refrigerated storage. Changes in muscle proteins during 9 weeks refrigerated storage of raw, smoked and marinated trout samples have been examined using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE and densitometric analysis revealed that intensity and the number of some protein bands were reduced while the bands of actin didn’t change significantly during marination and smoking of rainbow trout muscle. Myosin heavy chain (MHC) band with molecular weight of 205 kDa disappeared in the 9th week of smoking and marinating process. Subsequently, there were significant weekly changes in the protein percentages of samples caused by smoking and marinating processes (P<0.01). Protein percentages of the raw, smoked and marinated samples were determined as 18.34-18.82%, 23.83-24.70% and 16.29-17.76%, respectively

Changes of electrophoretic protein profiles of smoked and marinated rainbow trout (Oncorhynchus mykiss) during refrigerated storage

In this study, we aimed to determine the changes of electrophoretic protein profiles of smoked and marinated rainbow trout (Oncorhynchus mykiss) during refrigerated storage. Changes in muscle proteins during 9 weeks refrigerated storage of raw, smoked and marinated trout samples have been examined using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE and densitometric analysis revealed that intensity and the number of some protein bands were reduced while the bands of actin didn’t change significantly during marination and smoking of rainbow trout muscle. Myosin heavy chain (MHC) band with molecular weight of 205 kDa disappeared in the 9th week of smoking and marinating process. Subsequently, there were significant weekly changes in the protein percentages of samples caused by smoking and marinating processes (P<0.01). Protein percentages of the raw, smoked and marinated samples were determined as 18.34-18.82%, 23.83-24.70% and 16.29-17.76%, respectively

Haematological values of three Antarctic penguins: gentoo (Pygoscelis papua), Adélie (P. adeliae) and chinstrap (P. antarcticus)

It is established that haematological and biochemical parameters provide important data to assess the physiological condition and health status of wild birds. To undertake conservation physiology or ecophysiology work, it is therefore essential to establish baseline physiological parameters and how these parameters change with age and life history events. In this work, we determined and compared baseline haematology and serum biochemistry between adults and chicks of three Antarctic penguin species of the genus Pygoscelis : gentoo ( P. papua ), Adelie ( P. adeliae ) and chinstrap ( P. antarcticus ). Differences in adults among species were observed in haemoglobin and biochemical parameters such as total proteins, glucose and alkaline phosphatase activity. In addition, differences between adults and chicks in haematocrit, haemoglobin, total proteins and glucose concentration were determined. Moreover, we evaluated the electrophoretic protein profiles between adults and chicks of the genus Pygoscelis , and a conserved protein pattern was observed among species and ages in the genus. Altogether, the results suggest that biochemical and haematological differences among pygoscelids may be related to the nutritional status and energetic expenditure during breeding as well as their feeding habits and development stage. Keywords : Antarctic; haematology; physiology; Pygoscelis ; penguins; serum biochemistry. (Published: 15 June 2015) Citation: Polar Research 2015, 34 , 25718, http://dx.doi.org/10.3402/polar.v34.25718

Preparation andIn VitroRelease of Drug-Loaded Microparticles for Oral Delivery Using Wholegrain Sorghum Kafirin Protein

Kafirin microparticles have been proposed as an oral nutraceutical and drug delivery system. This study investigates microparticles formed with kafirin extracted from white and raw versus cooked red sorghum grains as an oral delivery system. Targeted delivery to the colon would be beneficial for medication such as prednisolone, which is used in the management of inflammatory bowel disease. Therefore, prednisolone was loaded into microparticles of kafirin from the different sources using phase separation. Differences were observed in the protein content,in vitroprotein digestibility, and protein electrophoretic profile of the various sources of sorghum grains, kafirin extracts, and kafirin microparticles. For all of the formulations, the majority of the loaded prednisolone was not released inin vitroconditions simulating the upper gastrointestinal tract, indicating that most of the encapsulated drug could reach the target area of the lower gastrointestinal tract. This suggests that these kafirin microparticles may have potential as a colon-targeted nutraceutical and drug delivery system.

