Characteristics of size-exclusion chromatography enriched porcine follicular fluid extracellular vesicles.

Extracellular vesicles (EVs) are membrane-bound nanoparticles that are released by different cell types and play a crucial role in the intercellular communication. They carry various biomolecular compounds such as DNA, RNA, proteins, and lipids. Given that EVs are a new element of the communication within the ovarian follicle, extensive research is needed to optimize method of their isolation. The aim of the study was to assess size-exclusion chromatography (SEC) as a tool for effective EVs isolation from porcine ovarian follicular fluid. The characterization of EVs was performed by nanoparticle tracking analysis, transmission electron microscopy, atomic force microscopy, mass spectrometry and Western blot. We determined EVs concentration, size distribution, zeta potential, morphology, purity, and marker proteins. Our results show that SEC is an effective method for isolation of EVs from porcine follicular fluid. They displayed predominantly exosome properties with sufficient purity and possibility for further functional analyses, including proteomics.

Characterization and function of extracellular vesicles in a canine mammary tumour cell line: Ultracentrifugation versus size exclusion chromatography.

Extracellular vesicles (EVs) are cell-derived membrane-bound vesicles involved in many biological processes such as tumour progression. For years, ultracentrifugation (UC) has been considered the gold standard for EV isolation but limited purity and integrity allowed the diffusion of alternative techniques. In this study, EVs were isolated from a canine mammary tumour cell line using UC and size exclusion chromatography (SEC) and analysed for size and concentration by nanoparticle tracking analysis (NTA) and for protein expression by western blot (WB). EV autocrine effect on cell proliferation, migration and invasiveness was then evaluated in vitro. In all samples, particles were in the EV size range (50-1000 nm), with a higher concentration in UC than in SEC samples (1011 and 1010 particles/ml respectively), and expressed EV markers (Alix, CD9). Functional assays did not show statistically significant difference among conditions, but EV treatment slightly increased cell proliferation and invasiveness and treatment with SEC-isolated EVs slightly enhanced cell migration compared to UC-isolated EVs. In conclusion, the main differences between the two isolation techniques are the quantity of the final EV-product and slight differences on EV functionality, which should be further explored to better highlight the real autocrine effect of tumoral EVs.

METALLOTHIONEIN EXPRESSION IN A PARASITIC CRUSTACEAN, LAMPROGLENA CLARIAE (CRUSTACEA: COPEPODA), ON CLARIAS GARIEPINUS (TELOESTEI: CLARIIDAE) CORRESPONDS TO WATER QUALITY.

Globally, parasites are sensitive toward environmental changes, and, in some cases, they are even more sensitive than their hosts. However, there is limited knowledge on the physiological responses of parasites and their effects on their hosts in relation to environmental degradation. In this study, metallothioneins (MTs) were isolated and compared between the ectoparasite Lamproglena clariae and its host fish Clarias gariepinus. Differences in the levels of MTs in the parasite and host were compared to physicochemical water quality variables and metals to determine if MT expression was linked with changes in water quality. Clarias gariepinus individuals were sampled from 2 sites of differing water quality along the Vaal River using gill nets and assessed for L. clariae. Gill, muscle, and liver tissue of the host and L. clariae were collected and stored in liquid nitrogen for analysis of MT. Water and sediment samples were collected for metal analysis by inductively coupled plasma-optical emission spectrometry and inductively coupled plasma-mass spectrometry. Nutrient levels and water hardness in water samples were assessed using spectrophotometry. MTs were quantified using spectrophotometry and size exclusion chromatography in the host and parasite, respectively. Infections by L. clariae differed between sites, with higher parasite intensity at the unpolluted Vaal Dam site. Concentrations of MT in host tissues and L. clariae were significantly higher at the polluted site, below the Vaal River Barrage, compared to the Vaal Dam site. Parasite MT concentrations were significantly lower compared to concentrations in the liver and gill tissue of C. gariepinus individuals. In conclusion, differences in the concentrations of MT and infection biology of L. clariae reflected the state of the environment and support the usefulness of this parasite and other Lamproglena spp. as bioindicators.

Investigation of the consequences of ultrasound on the physicochemical, emulsification, and gelatinization characteristics of citric acid-treated whey protein isolate.

