Gel Electrophoresis Of Proteins Research Articles

Analysis of variable major protein antigenic variation in the relapsing fever spirochete, Borrelia miyamotoi, in response to polyclonal antibody selection pressure.

Borrelia miyamotoi is a tick-transmitted spirochete that is genetically grouped with relapsing fever Borrelia and possesses multiple archived pseudogenes that encode variable major proteins (Vmps). Vmps are divided into two groups based on molecular size; variable large proteins (Vlps) and variable small proteins (Vsps). Relapsing fever Borrelia undergo Vmp gene conversion at a single expression locus to generate new serotypes by antigenic switching which is the basis for immune evasion that causes relapsing fever in patients. This study focused on B. miyamotoi vmp expression when spirochetes were subjected to antibody killing selection pressure. We incubated a low passage parent strain with mouse anti-B. miyamotoi polyclonal antiserum which killed the majority population, however, antibody-resistant reisolates were recovered. PCR analysis of the gene expression locus in the reisolates showed vsp1 was replaced by Vlp-encoded genes. Gel electrophoresis protein profiles and immunoblots of the reisolates revealed additional Vlps indicating that new serotype populations were selected by antibody pressure. Sequencing of amplicons from the expression locus of the reisolates confirmed the presence of a predominant majority serotype population with minority variants. These findings confirm previous work demonstrating gene conversion in B. miyamotoi and that multiple serotype populations expressing different vmps arise when subjected to antibody selection. The findings also provide evidence for spontaneous serotype variation emerging from culture growth in the absence of antibody pressure. Validation and determination of the type, number, and frequency of serotype variants that arise during animal infections await further investigations.

Lectin-Based Affinity Enrichment and Characterization of N-Glycoproteins from Human Tear Film by Mass Spectrometry.

The glycosylation of proteins is one of the most common post-translational modifications (PTMs) and plays important regulatory functions in diverse biological processes such as protein stability or cell signaling. Accordingly, glycoproteins are also a consistent part of the human tear film proteome, maintaining the proper function of the ocular surface and forming the first defense barrier of the ocular immune system. Irregularities in the glycoproteomic composition of tear film might promote the development of chronic eye diseases, indicating glycoproteins as a valuable source for biomarker discovery or drug target identification. Therefore, the present study aimed to develop a lectin-based affinity method for the enrichment and concentration of tear glycoproteins/glycopeptides and to characterize their specific N-glycosylation sites by high-resolution mass spectrometry (MS). For method development and evaluation, we first accumulated native glycoproteins from human tear sample pools and assessed the enrichment efficiency of different lectin column systems by 1D gel electrophoresis and specific protein stainings (Coomassie and glycoproteins). The best-performing multi-lectin column system (comprising the four lectins ConA, JAC, WGA, and UEA I, termed 4L) was applied to glycopeptide enrichment from human tear sample digests, followed by MS-based detection and localization of their specific N-glycosylation sites. As the main result, our study identified a total of 26 N glycosylation sites of 11 N-glycoproteins in the tear sample pools of healthy individuals (n = 3 biological sample pools). Amongst others, we identified tear film proteins lactotransferrin (N497 and N642, LTF), Ig heavy chain constant α-1 (N144 and 340, IGHA1), prolactin-inducible protein (N105, PIP), and extracellular lacritin (N105, LACRT) as highly reliable and significant N glycoproteins, already associated with the pathogenesis of various chronic eye diseases such as dry eye syndrome (DES). In conclusion, the results of the present study will serve as an important tear film N-glycoprotein catalog for future studies focusing on human tear film and ocular surface-related inflammatory diseases.

