Sulfate-polyacrylamide Gel Electrophoresis Profiles Research Articles

Innovative multistep modifications of β-Lactoglobulin for enhanced emulsifying and antioxidant activities

This aim of this study was to investigate the effects of limited enzymatic hydrolysis (H), glycation (G), and amyloid fibril (F) modification on the emulsifying and antioxidant activities of β-Lactoglobulin (β-Lg). Changes in the physicochemical properties of limited enzymatic hydrolysis-modified β-Lg (Hβ-Lg), glycation by maltodextrin (Mal) -modified Hβ-Lg (GHβ-Lg), and amyloid fibril-modified GHβ-Lg (FGHβ-Lg) were observed through free amino acid content, sodium dodecyl sulfate‒polyacrylamide gel electrophoresis profiles and ThT fluorescence intensity. The structural changes and microscopic morphology of the modified proteins were determined by circular dichroism, Fourier transform infrared spectroscopy and transmission electron microscopy. Circular dichroism analysis showed that the β-structure content of FHβ-Lg increased, while the secondary structure of FGHβ-Lg was preserved. Finally, the functional activities of the modified proteins were evaluated. Comparing β-Lg with FGHβ-Lg or GHβ-Lg, the emulsifying activity of FGHβ-Lg increased from 9.38 m2/g to 24.37 m2/g, while the emulsifying stability of GHβ-Lg increased from 48.93 min to 131.22 min. Finally, the antioxidant activities of the modified proteins were determined by reducing power (β-Lg: 0.17, FGHβ-Lg: 0.27), ABTS·+ scavenging activity (β-Lg: 75.91%, FGHβ-Lg: 86.33%), Fe2+ chelating activity (β-Lg: 54.30%, FGHβ-Lg: 75.49%), and DPPH scavenging ability (β-Lg: 67.43%, FGHβ-Lg: 77.13%). Overall, our findings suggest that FGHβ-Lg has great potential in the development of protein emulsifiers with high antioxidant activities.

Characterization and In Vitro Efficacy against Listeria monocytogenes of a Newly Isolated Bacteriophage, ɸIZSAM-1.

Listeria monocytogenes is a bacterial pathogen responsible of listeriosis, a disease that in humans is often related to the contamination of ready-to-eat foods. Phages are candidate biodecontaminants of pathogenic bacteria thanks to their ability to lyse prokaryotes while being safe for eukaryotic cells. In this study, ΦIZSAM-1 was isolated from the drain-waters of an Italian blue cheese plant and showed lytic activity against antimicrobial resistant Listeria monocytogenes strains. This phage was subjected to purification and in vitro efficacy tests. The results showed that at multiplicities of infection (MOIs) ≤ 1, phages were able to keep Listeria monocytogenes at low optical density values up to 8 h, with bacterial counts ranging from 1.02 to 3.96 log10 units lower than the control. Besides, ΦIZSAM-1 was further characterized, showing 25 principal proteins (sodium dodecyl sulfate polyacrylamide gel electrophoresis profile) and a genome of approximately 50 kilo base pairs. Moreover, this study describes a new approach to phage isolation for applications in Listeria monocytogenes biocontrol in food production. In particular, the authors believe that the selection of phages from the same environments where pathogens live could represent a new approach to successfully integrating the control measures in an innovative, cost effective, safe and environmentally friendly way.

Inhibitory effect of Nigella sativa oil against Listeria monocytogenes and Salmonella Enteritidis inoculated in minced beef meat