The effect of ultrasound (US) pretreatment (0, 200, 400, 600, and 800 W) on the physicochemical, emulsification, and gelatinization characteristics of citric acid (CA)-treated whey protein isolate (WPI) was investigated. Size exclusion chromatography demonstrated that when compared with untreated WPI, US pretreatment promoted production of more molecular polymers in the CA-treated WPI. There was a reduction in particle size of CA-treated WPI with the increase of US power (0-800 W), whereas its free sulfhydryl content, surface hydrophobicity, and intrinsic fluorescence strength increased. Furthermore, compared with untreated WPI, emulsifying ability index and emulsifying stability index of CA-treated WPI were increased by 14.04% and 10.10%, respectively, at 800 W. Accordingly, US pretreatment promoted the gel formation of CA-treated WPI, and its gel hardness was increased by 28.0% with US power ranging from 0 to 800 W. Therefore, US and CA treatment can be considered as an effective way to improve the emulsifying and gelatinization characteristics of WPI.

Comparison of isolation methods using commercially available kits for obtaining extracellular vesicles from cow milk.

Extracellular vesicles (EV) are important for delivering biologically active substances to facilitate cell-to-cell communication. Milk-derived EV are widely known because of their potential for immune enhancement. However, procedures for isolating milk-derived EV have not been fully established. To obtain pure milk-derived EV and accurately reveal their function, such procedures must be established. The aim of the present study was to compare methods using commercially available kits for isolating milk-derived EV. Initially, we investigated procedures to remove casein, which is the major obstacle in determining milk-derived EV purity. We separated whey using centrifugation only, acetic acid precipitation, and EDTA precipitation. Then, we isolated milk-derived EV by ultracentrifugation, membrane affinity column, size exclusion chromatography (SEC), polymer-based isolation, or phosphatidylserine-affinity isolation. Using EV count per milligram of protein, which is a good indicator of purity, we determined that acetic acid precipitation was the best method for removing casein. Using nanoparticle tracking analysis, protein quantity analysis, and RNA quantity analysis, we comprehensively compared each isolation method for its purity and yield. We found that SEC-based qEV column (Izon Science) could collect purer milk-derived EV at higher quantities. Thus, a combination of acetic acid precipitation and qEV can effectively isolate high amounts of pure extracellular vesicles from bovine milk.

In vitro anthelmintic activity of the crude hydroalcoholic extract of Piper cubeba fruits and isolated natural products against gastrointestinal nematodes in sheep.

This study describes the in vitro anthelmintic activity of a hydroalcoholic extract from the fruit of Piper cubeba and its major isolated components against the eggs and larvae of gastrointestinal nematodes obtained from naturally-infected ovines. In vitro anthelmintic activity was evaluated using the egg hatch test (EHT), larval development test (LDT) and L3 migration inhibition test (LMT). The extract showed ovicidal and larvicidal activity, with an EC50 of 200 µg/mL and 83.00 µg/mL in the EHT and LDT, respectively. The extract inhibited 100% of larval migration at the lowest tested concentration (95 µg/mL). The crude extract was purified using successive silica gel chromatographic columns, which revealed the lignans hinokinin, cubebin and dihydrocubebin as the major compounds that were present, which were then used in in vitro tests. Cubebin, dihydrocubebin and hinokinin showed higher activity than the crude extract, with an EC50 for ovicidal activity of 150.00 µg/mL, 186.70 µg/mL and 68.38 µg/mL, respectively. In the LDT, cubebin presented an EC50 of 14.89 µg/mL and dihydrocubebin of 30.75 µg/mL. Hinokinin inhibited 100% the larval development at all concentrations evaluated. In the LMT, dihydrocubebin inhibited 100% the larval migration in all concentrations evaluated while cubebin and hinokinin showed EC50 values of 0.89 µg/mL and 0.34 µg/mL, respectively. P. cubeba extract is rich in several classes of active compounds, but here we demonstrate that the described anthelmintic activity may be related to the presence of these lignans, which are present in larger concentrations than other components of the extract. Our results demonstrate for first time the anthelmintic activity against gastrointestinal nematodes in sheep for this class of special metabolites that are present in P. cubeba fruit. However, future detailed studies are needed to evaluate the effectiveness of P. cubeba fruits extract and active lignans in in vivo tests.

Recombinant expression of porcine spermadhesin AWN and its phospholipid interaction: Indication for a novel lipid binding property.