Crystalline protein profiling and cry gene detection in Bacillus thuringiensis strains isolated during epizootics in Cydia pomonella L.

Abstract The composition of Bacillus thuringiensis crystalline inclusions was characterized in 18 strains: 12 isolates were obtained from the intestinal tract of Cydia pomonella larvae during epizootics, 2 isolates were cultured from Leucoma salicis larvae taken from their natural populations, and 4 reference strains. The number and molecular mass of B. thuringiensis crystalline proteins (Cry and Cyt) was estimated by the sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The crystals contained 1-8 proteins with molecular masses of 36-155 kDa. The toxin profiles differed both quantatively and qualitatively. The B. thuringiensis MPU B9 isolate had the highest number and diversity of Cry toxins. The analysis of crystal composition by SDS-PAGE was insufficient to detect groups and subgroups of Cry proteins. We identified 20 groups and 3 subgroups of Cry and Cyt crystalline toxins. Only one epizootic strain harboured cry25. In single reference strains, the cry1H, cry10 and cry25 genes were found. We did not find any correlation between the occurrence of cry genes and electrophoretic protein profiles of crystalline toxins.

PLASMA ELECTROPHORETIC PROFILES AND HEMOGLOBIN BINDING PROTEIN REFERENCE INTERVALS IN THE EASTERN BOX TURTLE (TERRAPENE CAROLINA CAROLINA) AND INFLUENCES OF AGE, SEX, SEASON, AND LOCATION

Evaluation of plasma electrophoretic profiles and acute phase protein concentrations may play a valuable role in health assessment of reptiles; however, little is known about reference intervals in free-ranging eastern box turtles (Terrapene carolina carolina). The purpose of this study was to establish reference intervals of protein electrophoretic profiles and hemoglobin binding protein ([HBP] as determined by a haptoglobin assay) in free-ranging eastern box turtles and to assess any possible correlations between varying age class (adults vs. juvenile), sex (male, female, or unknown), season (spring, summer, or fall), or location (Tennessee vs. Illinois). Blood samples were obtained from 324 eastern box turtles from 2010 to 2012 at three sites in Illinois and one site in Tennessee, USA. Significant differences were observed with total protein (sex, season, state, Illinois location), albumin (age class, season, state, Illinois location), α-1 globulins (sex, season, Illinois location), α-2 globulins (sex, season, state, Illinois location), β globulins (age class, sex, season, state, Illinois location), γ globulins (sex, season state, Illinois location), and hemoglobin binding protein (age class, sex, state, Illinois location). The use of electrophoretic profiles and acute phase proteins is a relatively new concept in reptilian medicine, and this study allowed for establishment of references intervals in the eastern box turtle and emphasized differences that occured based on age, sex, season, and location. Future research in this area can now build on these data to determine changes in population health over time or alterations due to specific environmental or disease threats.

Use of an enzyme-assisted method to improve protein extraction from olive leaves

The improvement of protein extraction from olive leaves using an enzyme-assisted protocol has been investigated. Using a cellulase enzyme (Celluclast® 1.5L), different parameters that affect the extraction process, such as the influence and amount of organic solvent, enzyme amount, pH and extraction temperature and time, were optimised. The influence of these factors was examined using the standard Bradford assay and the extracted proteins were characterised by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE). The optimum extraction parameters were: 30% acetonitrile, 5% (v/v) Celluclast® 1.5L at pH 5.0 and 55°C for 15min. Under these conditions, several protein extracts from olive leaves of different genetic variety (with a total protein amount comprised between 1.87 and 6.64mgg−1) were analysed and compared by SDS–PAGE, showing differences in their electrophoretic protein profiles. The developed enzyme-assisted extraction method has shown a faster extraction, higher recovery and reduced solvent usage with respect to the use of the non-enzymatic methods described in literature.