The control of pathogenic bacteria and oxidative factors is important to keep microbiological and physicochemical traits of meat products. In this study, cold-pressed black cumin seed oil (BCSO) was incorporated (at levels of 1, 2, and 4%, w/w) into ground beef meat (GBM) to test the antimicrobial activity of BCSO on the growth inhibition of food-borne pathogens (Listeria monocytogenes Scott A and Salmonella Enteritidis PT4) artificially inoculated (~4.5 log CFU/g) in air-packed GBM during storage at 4 °C for 15 days. BCSO was analyzed for fatty acids, sterols, tocopherols, and thymoquinone using gas chromatography and high-performance liquid chromatography. BCSO induced higher inhibition zone (15–17 mm) against both pathogenic bacteria as compared with the control. The BCSO supplementation retarded the total bacterial count, while the food-borne pathogens were unable to grow in BCSO-supplemented GBM during cold storage for 15 days. After 15 days, S. Enteritidis PT4 was more resistant than L. monocytogenes Scott A in GBM supplemented with 4% BCSO. In general, the shelf life of BCSO-supplemented GBM was extended under refrigerated conditions with low microbial loads. BCSO-supplemented GBM also improved oxidative stability and sodium dodecyl sulfate polyacrylamide gel electrophoresis profile.

Identification of peanut seed prolamins with an antifungal role by 2D-GE and drought treatment.

This work revealed peanut seed prolamins likely displaying a defensive role besides the known nitrogen storage. Drought stress and proteomic approaches were used in varieties of peanuts to explore the prolamin member in association with a test against Aspergillus flavus spore germination. The stress effect was showed by aerial biomass, leaf content of malondialdehyde, and seed contamination by A. flavus. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles were not informative for the antifungal polypeptides. From two-dimensional gel electrophoresis, the suspected polypeptides were those with pI 5.45-5.75 and sizes of 22.0-30.5kDa specifically in Spanish-type peanuts. Regarding to the drought effect in most of these peanuts, the spot peak volume analysis deduced three novel prolamin-related antifungal polypeptides at pI 5.75-5.8 with 30.5, 27.5-28.5, and 22.0-22.5kDa, which was confirmed after isoelectric purification at pH 5.60. The data could not yet conclude their correlation with resistance to drought and to seed infection by A. flavus.

Size measuring techniques as tool to monitor pea proteins intramolecular crosslinking by transglutaminase treatment

In this work, techniques for monitoring the intramolecular transglutaminase cross-links of pea proteins, based on protein size determination, were developed. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis profiles of transglutaminase-treated low concentration (0.01% w/w) pea albumin samples, compared to the untreated one (control), showed a higher electrophoretic migration of the major albumin fraction band (26kDa), reflecting a decrease in protein size. This protein size decrease was confirmed, after DEAE column purification, by dynamic light scattering (DLS) where the hydrodynamic radius of treated samples appears to be reduced compared to the control one.

Characterization of intergeneric hybrid between common wheat andAegilops sharonensis(Eig) and transfer of alien high molecular weight glutenin subunits into wheat

Aegilops sharonensis (Sharon goatgrass) is a valuable source of novel high molecular weight glutenin subunits, resistance to wheat rust, powdery mildew, and insect pests. In this study, we successfully hybridized Ae. sharonensis as the pollen parent to common wheat and obtained backcross derivatives. F1 intergeneric hybrids were verified using morphological observation and cytological and molecular analyses. The phenotypes of the hybrid plants were intermediate between Ae. sharonensis and common wheat. Observations of mitosis in root tip cells and meiosis in pollen mother cells revealed that the F1 hybrids possessed 28 chromosomes. Chromosome pairing at metaphase I of the pollen mother cells in the F1 hybrid plants was low, and the meiotic configuration was 25.94 I + 1.03 II (rod). Two pairs of primers were screened out from 150 simple sequence repeat markers, and primer WMC634 was used to identified the presence of the genome of Ae. sharonensis. Sequencing results showed that the F1 hybrids contained the Ssh genome of Ae. sharonensis. The sodium dodecyl sulfate polyacrylamide gel electrophoresis profile showed that the alien high molecular weight glutenin subunits of Ae. sharonensis were transferred into the F1 and backcross derivatives. The new wheat- Ae. sharonensis derivatives that we have produced will be valuable for increasing resistance to various diseases of wheat and for improving the quality of bread wheat.