AWN is a porcine (Sus scrofa domestica) seminal plasma protein and has been linked to a variety of processes related to fertilization. To acquire the protein in sufficient amount and purity for functional studies, we established its recombinant expression in E. coli and a three-step purification protocol based on different chromatographies. The test for AWN-phospholipid interaction revealed phosphatidic acid and cardiolipin as potential binding partners. As phosphatidic acid is surmised to play a role in cation-induced membrane destabilization and fusion events, we propose a membrane protective function of the presented binding affinity. Further studies with recombinant AWN will allow new insights into the mechanism of sperm-spermadhesin interaction and might provide new approaches for artificial reproduction techniques.

Enhancement of secondary metabolites from Bacillus Licheniformis XY-52 on immune response and expression of some immune-related genes in common carp, Cyprinus carpio.

This study was undertaken to isolate active secondary metabolites from immunostimulatory Bacillus Licheniformis XY-52 and evaluate their activities at 0%, 0.5%, 1.0%, and 2.0% doses supplementation with feed on immune response in common carp at weeks 1, 2, and 3. By applying chromatography techniques and successive recrystallization, two purified metabolites were obtained and identified by spectral data (mass spectrometry and nuclear magnetic resonance) as: Cyclo-(Phe-Tyr) and Cyclo-(Phe-Gly). The results revealed that humoral innate immune parameters (lysozyme activity, phagocytic activity and bactericidal activity) were significantly (P < 0.05) increased after feeding on the two active compounds-supplemented diet. Furthermore, administration of the two active compounds significantly (P < 0.05) up regulated IL-1ß, Type 1 IFN, IFN g2b, IL10 and TNF-α gene expression. Heat shock protein 70 (HSP70) gene expression was significantly (P < 0.05) lower as compared to control group at the end of trial. Common carp fed with the two compounds had higher survival rates (69.3%) compared to the controls (32.0%) after challenged with the pathogen, Aeromonas hydrophila. The present study indicates that the two isolated active compounds could positively influence immune response and enhance disease resistance of common carp against A. hydrophila infection.

Characterization of incomplete vitellogenin (VgC) in the Indian freshwater murrel, Channa punctatus (Bloch).

A novel incomplete vitellogenin (VgC) was purified from the plasma of estradiol-treated male murrel, Channa punctatus, by gel filtration chromatography. The native mass of VgC protein was 180 kDa, and it resolved as a single peptide of 100 kDa on SDS-PAGE. The peptide on subjecting to matrix-assisted laser desorption/ionization-time of flight produced a peptide mass fingerprint. On tandem mass spectrometry, some of these peptides showed mass to charge (m/z) ratio and amino acid sequence similarity with VgC peptides of other teleosts. Phylogenetic analysis revealed a similarity of murrel VgC with fish species of the order Perciformes. Semi-quantitative RT-PCR assay was developed to study expression of vgc gene at variable levels of estradiol exposure. Presence of VgC in males indicates that fish has been exposed to estrogens; hence, it can be used as a biomarker for estrogenic exposure.

Chemical characterization of milk oligosaccharides of the common brushtail possum (Trichosurus vulpecula).