Use of the Western blot technique to identify the immunogenic proteins of Borrelia burgdorferi for developing a Lyme disease vaccine

BackgroundLyme disease is a serious infectious disease having a restricted worldwide distribution for which there is no vaccine available for human use. ObjectiveThis study was designed to determine common reactive antigens involved in Borrelia burgdorferi (Bb) infection that are recognized in mammalian sera that may be useful for vaccine development. MethodsBlood samples were collected from patients with documented Lyme disease, and from rabbits and mice experimentally infected with either tick-transmitted or culture-grown Borrelia burgdorferi. All samples were then processed for sera. For performing the Western blots, sonicated Bb organisms (whole cell lysates) and protein ladders were separated by protein gel electrophoresis. Immune reactivities of the electrophoresed proteins with the serum samples were then probed with anti-HRP IgG reagent. ResultsRabbit, mouse and human sera consistently reacted with the 41 kDa band of Bb which corresponded to the flagellin protein – the major protein component of this organism’s periplasmic flagella, also known as axial filaments or fibrils. Various other Bb antigens of wide molecular weight ranges were also recognized by rabbit and human sera, and less frequently with mouse sera. ConclusionThe strong immune response to the 41 kDa flagellin protein by the different mammalian species suggests the utility of a possible vaccine targeting this protein, although other proteins may also be appropriate, for preventing Lyme disease following a bite from an infected tick.

Biochemical properties and heavy metal analysis of a commercial fish Opisthopterus tardoore

Aim: The study was designed to evaluate various biochemical parameters, protein expression, antioxidant enzymes and heavy metals by testing an edible fish Opisthopterus tardoore. Methodology: The experimental fish were collected from five major fish markets of Bhubaneswar, India and distribution of carbohydrate, lipid and protein content were assessed in muscle tissues. To enhance the understanding of molecular mechanism, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of crude proteins was conducted. Catalase (CAT) and guaiacol peroxidase (GPX) activities were determined both spectrophotometrically and by native gel electrophoresis. For heavy metal analysis, three toxic elements viz.,chromium, cadmium and lead were examined by atomic absorption spectrophotometer. Results: The muscle tissues showed variation in the concentrations of carbohydrate (0.17 ± 0.02 to 0.28 ± 0.04 % f.wt.), lipid (3.48 ± 0.27 to 6.62 ± 0.44 % d. wt.) and protein (1.56 ± 0.11 to 2.92 ± 0.16 % f. wt.) with respect to the markets from where fish samples were collected. The protein profiling study revealed the disappearance as well as de novo synthesis of some proteins. The activities of CAT and GPX were determined for establishing a possible bioindicator of metal toxicity in fish. A rapid disturbance in their enzymatic actions was noticed which was also confirmed through native polyacrylamide gel electrophoresis with respect to fish samples. The heavy metals analysis manifested a varying level of accumulation of Cr whereas Pb and Cd were found below the detection limits. Interpretation: The biochemical disturbances and accumulation of Cr in muscle tissues of O. tardoore reflect the pollution status of nearby coastal areas of the Bay of Bengal, Odisha. Key words: Biochemical analysis, Chromium toxicity, Guaiacol peroxidase, Opisthopterus tardoore, Protein expression

Metal-binding proteins and cross-linking in the defensive glue of the slug Arion subfuscus.

The role of metals in forming the primary cross-links in slug glue was investigated. Several metal-binding proteins were identified in the defensive glue produced by the slug Arion subfuscus. Notably, the C-lectins that are unique to the glue are iron-binding proteins. This is unusual for C-lectins. Dissociating these proteins from iron does not affect the glue's stiffness. Similarly, several proteins that can bind to zinc were identified, but dissociating the proteins from zinc did not weaken the glue. These results suggest that metal coordination is not involved in the primary cross-links of this hydrogel glue. The stable cross-links that provide stiffness are more likely to be created by a catalytic event involving protein oxidation. Cross-linking was unexpectedly difficult to prevent. Collecting the glue into a large volume of ice-cold buffer with reagents aimed at inhibiting oxidative cross-linking caused a slight loss of cross-linking, as demonstrated by the appearance of uncross-linked proteins in native gel electrophoresis. Notable among these was a protein that is normally heavily oxidized (asmp165). Nevertheless, this effect was not large, suggesting that the primary cross-links form before secretion.