Physicochemical and functional properties of rapeseed protein isolate: influence of antinutrient removal with acidified organic solvents from rapeseed meal.

The presence of antinutritional constituents in rapeseed protein products (RPI), such as polyphenols, phytates, allyl isothiocyanates, and glucosinolates, is a formidable constraint. The effect of antinutrient removal from rapeseed meal with an organic solvent mixture (methanol/acetone, 1:1 v/v, combined with an acid (hydrochloric, acetic, perchloric, trichloroacetic, phosphoric)) on the physicochemical and functional properties of RPI was investigated. The extraction resulted in a substantial reduction of antinutrients from RPI, especially polyphenols and phytates, with concomitant decreases in protein yield and solubility. Treatment harbored significant improvement in the degree of whiteness, which was highest in the perchloric acid case. Surface hydrophobicity and free sulfhydryl group of RPI changed considerably, with perchloric acid-treated samples showing higher values, whereas the disulfide content remarkably increased in trichloroacetic acid- and phosphoric acid-treated samples, signifying aggregation. Intrinsic emission fluorescence and FTIR spectra showed significant changes in proteins' tertiary and secondary conformations, and the changes were more pronounced in samples treated with higher concentrations of acids. No appreciable alteration appeared among the electrophoretic profiles of proteins from pristine meal and those treated with lower levels of acids. Interfacial surface properties of proteins were variably improved by the solvent extraction, whereas the converse was true for their extent of denaturation. The results suggest that the physicochemical and conformational properties of RPI are closely related to its functional properties.

Humoral immune response in Oreochromis niloticus: modified direct agglutination test

Understanding the functional mechanisms of the humoral and cellular immune responses among teleost broadens the possibilities for intervention and improvement of vaccination methods. The aim of this study was to evaluate the dynamics of the humoral immune response, hematological biochemical changes, and electrophoretic profile of proteins in Oreochromis niloticus, when submitted to antigen stimulation using sheep erythrocytes in a single dose and with booster doses. Seventy O. niloticus were distributed in two groups (n = 35): one received a single dose and the other received two doses of antigen. Evaluations were made at the following predetermined times: zero (pre-immune, i.e., before inoculation with the first injection of sheep erythrocytes, negative control) and on 7th, 14th, 21st, and 28th days after the first immunization to evaluate the hemogram, biochemistry, and electrophoresis of proteins and direct hemagglutination. Twenty-eight days after antigen inoculation, it was observed that there had been an increase in the serum antibody titer and fractions gamma globulins after second antigen stimulation. The results suggest that teleost fish have the capacity to produce antibodies from a single stimulation, and a second antibody production wave after second stimulation.

Brevetoxin exposure, superoxide dismutase activity and plasma protein electrophoretic profiles in wild-caught Kemp's ridley sea turtles (Lepidochelys kempii) in southwest Florida

Because of their vulnerable population status, assessing exposure levels and impacts of toxins on the health status of Gulf of Mexico marine turtle populations is critical. From 2011 to 2013, two large blooms of the red tide dinoflagellate, Karenia brevis, occurred along the west coast of Florida USA (from October 2011 to January 2012 and October 2012 to April 2013). Other than recovery of stranded individuals, it is unknown how harmful algal blooms affected the Kemp's ridley sea turtles (Lepidochelys kempii) inhabiting the affected coastal waters. It is essential to gather information regarding brevetoxin exposure in these turtles to determine if it poses a threat to marine turtle health and survival. From April 2012 to May 2013, we collected blood from 13 immature Kemp's ridley turtles captured in the Pine Island Sound region of the Charlotte Harbor estuary. Nine turtles were sampled immediately after or during the red tide events (bloom group) while four turtles were sampled between the events (non-bloom group). Plasma was analyzed for total brevetoxins (reported as ng PbTx-3eq/mL), superoxide dismutase (SOD) activity, total protein concentration and protein electrophoretic profiles (albumin, alpha-, beta- and gamma-globulins). Brevetoxin concentrations ranged from 7.0 to 33.8ng PbTx-3eq/mL. Plasma brevetoxin concentrations in the nine turtles sampled during or immediately after the red tide events were significantly higher (by 59%, P=0.04) than turtles sampled between events. No significant correlations were observed between plasma brevetoxin concentrations and plasma proteins or SOD activity, most likely due to the small sample size; however alpha-globulins tended to increase with increasing brevetoxin concentrations in the bloom group. Smaller (carapace length and mass) bloom turtles had higher plasma brevetoxin concentrations than larger bloom turtles, possibly due to a growth dilution effect with increasing size. The research presented here improves the current understanding of potential impacts of environmental brevetoxin exposure on marine turtle health and survival.