Structural characterizations of marsupial milk oligosaccharides have been performed in only three species: the tammar wallaby, the red kangaroo and the koala. To clarify the homology and heterogeneity of milk oligosaccharides among marsupials, 21 oligosaccharides of the milk carbohydrate fraction of the common brushtail possum were characterized in this study. Neutral and acidic oligosaccharides were separated from the carbohydrate fraction of mid-lactation milk and characterized by (1)H-nuclear magnetic resonance spectroscopy and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The structures of the 7 neutral oligosaccharides were Gal(ß1-3)Gal(ß1-4)Glc (3'-galactosyllactose), Gal(ß1-3)Gal(ß1-3)Gal(ß1-4)Glc (3", 3'-digalactosyllactose), Gal(ß1-3)Gal(ß1-3)Gal(ß1-3)Gal(ß1-4)Glc, Gal(ß1-3)Gal(ß1-3)Gal(ß1-3)Gal(ß1-3)Gal(ß1-4)Glc, Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)Glc (lacto-N-novopentaose I), Gal(ß1-3)Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)Glc (galactosyl lacto-N-novopentaose I), Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-3)Gal(ß1-4)Glc (galactosyl lacto-N-novopentaose II). The structures of the 14 acidic oligosaccharides detected were Neu5Ac(α2-3)Gal(ß1-3)Gal(ß1-4)Glc (sialyl 3'-galactosyllactose), Gal(ß1-3)(O-3-sulfate)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)Glc (lacto-N-novopentaose I sulfate a) Gal(ß1-3)[Gal(ß1-4)(O-3-sulfate)GlcNAc(ß1-6)]Gal(ß1-4)Glc (lacto-N-novopentaose I sulfate b), Neu5Ac(α2-3)Gal(ß1-3)Gal(ß1-3)Gal(ß1-4)Glc, Neu5Ac(α2-3)Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)Glc (sialyl lacto-N-novopentaose a), Gal(ß1-3)(-3-O-sulfate)Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)Glc, Gal(ß1-3)Gal(ß1-3)[Gal(ß1-4)(-3-O-sulfate)GlcNAc(ß1-6)]Gal(ß1-4)Glc, Gal(ß1-3)[Neu5Ac(α2-6)Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)Glc (sialyl lacto-N-novopentaose b), Neu5Ac(α2-3)Gal(ß1-3)Gal(ß1-3)Gal(ß1-3)Gal(ß1-4)Glc, Gal(ß1-3)(-3-O-sulphate)Gal(ß1-3)Gal(ß1-3)Gal(ß1-3)Gal(ß1-4)Glc, Neu5Ac(α2-3)Gal(ß1-3)Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)Glc, Gal(ß1-3)(-3-O-sulphate)Gal(ß1-3)Gal(ß1-3)[Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)Glc, Gal(ß1-3)Gal(ß1-3)Gal(ß1-3)[Gal(ß1-4)(-3-O-sulphate)GlcNAc(ß1-6)]Gal(ß1-4)Glc and Gal(ß1-3)Gal(ß1-3)[Neu5Ac(α2-6)Gal(ß1-4)GlcNAc(ß1-6)]Gal(ß1-4)Glc (galactosyl sialyl lacto-N-novopentaose b). No fucosyl oligosaccharides were detected. Galactosyl lacto-N-novopentaose II, lacto-N-novopentaose I sulfate a, lacto-N-novopentaose I sulfate b and galactosyl sialyl lacto-N-novopentaose b are novel oligosaccharides. The results are compared with those of previous studies on marsupial milk oligosaccharides.

Isolation and characterization of chicken bile matrix metalloproteinase.

Avian bile is rich in matrix metalloproteinases (MMP), the enzymes that cleave extracellular matrix proteins such as collagens and proteoglycans. Changes in bile MMP expression have been correlated with hepatic and gall bladder pathologies, but the significance of their expression in normal, healthy bile is not understood. We hypothesized that the MMP in bile may aid the digestion of native collagens that are resistant to conventional gastric proteases. Hence, the objective of this study was to characterize the bile MMP and check its regulation in association with dietary factors. We used substrate zymography, azocoll protease assay, and gelatin affinity chromatography to identify and purify the MMP from chicken bile. Using zymography and SDS PAGE, 5 bands at 70, 64, 58, 50, and 42 kDa were detected. The bands corresponding to 64, 50, and 42 kDa were identified as MMP2 using trypsin in-gel digestion and matrix-assisted laser desorption time-of-flight mass spectrometry and peptide mass fingerprinting. Chickens fed diets containing gelatin supplements showed higher levels of MMP expression in the bile by both azocoll assay and zymography. We conclude that the bile MMP may be associated with the digestion of collagens and other extracellular matrix proteins in avian diets.

Purification and properties of a monomeric lactate dehydrogenase from yak Hypoderma sinense larva.

The objective of the present study was to study the characteristics of lactate dehydrogenase (LDH) from Hypoderma sinense larva. H. sinense larvae were collected from yak (Bos grunniens) and identified by a PCR-RFLP method. Analysis of LDH activity showed that the total LDH activity in H. sinense larva was negatively correlated with the length of larva. Polyacrylamide gel electrophoresis of the extracts of H. sinense larvae revealed one band of LDH, which was then purified by affinity chromatography and gel filtration. This enzyme showed an approximately 36 kDa band on SDS-gel under both reducing and non-reducing conditions, in addition, size exclusion chromatography analysis showed that its molecular weight was smaller than bovine serum albumin (67 kDa), indicating that it contains only one subunit. Michaelis constants (Km) values assay revealed that LDH from H. sinense larva showed significantly lower Km for lactate than other animals. LDH of H. sinense larva was stable at 60 °C for 15 min, and also exhibited high catalytic efficiency in a wide range of pH. HgCl2 at the concentration of 0.1mM significantly decreased the activity of LDH from H. sinense larva but not at the concentration of 0.01 mM. The results of the present study demonstrate that LDH from H. sinense larva is a thermal stable and pH insensitive enzyme suitable for catalyzing both forward and reverse reactions.

