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Heparin-binding proteins (HBP) from seminal plasma have been expected to participate in modulation of the acrosomal reaction, and have been correlated with fertility in some species. However, they have not been described in the dog. The aim of this study was to document the HBPs of canine seminal plasma. Six pooled samples of seminal plasma from three crossbred dogs were used. The HBPs were isolated by heparin affinity chromatography and the fractions recovered were pooled. One-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was carried out on 12 and 18% vertical minigels. The stained gels were scanned and the molecular weight (kDa) values for each band within a lane were calculated by image analysis software. The electrophoresis analysis of the pooled eluded fractions identified 19 bands, with molecular weights varying from 61.5 to 5.2 kDa. Previous studies, using one-dimensional SDS-PAGE, identified two bands (67 and 58.6 kDa), which were positively correlated with some semen parameters (sperm motility, sperm vigor, percentage of morphologically normal sperm and plasma membrane integrity). The 61.5 kDa band detected in the present study apparently corresponded to the 58.6 kDa band identified previously. Canine seminal plasma contained HBP; since HBP modulate the acrosome reaction in other species, they may have the same function in the dog. Further studies are necessary to better characterize this protein and determine if it is associated with fertility in the dog.
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PMID:Herapin-binding proteins of canine seminal plasma. 1676 6

Hyaluronidases play an important role in gamete interaction and fertility in mammals. The objectives of the present study were to investigate multiple forms of the enzyme in boar reproductive tract using electrophoretic methods. Two forms of hyaluronidase (EC 3.2.1.35) were detected in boar seminal plasma (relative molecular masses of 55,000 and 65,000) using hyaluronic acid-substrate polyacrylamide gel electrophoresis in the presence of SDS. These two forms can be separated by means of affinity chromatography on Heparin-Sepharose. They differ, besides their affinity to heparin, also in the pH optimum of their enzymatic activity. The form with relative molecular mass of 55,000 was active both at the acidic (pH 3.7) and the neutral pH (pH 7.4) and was bound to immobilized heparin. The second form (relative molecular mass 65,000) was active only at acidic pH and did not interact with heparin. The same acidic-active form (65,000) was found in seminal vesicle fluids. The hyaluronidase form which is active both at the acidic and the neutral pH (51,000) was detected in epididymal fluid. In the detergent extracts of boar sperm, three active forms of the enzyme were found (relative molecular masses 55,000, 70,000 and 80,000). The form of relative molecular mass 55,000 was active in a wide range of pH (pH 3-8). The forms of relative molecular masses 70,000 and 80,000 were active only at neutral pH.
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PMID:Preliminary characterization of multiple hyaluronidase forms in boar reproductive tract. 1788 Oct 45

The objective was to determine the effects of oviductal proteins on sperm function. Abbatoir-derived buffalo oviducts were flushed with PBS; the fluid recovered (protein concentration, 2.3 mg/mL; average of 3.5 mg protein/oviduct) was centrifuged, dialyzed, and clarified, and the supernatant applied to a Heparin-Sepharose affinity column. Unbound fractions were collected and bound proteins were separately eluted (with elution buffer). Eight distinct protein bands (from 12 to 177 kDa) in the H-unbound fraction and 15 distinct protein bands (from 12 to 165 kDa) in the H-bound fraction were detected in SDS-PAGE. Semen from four buffalo bulls was divided into three parts: Parts 1 and 2 were treated with the heparin binding (H-bound) and non-heparin binding (H-unbound) oviductal proteins, respectively, whereas Part 3 remained as an untreated control. Equilibrated and frozen-thawed semen was assessed for motility, viability, intact acrosome percentage, mucus penetration distance, and hypo-osmotic swelling test. The H-bound oviductal fluid proteins enhanced (P<0.05) the proportion of sperm that were progressively motile, alive, had an intact acrosome and functional plasma membrane (hypo-osmotic swelling test), as well as the distance covered in the cervical mucus sperm penetration test during cryopreservation. Addition of the H-unbound oviductal protein fraction did not increase sperm motility and penetration distance but increased (P<0.05) the proportion of sperm that were live, had an intact acrosome, and functional plasma membrane (hypo-osmotic swelling test). We concluded that the H-bound fraction of buffalo oviductal fluid protein(s) maintained sperm motility, viability and membrane integrity during cryopreservation, whereas the H-unbound proteins maintained sperm viability and membrane integrity.
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PMID:Effect of oviductal fluid proteins on buffalo sperm characteristics during cryopreservation. 1835 71