Analysis of Circulating Immune Complexes in the Serum of Clinically Healthy Horses

The aim of this study was to determine the level of circulating immune complexes (CIC) in equine serum and the electrophoretic properties of the protein fraction thereof. The study was carried out using 42 clinically healthy breeding horses of noble half-blood. The average extinction value of CIC's in equine serum was E450 nm = 0.278 ± 0.09 nm. Generally, 23 protein fractions were identified in the molecular mass range between 294 and 25 kDa. Eight fractions whose molecular masses were 137, 72, 65, 52, 42, 32, 28, and 26 kDa, respectively, appeared in all analyzed samples. In 20 of the analyzed serum an additional nine protein fractions whose molecular masses were 260, 110, 84, 78, 67, 62, 57, 45, and 39 kDa, respectively, were identified. Circulating immune complexes are a source of information about immunologic processes that take place in mammals. Determining the average values of CIC levels and identifying the electrophoretic profile of proteins in clinically healthy horses could be useful in research and diagnostics.

Electrophoretic profile of serum proteins from Dromedary camels

Electrophoretic pattern of serum protein and dialysed serum protein profiles were studied from apparently healthy camels on 10 and 15% SDS-PAGE in addition to total protein and immunoglobulin content. The electrophoretic pattern of camel serum protein revealed 7 prominent bands of molecular weights ranging from 28.21 kDa to 123.07 kDa on 10% SDS-PAGE and dialysed serum protein revealed 4 prominent bands of molecular weights ranging from 24.30 kDa to 110.87 kDa. The electrophoretic pattern of camel serum protein on 15% SDS PAGE revealed 8 prominent bands of molecular weights ranging from 0.005 to 121.04 kDa along with 5 bands of dialysed serum protein. There are about 7 lower molecular weight bands which may be of prealbumin and other lower molecular weights of proteins. The lower molecular weight bands may be of prealbumin. The band with higher molecular weight may be of different globulin fractions including immunoglobulins. Total protein and immunoglobulin content were low in present study suggesting that it is due to very hot temperature and limited grazing facility during summer.

Electrophoretic profile of serum proteins in dromedary camels

Electrophoretic pattern of serum protein and dialysed serum protein profiles were studied from apparently healthy camels on 10% and 15% SDS-PAGE in addition to total protein and immunoglobulin content. The electrophoretic pattern of camel serum protein revealed 7 prominent bands of molecular weights ranging from 28.21 kDa to 123.07 kDa on 10% SDS-PAGE and dialysed serum protein revealed 4 prominent bands of molecular weights ranging from 24.30 kDa to 110.87 kDa. The electrophoretic pattern of camel serum protein on 15% SDS PAGE revealed 8 prominent bands of molecular weights ranging from 0.005 to 121.04 kDa along with 5 bands of dialysed serum protein. There are about 7 lower molecular weight bands which may be of prealbumin and other lower molecular weight proteins. The lower molecular weight bands may be of pre albumin. The band with higher molecular weight may be of different globulin fractions including immunoglobulins. Total protein and immunoglobulin content were low in present study possibly due to very hot temperature and limited grazing facility during summer.