Antigenic components, isolation and partial characterization of excretion-secretion fraction of Paramphistomum cervi.

The immunogenic components of adult Paramphistomum cervi excretion-secretion (ES) fraction were revealed by SDS-PAGE and immunoblotting technique using sera from cattle naturally infected with P. cervi, Fasciola gigantica, strongylids, Trichuris sp., and Strongyloides sp. By SDS-PAGE, it was found that the ES fraction comprised 13 distinct protein bands. Immunoblotting analysis of these proteins exhibited nine prominent antigenic bands which were recognized by paramphistomosis antisera. These antigenic proteins had molecular weights ranging from 10-170 kDa. One antigenic protein band of 40 kDa was found to give a consistent reaction with sera from all infected cattle. Its diagnostic sensitivity, specificity and accuracy using this test were 100%, 98.9% and 99.3%, respectively. The positive and negative predictive values were 98% and 100%, respectively. The 40 kDa antigen was partially purified by gel filtration and ion-exchange chromatography. The antigenicity of 40 kDa protein for diagnosis of P. cervi infection was confirmed by immunoblotting and indirect ELISA (at 1:78,125 dilution) using a pool of sera and individual serum samples from infected cattle. The present findings suggest that the 40 kDa protein may be used as a diagnostic antigen for paramphistomosis.

Characterization of vitellogenin and its derived yolk proteins in cloudy catshark (Scyliorhinus torazame).

Elasmobranchs (sharks and rays) exhibit unique reproductive characteristics and, in contrast to the situation in teleosts, very little is known about the identity, structure and physical characteristics of their egg yolk proteins. The aims of this study were to (1) detect and purify the vitellogenin (Vtg; egg yolk precursor) and yolk proteins (YPs) of the cloudy catshark (Scyliorhinus torazame), (2) examine the relationships between Vtg and YPs and (3) characterize and classify the deduced primary structure of the Vtg transcript (vtg). The apparent molecular weights of purified Vtg and putative Vtg-related YPs (lipovitellin: Lv, phosvitin: Pv) were determined by gel filtration and were ~560, >669 and ~58 kDa, respectively. Following SDS-PAGE, these purified products (i.e., Vtg, Lv and Pv) appeared as bands of ~210, ~110 and ~22 kDa, respectively. On Western blots, antisera against purified Vtg, Lv and Pv recognized the ~210 kDa Vtg band. Catshark Pv, in contrast to teleost Pvs, had a very low serine content. The catshark Vtg cDNA sequence (vtg) appeared to contain an open-reading frame consisting of domains encoding Lv, Pv and ß'-component (ß'-c). A phylogenetic analysis, with a consideration of genome duplication events, placed catshark vtg into the 'vtgAB type.' It is concluded that at least a single major type of Vtg protein, which is transcribed and translated from catshark vtgAB gene, is the precursor of three egg yolk proteins (Lv, Pv and ß'-c) in catshark.

Isolation and partial characterization of Asian sea bass (Lates calcarifer) Vitellogenin.

A study was conducted to isolate, partial characterize Asian sea bass (Lates calcarifer) vitellogenin (vtg). Two-year-old juvenile L. calcarifer (n = 10) were given three intraperitoneal injections of 17-ß estradiol (E2) at a dose of 2 mg/kg body weight to induce vitellogenesis. Blood was collected 3 days after the last injection, and plasma was purified through gel filtration chromatography. A broad single symmetrical peak consisting of vtg molecule was produced. Protein concentration was 0.059 mg/ml as determined by Bradfrod assay using bovine serum albumin as a standard. The protein appeared as one circulating form in Native PAGE considering the dimeric form of putative vtg with molecular weight of 545 kDa. In SDS-PAGE under reducing conditions, two major bands appeared at 232.86 and 118.80 kDa and minor bands at 100.60, 85.80 and 39.92 kDa, respectively. The purified vtg was used to generate a polyclonal antibody, and the specificity of antibody was assessed by Western blot analysis. Two major bands were immunoreacted, but no cross-reactivity was observed with plasma from non-induced males. The protein was characterized as phosphoglycolipoprotein as it positively stained for the presence of lipid, phosphorus and carbohydrate using Sudan Black B, methyl green and periodic acid/Schiff reagent solution, respectively. The amino acid composition was analyzed by high sensitivity amino acid analysis that showed high percentage of non-polar amino acids (~48 %). The results suggest the potential utilization of vtg as a basis tool to further study about reproductive physiology of this important economical species.