Early postmortem muscle proteome and metabolome of beef longissimus thoracis muscle classified by pH at 6 hours postmortem

The objective was to identify metabolome and proteome differences at 1 h and 1 d postmortem between longissimus thoracis (LT) muscle classified based on 6 h pH values. Twenty beef LT rib sections were sorted based on 6 h postmortem pH values into low (LpH; pH < 5.55; n = 9) and high (HpH; pH > 5.84; n = 8) pH classifications. Warner-Bratzler shear force (WBSF), desmin degradation, and calpain-1 autolysis were measured. Two-dimensional difference in gel electrophoresis (3–10, 4–7, and 6–9 pH range) and Tandem mass tagging (TMT) protein analyses were employed to determine how the sarcoplasmic protein profile varied across pH classification. Non-targeted metabolomic analyses were conducted on extracts prepared at 1 h and 1 d postmortem. The LpH classification had a lower WBSF value at 1 d postmortem, which was explained by greater calpain-1 autolysis and desmin degradation at 1 d postmortem. Proteome and metabolome analysis revealed a phenotype that promotes more rapid energy metabolism in the LpH group. Proteome and metabolome analyses identified energy production, apoptotic, calcium homeostasis, and proteasome systems influencing pH classifications that could explain the observed pH, proteolysis, and beef tenderness differences. SignificanceThis study is the first to identify proteomic and metabolomic variations early (1 h and 1 day) postmortem that are linked to differences in early (6 h) postmortem pH values and to tenderness differences at 1 day postmortem. This study integrates postmortem biochemical features (protein degradation, proteome, and metabolome variations) to postmortem pH decline and eating quality of beef steaks. Potential biomarkers of more rapid postmortem metabolism linked to earlier tenderization in beef are suggested. Identification of these biochemical features will assist in predicting the eating quality of beef products.

Electromigration Dispersion in Sodium Dodecyl Sulfate Capillary Gel Electrophoresis of Proteins

The electromigration dispersion of the light- and heavy-chain subunit peaks of the therapeutic monoclonal antibody omalizumab was investigated in sodium dodecyl sulfate capillary gel electrophoresis (SDS-CGE) using borate cross-linked dextran sieving matrices. Increasing boric acid content (340-640 mM) caused electromigration dispersion shifts for both low (2%)- and high (10%)-dextran-concentration gels in all gel-buffer compositions. In case of the heavy-chain fragment, elevated borate concentrations resulted in decreasing tailing and increasing fronting with the use of higher- and lower-dextran-concentration gels, respectively. The light-chain fragment, on the other hand, exhibited increased fronting with increasing borate concentration for both dextran concentrations examined in this study. Increase of the glycerol ingredient level in the gel-buffer system caused the same effect as the increasing borate concentration in both dextran concentrations. The detected electromigration dispersion was considered as the result of the formation of monomeric and dimeric glycerol-borate complexes as co-ionic constituents, migrating slower than that of the unconjugated tetrahydroxyborate. In addition, complexation of the tetrahydroxyborate anion with the glucose building blocks of the dextran polymer decreased its mobility to practically zero, contributing to further decrease in the resultant effective mobility of the co-ionic species. We suggest that the observed fronting and/or tailing peak shapes of the monoclonal antibody fragments in SDS-CGE at increasing boric acid concentrations can be considered as the result of multiple effects including changes in pH, sieving matrix pore size, viscosity, and the mobility variation of the co-ionic borate adducts with the gel-buffer ingredients. While electromigration dispersion-mediated band broadening, in general, can be minimized via matching the effective mobility of the co-ionic species to the analyte molecules of interest, in case of borate cross-linked dextran gels, optimization of the boric acid concentration required special consideration of its gel cross-linking function. For the light- and heavy-chain fragments of the IgG analyte, best peak shapes were attained with the use of 10% dextran/340 mM boric acid and 10% dextran/640 mM boric acid-containing gel-buffer systems, respectively. Based on this observation, here we introduce the concept of borate-gradient-mediated transient mobility matching in SDS-CGE of proteins. This novel approach resulted in close to optimal peak shapes for the distantly migrating IgG subunits within a single run, as well as unraveled the long-sought possible solution to perform capillary pore-size-gradient gel electrophoresis.