Comparative electrophoretic profile of proteins and esterases in healthy silkworm larvae ( Bombyx mori Lineu, 1758) and infected with nucleopolyhedrovirus

Total proteins and esterases from silk gland extracts of Bombyx mori silkworm were characterized and compared with electrophoretic profiles of prepared extracts with silkworm glands infected with nucleopolyhedrovirus (BmNPV). SDS-PAGE (7%) gels was used for the total proteins, and 10% native PAGE for esterases. In the silk glands extracts of healthy silkworms, it was observed seven protein zones with molecular weight varying between 10 kDa (P1) and 60 kDa (P7). In the infected silkworms, a new zone named P8 (90 kDa) was also detected. Esterases activity at 5 th instar larvae underwent changes after the infection with BmNPV, since there was a reduction (EST-6 and EST-7) and an increase (EST-8) in the intensity of the regions of esterases activity, and specificity of EST-9 to β-naphthyl acetate. Those alterations observed in the expression of genes after the infection with the nucleoplyhedrovirus can be used as markers to detect infections in B. mori.

Post-testicular sperm maturation and identification of an epididymal protein in the Japanese quail (Coturnix coturnix japonica)

The role of the avian epididymis in post-testicular development and capacitation was examined to assess whether avian spermatozoa undergo any processes similar to those characteristic of mammalian sperm development. We found no evidence of a need for quail sperm to undergo capacitation and 90% of testicular sperm could bind to a perivitelline membrane and acrosome react. However, computer-assisted sperm analysis showed that 20% of testicular sperm from the quail were capable of movement and only about 12% of the motile sperm would have a curvilinear velocity greater than the mean for sperm from the distal epididymis. Nevertheless, epididymal transit was associated with increases in mean sperm velocity and the proportion of motile sperm. Together, these findings explain why earlier workers have achieved some fertilizations following inseminations of testicular spermatozoa and also demonstrate the need for some epididymal maturation of avian spermatozoa. Analysis of the electrophoretic profile of quail epididymal luminal proteins revealed that only one major protein (∼16 kDa) is secreted by the epididymis and it was virtually the only protein secreted by the ipsilateral epididymis following unilateral orchidectomy. Mass spectrometry showed that this protein is hemoglobin; this finding was confirmed using anti-hemoglobin antibodies. It is suggested that hemoglobin may support sperm metabolism in the quail epididymis, aid in motility, and/or serve as an antioxidant.

Phenetic analysis of the complex Senna fabrisii—S. trichosepala (Leguminosae, Caesalpiniodeae, Aphyllae) based on morphological characters and seed protein electrophoretic profiles

Multivariate and univariate analyses based on morphological characters and seed soluble protein electrophoretic profiles were carried out in order to understand the taxonomic status within the complex S. fabrisii-S. trichosepala. Twenty morphological characters from herbarium specimens were recorded and analyzed by ANOVA, principal component and cluster analysis. The seed protein electrophoretic analysis including cluster analysis was based on three populations: one of S. fabrisii and two of S. trichosepala. The results showed that both taxa differed only by three morphological features; in parallel, the CPA and CA demonstrate that no clearly separated group can be recognized. The seed protein electrophoretic profiles show a high degree of similarity. Based on these results, and due to the variability observed, the inclusion of S. fabrisii in the synonymy of S. trichosepala is proposed.

ADMINISTRAÇÃO DE PROPILENOGLICOL, COBALTO E VITAMINA B12 ÀS OVELHAS E SEUS REFLEXOS SOBRE O PERFIL ELETROFORÉTICO DAS PROTEÍNAS SÉRICAS NAS SUAS CRIAS.