Proteomic identification of camel seminal plasma: purification of ß-nerve growth factor.

The camel seminal plasma contains a diverse array of components including lipids, carbohydrates, peptides, ions and proteins. These are essential for maintaining normal physiology of spermatozoa and are secreted mainly from the prostrate, epidydimis and bulbo-urethral glands of reproductive system. The protein profiles of camel seminal plasma were resolved by two-dimensional gel electrophoresis (2D-PAGE). The majority of the protein was found in acidic regions below pI 7.0 and the 19 brightly stained proteins were identified by MALDI-TOF/MS analysis. On the basis of proteomic profiles, ß-nerve growth factor (ß-NGF) was purified by ion-exchange and gel filtration chromatography and identified by SDS-PAGE and MALDI-TOF/MS analysis. It was further confirmed by western blotting experiments using rabbit anti-ß-NGF primary antibody.

Cryopreservation with α-tocopherol and Sephadex filtration improved the quality of boar sperm.

The objectives were to evaluate postthaw sperm quality and the response to an inducer of in vitro sperm capacitation in boar sperm, cryopreserved with (T) or without (C) α-tocopherol. Boar sperm frozen in 0.2-mL pellets were thawed and washed (W) or selected by three methods: Percoll discontinuous gradient (PS) or Sephadex (Sigma-Aldrich, St. Louis, MO, USA) (neutral [S] or with ion exchange [S+IO] columns). All separation methods enhanced sperm motility, plasma membrane integrity, and functionality and acrosome integrity for both C and T samples (P < 0.05). The best results were obtained with S and ionic Sephadex column. There was a decrease (P < 0.05) in capacitation-like changes in C samples separated with Sephadex (W: 19 ± 0.9%, PS: 22 ± 2.5%, S: 17 ± 1.2%, and S+IO: 17 ± 2.0%). Cryopreservation with α-tocopherol decreased (P < 0.05) the percentage of cryocapacitated sperm (W: 14 ± 0.7%, PS: 14 ± 1.0%, S: 13 ± 1.0%, and S+IO: 14 ± 0.9%) compared with C samples, without differences among selection techniques. Freezing with α-tocopherol and subsequent selection decreased lipid peroxidation (W: 20.79 ± 2.64 nmol thiobarbituric acid reactive substances (TBARS)/10(8) sperm; PS: 13.15 ± 2.39 nmol TBARS/10(8) sperm; S: 13.20 ± 2.18 nmol TBARS/10(8) sperm, and S+IO: 13.62 ± 2.76 nmol TBARS/10(8) sperm), with respect to washed and selected C samples (W: 37.69 ± 5.34 nmol TBARS/10(8) sperm, PS: 25.61 ± 5.85 nmol TBARS/10(8) sperm, S: 19.16 ± 3.28 nmol TBARS/10(8) sperm, and S+IO: 22.16 ± 6.09 nmol TBARS/10(8) sperm). In vitro capacitation levels were significantly higher for neutral Sephadex-selected T samples in comparison with C and unselected samples. These results were confirmed with a follicular fluid-induced acrosome reaction. In conclusion, cryopreserved sperm with α-tocopherol and subsequent Sephadex selection, improved postthaw quality and functionality of boar sperm, which could be useful for assisted reproductive techniques.

Monoclonal antibodies recognizing mucus immunoglobulin and surface immunoglobulin-positive cells of flounder (Paralichthys olivaceus).