Serine protease inhibitors (serpins) regulate coagulation and inflammation. Heparin, a glycosaminoglycan, is an important cofactor for modulation of the inhibitory function of mammalian serpins. The secreted myxoma viral serpin, Serp-1 exerts profound anti-inflammatory activity in a wide range of animal models. Serp-1 anti-inflammatory and anti-atherogenic activity is dependent upon inhibition of the uPA / uPA receptor thrombolytic complex. We demonstrate here that heparin binds to Serp-1 and enhances Serp-1 inhibition of thrombin, a human pro-thrombotic serine protease, in vitro, altering inhibitory activity to a more predominant anti-thrombotic activity. Heparin also facilitates the simultaneous thrombin-mediated cleavage of Serp-1 and prevents formation of a serpin-typical SDS-resistant complex, implying mutual neutralization of Serp-1 and thrombin. In a cell-based assay, heparin facilitates Serp-1 reversal of cellular activation by stabilizing cellular membrane fluidity in thrombin-activated monocytes. In conclusion, heparin and other GAGs serve as cofactors enhancing Serp-1 regulation of local thrombotic and inflammatory pathways.
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PMID:Heparin alters viral serpin, serp-1, anti-thrombolytic activity to anti-thrombotic activity. 1894 70

Antithrombin was purified from Bothrops jararaca plasma by affinity chromatography using HiTrap Heparin HP column, and its molecular weight, amino-terminal sequence, carbohydrate content, isoelectric point, inhibition of bovine thrombin, and immunological properties were studied and compared with previously described antithrombins. B. jararaca antithrombin is a single-chain glycoprotein with a total carbohydrate content of 18%. The molecular weight from SDS-PAGE was 61 kDa and the inhibitor exhibited an acidic isoelectric point (4.5). The amino-terminal sequence has been determined as His-Glu-Ser-Ser-Val-Gln-Asp-Ile-Ile-Thr, which is highly homologous to the terminal sequences of other animal antithrombins, indicating high amino acid conservation among several animals. Immunological cross-reactivity was observed among fish, frog, chicken, human, non-venomous snake and B. jararaca antithrombins. B. jararaca antithrombin showed inhibitory activity upon human and B. jararaca coagulation and amidolytic substrate S-2238.
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PMID:Bothrops jararaca antithrombin: isolation, characterization and comparison with other animal antithrombins. 1904 27

Venoms from the bee Apis mellifera, the caterpillar Lonomia achelous, the spiders Lycosa sp. and Phoneutria nigriventer, the scorpions Tityus bahiensis and Tityus serrulatus, and the snakes Bothrops alternatus, Bothrops jararaca, Bothrops jararacussu, Bothrops moojeni, Bothrops neuwiedi, Crotalus durissus terrificus, and Lachesis muta were assayed (800mug/mL) for activity against Staphylococcus aureus. Venoms from B. jararaca and B. jararacussu showed the highest S. aureus growth inhibition and also against other Gram-positive and Gram-negative bacteria. To characterize the microbicidal component(s) produced by B. jararaca, venom was fractionated through gel exclusion chromatography. The high molecular weight, anti-S. aureus P1 fraction was further resolved by anion exchange chromatography through Mono Q columns using a 0-0.5M NaCl gradient. Bactericidal Mono Q fractions P5 and P6 showed significant LAAO activity using l-leucine as substrate. These fractions were pooled and subjected to Heparin affinity chromatography, which rendered a single LAAO activity peak. The anti-S. aureus activity was abolished by catalase, suggesting that the effect is dependent on H(2)O(2) production. SDS-PAGE of isolated LAAO indicated the presence of three isoforms since deglycosylation with a recombinant N-glycanase rendered a single 38.2 kDa component. B. jararaca LAAO specific activity was 142.7 U/mg, based on the oxidation of l-leucine. The correlation between in vivo neutralization of lethal toxicity (ED(50)) and levels of horse therapeutic antibodies anti-LAAO measured by ELISA was investigated to predict the potency of Brazilian antibothropic antivenoms. Six horses were hyperimmunized with Bothrops venoms (50% from B. jararaca and 12.5% each from B. alternatus, B. jararacussu, B. neuwiedii and B. moojeni). To set up an indirect ELISA, B. jararaca LAAO and crude venom were used as antigens. Correlation coefficients (r) between ED(50) and ELISA antibody titers against B. jararaca venom and LAAO were 0.846 (p<0.001) and 0.747 (p<0.001), respectively. The hemolytic and leishmanicidal (anti-Leishmania amazonensis) activity of LAAO was also determined.
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PMID:Antigenic, microbicidal and antiparasitic properties of an l-amino acid oxidase isolated from Bothrops jararaca snake venom. 1910 83