This work was carried out to evaluate the influence of administration of propilene glycol, cobalt and vitamin B 12 , on the electrophoretic profile of serum proteins on sheep and their offspring. The study was conducted using 18 pregnant ewes which were randomly divided into groups at 30 days before the date scheduled for delivery in order to be given supplements until the date preceding the birth. The groups were as follows: Group 1 (G1/n=6) Control Group; Group 2 (G2/n=6) Cobalt and Vitamin B 12 (wich was received 1mg of cobalt orally daily and 2mg of vitamin B 12 , intramuscularly weeckly); and Group 3 (G3/n=6) Propylene Glycol (administration of 30mL of propylene glycol orally daily). We observed that mosto f the protein fractions vary with age development, particularly total serum protein, albumin, beta-globulin and gamma-globulin, and the determinant factor for these changes is colostrum intake. Furthermore, there was no influence of intake by sheep of the components studied on the profile of these variables in lambs. KEYWORDS: colostrum; electrophoresis; metabolic profile; ovine.

Comparison of total protein and phospholipase A2 levels in individual coralsnake venoms

Studies of differences or changes in venom protein levels or enzymatic activities have significance only if contrasted to the normal variations between individual snakes. This study involves the analysis and comparison of venom from 13 individual Texas coralsnakes (Micrurus tener tener) in order to detect differences in the volume, total protein concentration, electrophoretic profile, and PLA2 enzyme activity. A significant inverse correlation between venom volume and total protein concentration was found. Although the 13 venoms were indistinguishable from their electrophoretic protein profiles, phospholipase A2 enzymatic activities varied considerably.

Individual Variability in the Venom Proteome of Juvenile Bothrops jararaca Specimens

Snake venom proteomes/peptidomes are highly complex and subject to ontogenetic changes. Individual variation in the venom proteome of juvenile snakes is poorly known. We report the proteomic analysis of venoms from 21 juvenile specimens of Bothrops jararaca of different geographical origins and correlate it with the evaluation of important venom features. Individual venoms showed similar caseinolytic activities; however, their amidolytic activities were significantly different. Rather intriguingly, plasma coagulant activity showed remarkable variability among the venoms but not the prothrombin-activating activity. LC-MS analysis showed significant differences between venoms; however, an interesting finding was the ubiquitous presence of the tripeptide ZKW, an endogenous inhibitor of metalloproteinases. Electrophoretic profiles of proteins submitted to reduction showed significant variability in total proteins, glycoproteins, and in the subproteomes of proteinases. Moreover, identification of differential bands revealed variation in most B. jararaca toxin classes. Profiles of venoms analyzed under nonreducing conditions showed less individual variability and identification of proteins in a conserved band revealed the presence of metalloproteinases and l-amino acid oxidase as common components of these venoms. Taken together, our findings suggest that individual venom proteome variability in B. jararaca exists from a very early animal age and is not a result of ontogenetic and diet changes.

In vitro Gastrointestinal Digestion of Glabrous Canaryseed Proteins as Affected by Variety and Thermal Treatment

Glabrous or hairless canaryseed is a nutritional grain that could be a good addition to the diet if approved as a novel food. To assess the impact of thermal treatment on its digestibility; raw, roasted or boiled flours prepared from three different varieties of glabrous canaryseed were subjected to in vitro gastrointestinal digestion conditions and the effect on protein electrophoretic profiles was examined using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Roasting was done by dry-heat in an oven at 176 °C for 12 min whereas boiling was done in water at 98 °C for 12 min. SDS-PAGE showed approximately twenty-five protein bands in the undigested raw flour with molecular masses (MM) ranging from <14 kDa to >97 kDa. The dominant proteins had low MM, between the ranges of ~57 to 12 kDa. Roasting markedly altered the protein electrophoretic profile with the appearance of large molecular weight aggregates. Canaryseed proteins were more easily digested after thermal treatment and under sequential gastric-duodenal conditions than under gastric or duodenal conditions alone. Furthermore, roasting appeared to have a greater impact on in vitro protein digestibility than boiling.