Amphidiploids between tetraploid wheat and Aegilops sharonensis Eig exhibit variations in high-molecular-weight glutenin subunits

Wide crossing is a useful method for transferring traits of interest from wild Triticeae species to wheat. In this study, Aegilopssharonensis was successfully hybridized to three tetraploid wheat species with crossability varying from 0.86 to 1.76 %. F1 hybrids were verified by their display of intermediate characteristics in morphology, cytology, and sodium dodecyl sulfate–polyacrylamide gel electrophoresis profiles. The exogenous x- and y-type subunits of high-molecular-weight glutenin subunits of Ae.sharonensis were expressed in F1 hybrid seeds and showed identical electrophoretic mobilities to those originally present in Ae.sharonensis. In F2 hybrids, the x-type subunit migrated faster, indicating that a decrease in molecular mass had occurred. Cloning of the open reading frames of subunit variants revealed that the x-type subunit had protein sequence variations, including single residue mutations, and insertions and deletions involving one or more repeat motifs. The two deletions causing the size reduction of the exogenous x-subunits of Ae.sharonensis in the F2 seeds were most likely the result of slip-mismatching.

PROTEOMIC ANALYSIS OF VITREOUS BIOPSY TECHNIQUES

To compare vitreous biopsy methods using analysis platforms used in proteomics biomarker discovery. Vitreous biopsies from 10 eyes were collected sequentially using a 23-gauge needle and a 23-gauge vitreous cutter instrument. Paired specimens were evaluated by UV absorbance spectroscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and liquid chromatography tandem mass spectrometry (LC-MS/MS). The total protein concentration obtained with a needle and vitrectomy instrument biopsy averaged 1.10 mg/mL (standard error of the mean = 0.35) and 1.13 mg/mL (standard error of the mean = 0.25), respectively. In eight eyes with low or medium viscidity, there was a very high correlation (R = 0.934) between the biopsy methods. When data from 2 eyes with high viscidity vitreous were included, the correlation was reduced (R = 0.704). The molecular weight protein sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles of paired needle and vitreous cutter samples were similar, except for a minority of pairs with single band intensity variance. Using LC-MS/MS, equivalent peptides were identified with similar frequencies (R ≥ 0.90) in paired samples. Proteins and peptides collected from vitreous needle biopsies are nearly equivalent to those obtained from a vitreous cutter instrument. This study suggests both techniques may be used for most proteomic and biomarker discovery studies of vitreoretinal diseases, although a minority of proteins and peptides may differ in concentration.

Study on the protein fractions extracted from the muscle tissue of Pinctada martensii and their hydrolysis by pancreatin

SummaryPinctada martensii muscle proteins were separated into water‐soluble, salt‐soluble and insoluble protein fractions. The salt‐soluble protein fraction was the most abundant, comprising approximately 66.3% of the protein, followed by the water‐soluble and insoluble protein fractions in decreasing order. sodium dodecyl sulphate polyacrylamide gel electrophoresis profiles showed that 35‐ and 97‐kDa peptides represented the highest proportions of the water‐soluble and salt‐soluble protein fractions, respectively. The three protein fractions contained high levels of flavourful amino acids (Glu, Asp, Ala and Lys), with the water‐soluble protein fraction having the highest level (47.1%). The size exclusion chromatography profiles of the water‐soluble and salt‐soluble protein pancreatin hydrolysate demonstrated no significant differences in the molecular weight distributions in the >20 000, 5000–10 000 and 2000–5000 Da fractions, while those of the insoluble protein pancreatin hydrolysate were different. In conclusion, the results provided some basic information that would contribute to the utilisation of these protein fractions.