Obtaining of bioactive di‐ and tripeptides from enzymatic hydrolysis of soybean meal and its protein isolate using Alcalase® and Neutrase®

SummaryThe obtaining of bioactive di‐ and tripeptides using Alcalase® and Neutrase® enzymes in the hydrolysis of soybean meal (SM) and its protein isolate (SPI) was evaluated. An innovative system by fast LC‐MS/MS neutral loss screening andde novosequencing was used to identify bioactive peptides. Soy protein characterisation, gel electrophoresis and antioxidant activity of the obtained peptides were performed. Results achieved showed that the use of Alcalase® and SPI preparation potentiated the peptide breaking bonds and favoured the obtainment of bioactive peptides. The antioxidant activity of tested samples was significantly improved with enzymatic hydrolysis. LC‐MS/MS analyses identified nineteen peptides in SM and 51 in SPI, all obtained after hydrolysis with Alcalase® and, according to BIOPEP, with relevant bioactivities and expressive functional potential. Therefore, it is suggested that bioactive peptides achieved in this study can enable the development of new ingredients and provide greater added value to soy by‐products.

Effect of thermal and chemical treatments used for SARS-COV-2 inactivation in the measurement of saliva analytes.

The present study aims to assess the effects of thermal and chemical inactivating procedures, that can be used for SARS-CoV-2 inactivation, on different salivary analytes. SDS–Polyacrylamide Gel Electrophoresis (SDS-PAGE) protein profile and a panel of 25 specific biomarkers of oxidative status, stress, metabolism and tissue damage were evaluated in samples subjected to different treatments: thermal (65 °C or 92 °C) and chemical with detergents [sodium dodecyl sulphate (SDS), Triton X-100 or NP-40]. Salivary SDS-PAGE profile was most affected by heating at 92 °C, with three and two protein bands decreasing and increasing their expression levels, respectively. This treatment also affected the results of several enzymes, with some of them being also affected by heating at 65 °C and incubation with SDS. The use of Triton X-100 or NP-40 resulted in increased values of cortisol, triglycerides and glucose, not affecting the other tested biomarkers. The present results will help researchers and clinicians to select the best protocols to work in safe conditions with saliva, taking into account the target analyte planned to be measured.

Effect of Enterobacter cloacae toxin on immune cells isolated from Leukemia patients

Toxin extract affects Neutrophils and macrophages, which are phagocytic cells. A total of (180) samples, (50) urine and (130) peripheral blood have been obtained from leukemia patients referred to Medical city/ Baghdad Teaching Hospital/ Hematology center; all patients enrolled in this study were diagnosed by the physician according to clinical presentation and laboratory findings as leukemic patients. Investigate the role of Enterobacter cloacae toxins on phagocytic activity/THP-1cells and epithelial line/HBL-100 cells. THP-1 and HBL-100 cells have been kept in RPMI-1640 that have been supplemented by (10%) of the fetal bovine serum (100µg/mL) of the streptomycin and 100units/mL of the penicillin. The cells have been passaged with the use of the Trypsin-EDTA that has been re-seeded at a confluence of 80% twice every week, and then it has been incubated afterward at a temperature of (37C°). The results of toxin extract from Enterobacter cloacae revealed that (22) fraction/toxins were taken from (11) bacterial samples; these toxins were separated by gel electrophoresis for protein. (18) samples were positive results for gel electrophoresis of protein according to molecular weight: (13.3 KD for α-hemolysin), (70.5 KD for thiol), (75 KD for enterotoxin). This study demonstrates the cytotoxic activity of E. cloacae toxin by using tissue culture through their effect on suppressing the growth of "THP-1 cells" and HBL-100 cells". Keywords. Tissue culture; Toxin; THP-1 cells; HBL-100 cells; Enterobacter cloacae; Leukemia patients.