Mucus immunoglobulin (Ig) of flounder (Paralichthys olivaceus) was purified by the combination of salting-out, Sephacryl S-300 gel filtration chromatography and DEAE Sepharose chromatography. According to the SDS-PAGE and native-PAGE, the purified mucus Ig showed apparent molecular weights of 72 kDa (heavy chain) and 26 kDa (light chain), and a total molecular weight of 798 kDa, which indicated mucus IgM was in tetrameric form. Purified mucus Ig was used to immunize the Balb/C mice, nineteen hybridomas secreting monoclonal antibodies (mAbs) against flounder mucus Ig were obtained by indirect enzyme-linked immunosorbent assay, and three of them designated as 1A-M2, 1C-M10 and 3F-M9 were cloned by limiting dilution. In Western blotting, the three mAbs specifically reacted to the heavy (H) chain of mucus Ig, but not reacted with serum Ig of flounder, whereas mAb 2D8 against serum Ig previously produced could react with the H chain of both mucus and serum Ig, indicating the composition of the mucus and serum Ig H chains was different. Meanwhile, surface Ig positive (sIg+) lymphocytes in the peripheral blood, spleen, skin and gills of healthy flounder, were analyzed by flow cytometry using mAb 1A-M2 and mAb 2D8, and the results revealed that both mAbs were reactive with the sIg+ lymphocytes. The positive reactivity rates for mAb 1A-M2 were 38.64% in the peripheral blood, 23.6% in the spleen, 16.56% in the skin and 6.26% in the gills, while the positive reactivity rates for mAb 2D8 were 48.89%, 33.7%, 15% and 6.02%, respectively, suggesting mucus Ig was similar, but not identical, to serum Ig. These results generated important mucosal immunological information and gave a valuable insight into understanding the mucosal immunity in flounder.

Serum carbohydrate-binding IgM are present in Vietnamese striped catfish (Pangasianodon hypophthalmus) but not in North African catfish (Clarias gariepinus).

Pangasianodon hypophthalmus serum was fractionated by affinity chromatography on 12 different Sepharose-carbohydrate columns and proteins eluted by the corresponding sugar. Binding to the affinity matrices is dependent on Ca(2+) ions. Upon gel filtration using Superose-12, essentially one fraction was obtained, eluting as a protein with a molecular mass of about 900 kDa. SDS-PAGE in reducing conditions revealed the presence of large (72 kDa) subunits (H-chains) and one up to three small (24, 26 and/or 28-29 kDa) subunits (L-chains). The isolated proteins were shown to be IgM since they bind monoclonal anti-P. hypophthalmus IgM antibodies. Rabbit polyclonal anti-galactose-binding IgM only cross-react with some sugar-binding IgM. The H-chains of the anti-carbohydrate IgM are glycosylated. Circular dichroism studies revealed that the IgMs have an "all-ß" type of structure, and that Ca(2+) ions, though essential for carbohydrate-binding activity, are not required for the structural integrity of the molecules. In non-reducing SDS-PAGE, only monomers and halfmers were obtained, showing that there are no disulfide bonds linking the monomers, and that a disulfide bond connecting both H-chains within one monomer is only present in 45% of the molecules. Both the monomers and the halfmers display molecular mass heterogeneity which is indicative for redox forms at the level of the intradomain disulfide bonds. The native carbohydrate-binding IgMs agglutinate erythrocytes from different animals, as well as fish pathogenic bacteria. Similar proteins could not be isolated from another catfish, Clarias gariepinus.

Prokaryotic expression, purification, and refolding of leukocyte cell-derived chemotaxin 2 and its effect on gene expression of head kidney-derived macrophages of a teleost fish, ayu (Plecoglossus altivelis).

Leukocyte cell-derived chemotaxin 2 (LECT2) is reported to be an immunorelevant protein in ayu (Plecoglossus altivelis). In this study, ayu LECT2 mature peptide (aLECT2m) was expressed as insoluble inclusion bodies in Escherichia coli. The denatured recombinant aLECT2m (raLECT2m) was refolded by a size-exclusion chromatography refolding process achieved by using arginine-containing mobile phase and a decreasing urea gradient. The in vitro chemotactic activity assay showed that the refolded raLECT2m had the bioactivity. By using suppression subtractive hybridization (SSH) method, we further identified up-regulated genes in ayu macrophages treated with refolded raLECT2m. These genes were tightly involved in endocytosis, hydrolysis, transcriptional regulation, signal transduction, and so on. Moreover, real-time quantitative PCR (RT-qPCR) results confirmed that selected 10 genes expression was significantly up-regulated in refolded raLECT2m-treated ayu macrophages. This study provides a basis for further studies of the mechanism of cytokine LECT2 in fish immune responses.