Improvement of the quality and abatement of the biogenic amines of grass carp muscles by fermentation using mixed cultures

To improve the quality of processed grass carp (Ctenopharyngodon idellus) products and control the accumulation of hazardous substances therein, minced grass carp slices were salted for 6 h at room temperature and then inoculated with mixed starter cultures of Lactobacillus casei, Streptococcus lactis, Saccharomyces cerevisiae Hansen and Monascus anka and fermented for 12 h at 30 degrees C. The changes in some characteristics and biogenic amine contents of the fermented muscles were investigated. During the 12 h fermentation at 30 degrees C, muscles inoculated with mixed starter cultures showed a rapid decrease in pH from 6.0 to 5.1 and suppression of the growth of enterobacteria and pseudomonads. The fermented muscles exhibited better colour, appearance, flavour and overall acceptability than the control (P < 0.05). The changes in non-protein nitrogen and free amino acid contents of the fermented muscles and in their sodium dodecyl sulfate polyacrylamide gel electrophoresis profiles indicated that severe hydrolysis of muscle proteins occurred during fermentation. The accumulation of biogenic amines in the muscles was efficiently reduced by fermentation with mixed starter cultures. Fermentation with mixed starter cultures of L. casei, S. lactis, S. cerevisiae Hansen and M. anka significantly improved the characteristics of grass carp muscles and controlled the accumulation of biogenic amines.

Identification and pathogenicity to rainbow trout, Oncorhynchus mykiss (Walbaum), of some aeromonads

Twelve strains of fish pathogenic aeromonads were identified by 16S rRNA sequencing as Aeromonas bestiarum, A. hydrophila, A. hydrophila subsp. dhakensis, A. salmonicida subsp. salmonicida, A. sobria biovar sobria and A. veronii biovar sobria. Following intramuscular injection, A. hydrophila subsp. dhakensis caused dark liquefying, raised furuncle-like lesions in rainbow trout within 48 h. Extracellular products of all cultures contained gelatinase and lecithinase, and most revealed lipase. Congo red absorption and siderophore production was recorded, but not so the suicide phenomenon or slime production. Sodium dodecyl sulphate polyacrylamide gel electrophoresis profile of the outer membrane proteins (OMP) revealed 10-25 bands, of which major bands were seen in the region of 32.5-47.5 and 62-83 kDa. Marked heterogenicity of the OMP and whole cell protein (WCP) profiles within and among the species was observed. Polypeptides of 83-173 kDa were detected in the WCP profile of the cultures, but they were not expressed in OMP fractions.

Isolation and characterization of outer membrane proteins of Edwardsiella tarda and its application in immunoassays

Edwardsiella tarda is an important cause for hemorrhagic septicemia in fish and also for gastro intestinal infections in humans. Sodium lauryl sarcosinate extraction method was used for the isolation of outer membrane proteins (OMPs) from 16 isolates of E. tarda, recovered from snakeheads (Ophiocephalus punctatus). Sodium dodecyl sulphate polyacrylamide gel electrophoresis profiles of OMPs from E. tarda isolates had one major and common protein band of 44 kilodalton (kDa) and 9 other variable protein bands. Rabbit polyclonal antibodies (RPAbs) against OMPs of E. tarda ET-7 detected mainly one common 44 kDa protein in all isolates by western blotting. RPAbs strongly reacted with all isolates of E. tarda by indirect enzyme linked immunosorbent assay (ELISA). These polyclonal antibodies showed no cross reactivity with OMPs or whole cells of other selected aquatic bacteria, i.e., Escherichia coli, Pseudomonas fluorescens, Aeromonas hydrophila, Vibrio cholerae and Flavobacterium ferrugineum. This study indicated that RPAbs against OMPs is useful for detection of E. tarda by ELISA.