Synthetic Data Generation for the Development of 2D Gel Electrophoresis Protein Spot Models

Two-dimensional electrophoresis gels (2DE, 2DEG) are the result of the procedure of separating, based on two molecular properties, a protein mixture on gel. Separated similar proteins concentrate in groups, and these groups appear as dark spots in the captured gel image. Gel images are analyzed to detect distinct spots and determine their peak intensity, background, integrated intensity, and other attributes of interest. One of the approaches to parameterizing the protein spots is spot modeling. Spot parameters of interest are obtained after the spot is approximated by a mathematical model. The development of the modeling algorithm requires a rich, diverse, representative dataset. The primary goal of this research is to develop a method for generating a synthetic protein spot dataset that can be used to develop 2DEG image analysis algorithms. The secondary objective is to evaluate the usefulness of the created dataset by developing a neural-network-based protein spot reconstruction algorithm that provides parameterization and denoising functionalities. In this research, a spot modeling algorithm based on autoencoders is developed using only the created synthetic dataset. The algorithm is evaluated on real and synthetic data. Evaluation results show that the created synthetic dataset is effective for the development of protein spot models. The developed algorithm outperformed all baseline algorithms in all experimental cases.

Efficacy of gel electrophoresis for proteins and biotechnological products –an overview.

Nucleic acids or proteins electrophoresed within a matrix or gel that immersed in a buffer provides ions needed to carry a current and for pH maintenance at a relatively constant value. Sodium dodecyl sulphate–polyacrylamide gel electrophoresis&#x0D; &#x0D; (SDS-PAGE) utilizes SDS as an anionic detergent that causes protein denaturation that linearized protein molecules. Each molecule of SDS has the ability to binds to two amino acids. As a result, the ratio of charge to mass becomes constant for all denatured proteins in the mixture. The molecules of protein migrate toward the positive pole and separated in the gel depending only on their molecular weights. The chains of polyacrylamide are cross linked by N, N-methylene bisacrylamide comonomers and ammonium persulfate used as an initiator for polymerization as they act as radical source and N, N, N', N'- tetramethylethylenediamine (TEMED) used to catalyse the polymerization. Electrophoresis of proteins and nucleic acids by using agarose or polyacrylamide gels were illustrated in this review.

Study on Salicylanilide Analogue for Antioxidant Activity and Melanosis Prevention in Chinese White Shrimp (Fenneropenaeus chinensis)

ABSTRACT This study investigated the effects of salicylanilide analogue NBHE for shrimp preservation. The antioxidant activity of 4-Nitro-benzoic acid 3-(2-hydroxy-benzoylamino)-propyl ester (NBHE) was evaluated using the superoxide anion scavenging ability assay. The results showed that NBHE possessed a higher inhibitory effect than that of salicylic acid but weaker than that of kojic acid and ascorbic acid. The IC50 value for ascorbic acid, kojic acid, salicylic acid, and NBHE was calculated to be 9.88 ± 1.98 μg/mL, 63.10 ± 1.13 μg/mL, 328.94 ± 25.01 μg/mL, and 80.94 ± 4.20 μg/mL, respectively. The copper chelating activity of 80.04 ± 3.00% and 85.39 ± 2.97% was observed in the presence of 30 μg/mL and 35 μg/mL of NBHE, respectively. Fifty μM concentration of NBHE had no considerable cytotoxic effect in HaCaT cells. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of shrimp myofibrillar proteins (MP) showed that NBHE could effectively inhibit myofibrillar protein degradation and displayed obvious preservative effect.

Tissue Damage, Not Infection, Triggers Hepatic Unfolded Protein Response in an Experimental Rat Peritonitis Model.