L-amino acid oxidases (LAAOs) are widely distributed in snake venoms, which contribute to the toxicity of venoms. However, LAAO from Bungarus fasciatus (B. fasciatus) snake venom has not been isolated previously. In the present study, LAAO from B. fasciatus snake venom was purified by SP-Sepharose HP anion exchange chromatography followed by Heparin-Sepharose FF affinity chromatography procedure and the purified enzyme was named BF-LAAO. BF-LAAO presented an estimated molecular weight of 55kDa in SDS-PAGE and an apparent molecular weight of 70kDa in size-exclusion chromatography suggesting that BF-LAAO is a monomeric protein. Kinetics studies showed that BF-LAAO was very active against L-Tyr, L-Asp, L-Phe, L-Glu, L-Trp, L-His, L-Gln, L-Ile, L-Met, L-Leu and moderately active against L-Lys, L-Arg, L-Ala and L-Asn. BF-LAAO exhibited a cytotoxic effect on A549 cells and caused up to 41.2% apoptosis of A549 cells following 12h incubation period. In the mouse peritoneum, BF-LAAO provoked a marked increase in the number of neutrophils, lymphocytes and macrophages following injection. It also induced rabbit platelet aggregation in a dose-dependent manner. At 3h following injection, BF-LAAO elicited severe inflammation in the gastrocnemius muscles of mice, but failed to induce significant organ damage. In conclusion, the cytotoxic and proinflammatory activities of BF-LAAO could be the main cause of the local inflammation, which helps us to understand the pathogenesis of snakebite.
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PMID:Purification, characterization and biological activities of the L-amino acid oxidase from Bungarus fasciatus snake venom. 1939 76

The family B DNA polymerase gene from the archaeon Thermococcus marinus (Tma) contains a long open reading frame of 3,939 bp that encodes 1,312 amino acid residues. The gene is split by one intervening sequence that forms a continuous open reading frame with the two polymerase exteins. In this study, the Tma DNA polymerase gene both with (precursor form) and without (mature form) its intein was expressed in Escherichia coli, purified by heat treatment and HiTrap Heparin HP column chromatography and characterized. Primary sequence analysis of the mature Tma polymerase showed high sequence identity with DNA polymerases in the genus Thermococcus. The expressed precursor form was easily spliced during purification steps. The molecular mass of the purified Tma DNA polymerases is about 90 kDa, as estimated by SDS-PAGE. Both Tma DNA polymerases showed the same properties. PCR performed with this enzyme was found to be optimal in the presence of 50 mM Tris-HCl (pH 8.4), 40 mM KCl, 12.5 mM (NH(4))(2)SO(4,) 2 mM MgCl(2,) 0.05% Triton X-100 and 0.0075% BSA. Furthermore, long-range PCR and time-saving PCR were performed using various specific ratios of Taq and Tma DNA polymerases (Tma plus DNA polymerase).
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PMID:Characterization of DNA polymerase from the hyperthermophilic archaeon Thermococcus marinus and its application to PCR. 1941 77

In order to research the bioactivity of kallistatin (Kal), we obtained the recombinant Kal using Pichia pastoris expression system. Kal cDNA was amplified from pAAV-Kal and inserted into pPIC9 vector to generate a recombinant vector of pPIC9-Kal. Then, pPIC9-Kal was linearized and transformed into Pichia pastoris strain GS115 (His4) by electroporation. The positive transformants were selected by MD plate and confirmed by PCR. High level of Kal was obtained in BMMY medium (pH 7.0) after 96 hours induction of 29 degrees C and 2% methanol, with the highest yield of 14 mg/L in shake flask culture. Kal protein was purified from the supernatant with Phenyl Superose and Heparin Sepharose FF chromatograph. The recombinant Kal had a molecular weight of 58 kDa with 98% purity, showing by SDS-PAGE. Moreover, it had a high peroxidase activity (163+/-4) U/(mgmin), which could protect LX-2 cell against oxidation of H2O2. Recombinant Kal also effectively inhibited HUVEC proliferation. In this report, we successfully established the expression system using Pichia pastoris and obtained the bioactive recombinant human Kal. It lays a foundation for its further anti-cancer therapy.
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PMID:[High level expression of recombinant human kallistatin in Pichia pastoris and its bioactivity]. 2043 46

Because of the risk of suffering a stroke or heart attack, some athletes and their medical supervisors admitted having used anticoagulants (e.g. acetylsalicylic acid) in combination with doping with recombinant erythropoietins (rhEPO). Heparin is one of the oldest and cheapest anticoagulants. The anticoagulative effect of heparin is a result of the binding of heparin to the plasma protein antithrombin III and the subsequent inactivation of blood clotting factors (e.g. factor IIa, IXa, Xa, XIa, XIIa). Heparin-a polyanion-is known to interact with carrier ampholytes used in IEF-PAGE. Two different types of heparin pharmaceuticals are used for medical purposes: unfractionated heparins (UFH) and low molecular weight heparins (LMWH). Their influence on IEF- and SDS-PAGE was investigated. Only UFH had a profound impact on IEF-PAGE, leading to excessive smearing or complete abolishment of the EPO IEF-profile and shifting of acidic EPO-isoforms in the endogenous region of the gel. No such effect was observable for SDS-PAGE. Remedies include immunoaffinity purification of EPO before IEF-PAGE or the treatment of the urinary retentate with solid urea. A combined usage of IEF- and SDS-PAGE is recommended for confirming the presence of rhEPO in urine and for further analysis of smearing (and therefore suspicious) samples. This two-method approach is already in accordance with the technical document on EPO-analysis (TD2009EPO) of the World Anti-Doping Agency (WADA).
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PMID:The interference of heparin on IEF-PAGE of erythropoietins. 2120 88


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