Vitamin C prevents cigarette smoke induced oxidative damage of proteins and increased proteolysis

Aqueous extract of cigarette smoke (CS) contains some stable oxidants, which oxidize human plasma proteins, bovine serum albumin, amino acid homopolymers, and also cause extensive oxidative degradation of microsomal proteins. Similar observations are made when the aqueous extract of cigarette smoke is replaced by whole phase CS solution or whole phase cigarette smoke. CS-induced microsomal protein degradation is a two step process: (i) oxidation of proteins by the oxidants present in the CS and (ii) rapid proteolytic degradation of the oxidized proteins by proteases present in the microsomes. Using aqueous extract of CS equivalent to that produced from one-twentieth of a cigarette, the observed initial and postcigarette smoke treated values of different parameters of oxidative damage per milligram of microsomal proteins are respectively: 0.24 and 1.74 nmoles for carbonyl formation, 125.4 and 62.8 fluorescence units for tryptophan loss, 10.2 and 33.4 fluorescence units for bityrosine formation, and 58.3 and 12.2 nmoles for loss of protein thiols. When compared with sodium dodecyl sulphate polyacrylamide gel electrophoresis profiles of untreated microsomal proteins, the extent of microsomal protein degradation after treatment with whole phase CS solution or aqueous extract of CS is above 90%. Ascorbate (100 μM) almost completely prevents cigarette smoke–induced protein oxidation and thereby protects the microsomes from subsequent proteolytic degradation. Glutathione is partially effective, but other antioxidants including superoxide dismutase, catalase, vitamin E, probucol, β-carotene, mannitol, thiourea, and histidine are ineffective. The gas phase cigarette smoke contains unstable reactive oxygen species such as superoxide (O 2 −•) and hydrogen peroxide (H 2O 2) that can cause substantial oxidation of pure protein like albumin but is unable to produce significant oxidative damage of microsomal proteins. Gas phase cigarette smoke–induced albumin oxidation is not only inhibited by ascorbate and glutathione but also by superoxide dismutase, catalase and mannitol. The stable oxidants in the cigarette smoke are not present in the tobacco and are apparently produced by the interaction of O 2 −•/H 2O 2/OH of the gas phase with some components of the tar phase during/following the burning of tobacco.

A monoclonal antibody, HCL-2, raised against human luteal cells reacts with apolipoprotein-B and detects the uptake of low density lipoprotein by luteinizing granulosa cells.

A monoclonal antibody, HCL-2, was raised by immunizing mice against human luteal cells. HCL-2 reacted with luteal cells and villous trophoblasts. The sodium dodecyl sulphate-polyacrylamide gel electrophoresis profile of immunopurified antigens from corpus luteum, chorionic villi, and placenta showed the same main protein band, the molecular mass of which is >200 kDa. The sequence of a portion of the N-terminal region of the antigenic protein purified from placenta was identical to that of apolipoprotein-B. The antigen purified from human serum and low density lipoprotein (LDL) using HCL-2 showed the same protein band as that from corpus luteum. Furthermore, the amino acid sequence (20 amino acids) of the protein purified from serum was also identical to that of apolipoprotein-B. Thus, we concluded that HCL-2 antigen is apolipoprotein-B. Human luteinizing granulosa cells isolated from the patients undergoing in-vitro fertilization treatment were cultured in the medium containing lipoprotein-deficient serum with or without supplementation of LDL. Using HCL-2, apolipoprotein-B was immunocytochemically detected on granulosa cells only in the presence of LDL. These findings showed that the uptake of LDL by granulosa cells was detected by immunocytochemical staining of apolipoprotein-B, indicating that HCL-2 is useful for analysing dynamic utilization of LDL by ovarian cells.

Immunoglobulin A proteases in gram-negative bacteria isolated from human urinary tract infections.

Several strains of gram-negative bacteria (seven genera, eight species) isolated from patients with urinary tract infections were found to hydrolyze myeloma immunoglobulin A (IgA) protein. Human IgG and IgM and colostrum IgA were not degraded by these organisms. Examination of cleavage digests showed two fragments of different electrophoretic mobilities, with antigenic reactivity and sodium dodecyl sulfate polyacrylamide gel electrophoresis profiles consistent with their identification as Fc and Fab components. The immunoelectrophoresis patterns of cleavage digests suggested that the proteases responsible for this hydrolysis may be dissimilar in the specificity of their IgA cleavage sites.