BackgroundAbdominal surgery is an efficient treatment of intra-abdominal sepsis. Surgical trauma and peritoneal infection lead to the activation of multiple pathological pathways. The liver is particularly susceptible to injury under septic conditions. Liver function is impaired when pathological conditions induce endoplasmic reticulum (ER) stress. ER stress triggers the unfolded protein response (UPR), aiming at restoring ER homeostasis, or inducing cell death. In order to translate basic knowledge on ER function into the clinical setting, we aimed at dissecting the effect of surgery and peritoneal infection on the progression of ER stress/UPR and inflammatory markers in the liver in a clinically relevant experimental animal model.MethodsWistar rats underwent laparotomy followed by colon ascendens stent peritonitis (CASP) or surgery (sham) only. Liver damage (aspartate aminotransferase (AST), alanine aminotransferase (ALT) and De Ritis values), inflammatory and UPR markers were assessed in livers at 24, 48, 72, and 96 h postsurgery. Levels of inflammatory (IL-6, TNF-α, iNOS, and HO-1), UPR (XBP1, GRP78, CHOP), and apoptosis (BAX/Bcl-XL) mRNA were determined by qPCR. Splicing of XBP1 (XBP1s) was analyzed by gel electrophoresis, p-eIF2α and GRP78 protein levels using the western blots.ResultsAspartate aminotransferase levels were elevated 24 h after surgery and thereafter declined with different kinetics in sham and CASP groups. Compared with sham De Ritis ratios were significantly higher in the CASP group, at 48 and 96 h. CASP induced an inflammatory response after 48 h, evidenced by elevated levels of IL-6, TNF-α, iNOS, and HO-1. In contrast, UPR markers XBP1s, p-eIF2α, GRP78, XBP1, and CHOP did not increase in response to infection but paralleled the kinetics of AST and De Ritis ratios. We found that inflammatory markers were predominantly associated with CASP, while UPR markers were associated with surgery. However, in the CASP group, we found a stronger correlation between XBP1s, XBP1 and GRP78 with damage markers, suggesting a synergistic influence of inflammation on UPR in our model.ConclusionOur results indicate that independent mechanisms induce ER stress/UPR and the inflammatory response in the liver. While peritoneal infection predominantly triggers inflammatory responses, the conditions associated with organ damage are predominant triggers of the hepatic UPR.

Integration of the Physiology, Transcriptome and Proteome Reveals the Molecular Mechanism of Drought Tolerance in Cupressus gigantea

Drought stress can dramatically impair woody plant growth and restrict the geographical distribution of many tree species. To better understand the dynamics between the response and mechanism of Cupressus gigantea to drought and post-drought recovery, a comparative analysis was performed, relying on physiological measurements, RNA sequencing (RNA-Seq) and two-dimensional gel electrophoresis (2-DE) proteins. In this study, the analyses revealed that photosynthesis was seriously inhibited, while osmolyte contents, antioxidant enzyme activity and non-enzymatic antioxidant contents were all increased under drought stress in seedlings. Re-watering led to a recovery in most of the parameters analyzed, mainly the photosynthetic parameters and osmolyte contents. Transcriptomic and proteomic profiling suggested that most of the differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) were specifically altered, and a few were consistently altered. Drought induced a common reduction in the level of DEGs and DEPs associated with photosynthesis. Notably, DEGs and DEPs involved in reactive oxygen species (ROS) scavenging, such as ascorbate oxidase and superoxide dismutase (SOD), showed an inverse pattern under desiccation. This study may improve our understanding of the underlying molecular mechanisms of drought resistance in C. gigantea and paves the way for more detailed molecular analysis of the candidate genes.

Inhibition of endogenous protease activity and protection of histomorphical integrity during refrigerated storage of grass carp fillets by treatment with natural edible di‐ and tri‐carboxylic acids

SummaryIn this study, by dipping grass carp fillets with 10 mM di‐ and tri‐carboxylic acid solutions individually before storage at 4 °C for 10 days, the application potential of these acids in mitigating softening of fish was investigated through analysing shear force, pH, endogenous cathepsin activities, myofibrillar proteolysis and histomorphological characteristics. The results suggested that maleic acid, fumaric acid and tartaric acid were significantly effective against softening of grass carp muscle, as shown by shear force 26%–31% higher than that of control after storage. Acid treatments led to a decline in the pH of muscle. Besides, the inhibitory effect of these acids on cathepsins was demonstrated by activity reduction by 20%–90%. Further, sodium dodecyl sulphate–polyacrylamide gel electrophoresis and heat shock protein (HSP) analysis supported the protection of these acids against degradation and dissociation of myosin and actin through suppressing HSP loss. Moreover, histomorphological images with higher intensity of acridine orange fluorescence in muscle revealed the improvement of tissue integrity by acid treatments. The inhibitory potential of these acids in suppressing softening of fish muscle is particularly relevant to their capacity of mitigating cathepsin‐induced proteolysis and improving lysosome stability. Therefore, di‐ and tri‐carboxylic acids could play pleiotropic functions in the quality control of fish fillets.

Effects of Cathepsins on Gel Strength and Water-Holding Capacity of Myofibrillar Protein Gels from Bighead Carp (Aristichthys nobilis) under a Hydroxyl Radical-Generation Oxidizing System.

This study investigates the effects of cathepsins on the gel strength and water-holding capacity (WHC) of myofibrillar protein gels from bighead carp (Aristichthys nobilis) under a hydroxyl radical-generation oxidizing system. The myofibrillar proteins were divided into control group (with cathepsins) and E64 group (without cathepsins). The changes of cathepsin B and cathepsin L activities, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), protein oxidation (total sulfhydryl and carbonyl contents), and chemical interactions (nonspecific association, ionic bonds, hydrogen bonds, hydrophobic interactions, and disulfides) of myofibrillar protein and gels, as well as the gel strength and WHC of two groups under 0–100 mM H2O2, were measured. The results indicated that mild oxidation (10 mM H2O2) made a better gel strength and WHC. Cathepsin B and L activities decreased with increasing H2O2 concentrations but their effects on myofibrillar protein degradation still existed during 0.1–50 mM H2O2, which was expressed by higher carbonyl contents and ionic bonds at 0.1 and 50 mM H2O2, higher total sulfhydryl contents at 0 mM H2O2, and a lower intensity of MHC and actin of the control group than the E64 group. Besides more protein degradation, cathepsin proteolysis also resulted in lower gel strength and WHC in control gels than E64 gels under mild oxidation, which could be explained by lower hydrophobic interaction and moderate disulfides bonds between gel protein molecules of control gels.

Identification and Characterization of Differentially Expressed IgM Transcripts of Channel Catfish Vaccinated with Antigens of Virulent Aeromonas hydrophila

Channel catfish (Ictalurus punctatus) is the top species produced in US aquaculture and motile Aeromonas septicemia, caused by virulent Aeromonas hydrophila (vAh), is one of the most severe diseases that afflict catfish farms. Previously, vaccination of fish with extracellular proteins (ECP) of vAh was shown to produce a robust antibody-mediated immune response against vAh infection. In this study, we analyzed IgM transcripts that were differentially expressed in the head kidney and liver of ECP-immunized and mock-immunized (control) fish with emphasis on a variable domain of heavy chain. Quantitative PCR analysis indicated that immunized fish produced significantly more IgM transcripts than control fish. Full-length IgM heavy chain cDNA was cloned, which encoded typical IgM peptide, including signal peptide, variable domain (VH), constant domain (CH), and carboxyl terminal peptide. Great sequence diversity was revealed in a VH segment, with the third complementarity diversity region (CDR3) being most variable. Using germline VH gene grouping method, variants (clones) of VH characterized in this study belonged to nine VH families. The most unique variants (approximately 49%) were found in the VH2 family. Vaccinated fish apparently had more unique variants than in the control fish. There were 62% and 79% of unique variants in the head kidney and liver of vaccinated fish, respectively, while 44% and 27% unique variants in the head kidney and liver of control fish, respectively. Among the unique variants in VH2 family, approximately 87% of them were found in vaccinated fish. Two-dimensional gel electrophoresis of semi-purified IgM protein confirmed that matured IgM protein was as variable as IgM transcripts identified in this study, with isoelectric points crossing from 6 to 10. Results of this study provided insight into the molecular and genetic basis of antibody diversity and enriched our knowledge of the complex interplay between antigens and antibodies in Ictalurid catfish.

Alcalase treatment for removal of adhesiveness in greenfin horse-faced filefish (Thamnaconus septentrionalis) eggs

The greenfin horse-faced filefish Thamnaconus septentrionalis is a new marine culture species in East Asia in recent years. It has demersal and adhesive eggs. The artificial fertilization technique of T. septentrionalis has not been established, and commercial hatcheries currently rely on the collection of naturally fertilized eggs. It is impracticable to separate dead eggs from the egg mass, and a large number of waste and faecal matter adhere to the surface of fertilized eggs, causing excess bacterial and fungal loading in eggs incubation tanks. This study aimed to develop an effective method to remove the adhesiveness of fertilized eggs of T. septentrionalis. The effectiveness of tannic acid (0.5%, 0.2% and 0.1%), sodium hypochlorite (0.05%, 0.025% and 0.01%) and alcalase enzyme (4%, 2%, 1% and 0.5%) to eliminate the adhesiveness of T. septentrionalis eggs was evaluated. Eggs were exposed to different deadhesion solutions for up to 20 min, and the number of deadhesive eggs was counted. The results showed that only the alcalase enzyme could effectively remove the adhesiveness of eggs, and the deadhesion efficiency was positively associated with enzyme concentration. The deadhesion rate of the 0.5% alcalase enzyme treatment was more than 70%, while that of the 4%, 2% and 1% alcalase enzyme treatments were more than 90%. The hatching rates were not affected by alcalase enzyme treatment. The hatching rates of the 2% and 1% treatments were higher than those of the control groups. Ultrastructural analysis showed that the surface of eggs had a pore-canal structure, and the zona radiata externa was gradually degraded and finally peeled off during the deadhesion process. Similar to freshwater fishes with adhesive eggs, biochemical analysis showed that the adhesiveness of T. septentrionalis eggs was related to the presence of glycoproteins and mucopolysaccharides in the zona radiata externa. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of zona radiata externa proteins showed that the nonwater-activated eggs, water-activated eggs and adhesive fertilized eggs had same protein pattern, and only small peptide fragments were detected in the deadhesive fertilized eggs. This study provides valuble theoretical and technical support for improvement of hatching technology of filefish.

Schinus terebinthifolia Raddi leaf lectin is an antiangiogenic agent for Coturnix japonica embryos.

Angiogenesis (budding of new blood vessels) is involved in several processes, including the development of embryos and growth of tumors. Schinus terebinthifolia leaves express an antitumor lectin (SteLL). This work hypothesized that SteLL can interfere with the formation of a vascular network from preexisting vessels. To test this hypothesis, the effect of SteLL on the angiogenesis process was assessed using an in vivo model of yolk sac membrane of Coturnix japonica embryos. SteLL was isolated with purification factor of 46.6. As expected, polyacrylamide gel electrophoresis (PAGE) for native basic proteins confirmed the homogeneity and PAGE in presence of dodecyl sodium sulphate revealed a single 14-kDa polypeptide band. The fractal analysis by box counting and information dimension measurements indicated that SteLL at 1.35 mg/mL significantly decreased by ca. 12% the angiogenesis within the C. japonica yolk sac membrane regarding the control. The inhibition of the vascular network formation in the yolk sac membrane resulted in decreased blood supply to the embryos. Consequently, the area of embryos was significantly reduced by 9.2% regarding the control, which corroborated with the antiangiogenic activity of SteLL. The findings implicate SteLL as an antiangiogenic agent and add to the panel of biological activities of this lectin.