Identification of a new aspartic proteinase expressed by the outer chorionic cell layer of the equine placenta.
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The pregnancy-associated glycoproteins (PAGs) are placental antigens that were initially characterized as pregnancy markers in the maternal circulation of domestic ruminant species. They are members of the aspartic proteinase gene family, having greatest sequence identity with pepsinogens. However, some are not capable of functioning as enzymes. The PAGs are associated with a large gene family within the Artiodactyla order (cattle, camels, pigs). So far, no members of this family have been characterized in species outside this order. This report describes the cloning and initial characterization of a PAG-like protein (equine PAG or ePAG) expressed in the placenta of the horse and zebra (order Perrisodactyla). Equine PAG is a proteinase capable of degrading 14C-hemoglobin and catalyzing the removal of its own pro-peptide. The ePAG mRNA is restricted to the chorion both prior to implantation and in the term placenta. Equine PAG is secreted from cultured placental tissue as both a processed (mature) and unprocessed (zymogen) form. Equine PAG shares similar identity with the PAGs and pepsinogens and probably arose from a pepsinogen-like precursor that gained the ability to be expressed in the placenta. The promoter of the ePAG gene shares sequence identity with the promoter from a bovine PAG gene but not with promoters of other aspartic proteinases. Therefore, we hypothesize that ePAG is a remnant of the pepsinogen-like progenitor gene that was expanded within the Artiodactyla to create the large and highly diverse PAG family. (+info)
Intrafollicular content of luteinizing hormone receptor, alpha-inhibin, and aromatase in relation to follicular growth, estrous cycle stage, and oocyte competence for in vitro maturation in the mare.
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The intrafollicular content of LH receptor, alpha-inhibin, and aromatase are known good indicators of follicular status. We investigated the amounts of these proteins in granulosa and cumulus cells in relation to oocyte competence for in vitro maturation, follicular growth, and estrous cycle stage in the mare. Follicular punctures were performed 34 h after an injection of crude equine gonadotropins, either during the follicular phase, at the end of the follicular phase, or during the luteal phase. The cumulus-oocyte complex, granulosa cells, and follicular fluid of follicles larger than 5 mm were collected. The nuclear stage of the oocytes after in vitro culture was determined microscopically. Granulosa and cumulus cell amounts of LH receptor, alpha-inhibin, and aromatase were assessed by the semiquantitative Western blot method and image analysis. Follicular fluids were assayed for progesterone (P4) and estradiol-17beta (E2). The three factors were expressed in mural granulosa and cumulus cells from all follicles from the gonadotropin-independent growth period until the preovulatory stage. Considering all the follicles punctured, the amounts of LH receptor and alpha-inhibin in granulosa cells were not different for the three physiological stages studied. The amounts of aromatase in granulosa cells, as well as the E2:P4 ratios, were higher for follicles punctured during the follicular phase than for the two other groups (p < 0.05). Considering the data from the three groups, the E2:P4 ratio and the LH receptor and aromatase contents, but not alpha-inhibin, in granulosa cells increased with an increase in follicular diameter (p < 0.01). The E2:P4 ratios and the amounts of LH receptor, alpha-inhibin, and aromatase in granulosa cells were lower in follicles 5-9 mm in diameter than in larger ones (p < 0.05). In cumulus cells, the amounts of the three factors were different neither between the three groups nor between the follicular diameters. Although we could not establish any obvious relationship to oocyte competence for in vitro maturation, the influence of the follicle diameter on the content of LH receptors, alpha-inhibin, and aromatase in granulosa cells was similar to the influence of follicle diameter on oocyte competence. Therefore, one can hypothesize that, in the mare, there is a link between the acquisition of oocyte competence and the expression of these factors in the follicular cells. (+info)
Isolation and characterization of a novel Forssman active acidic glycosphingolipid with branched isoglobo-, ganglio-, and neolacto-series hybrid sugar chains.
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Equine kidney and spleen contain a Forssman active glycosphingolipid, and the structure of this glycolipid has been reported to be that of a globopentaosylceramide (GalNAcalpha-1,3GalNAcbeta-1,3Galalpha-1, 4Galbeta-1,4Glcbeta-1,1'Ceramide). We found that equine kidney contains several other anti-Forssman antibody-reactive glycosphingolipids. One of these acidic Forssman active glycosphingolipids was isolated and characterized by means of NMR, mass spectrometry, permethylation studies, and TLC-immunostaining. This glycolipid contains three moles of galactose, one mole of glucose, three moles of N-acetylgalactosamine, one mole of N-acetylglucosamine, and one mole of N-acetylneuraminic acid, and is stained on TLC with anti-Forssman antibodies and anti-GM2 ganglioside antibodies. HOHAHA and ROESY experiments and permethylation studies showed this glycolipid oligosaccharide to be branched at the innermost galactose; one chain has an isoglobo structure with a terminal Forssman disaccharide and the other chain is branched through the linkage of N-acetylglucosaminebeta-1,6 to the inner galactose. The nonreducing end of the GM2 trisaccharide is linked to this glucosamine. The structure of the oligosaccharide of the glycolipid presented here is a novel type, having branched isoglobo-, ganglio-, and neolacto-series oligosaccharides. Mass spectrometric analyses indicated the ceramide moiety of the glycolipid to be composed predominantly of hydroxy fatty acids (C20:0, C22:0, C23:0, C24:0, and C25:0) and hydroxysphinganine. GalNAcalpha-1,3GalNAcbeta-1,3Galalpha-1,3[GalNAcbet a-1, 4(NeuAcalpha-2,3)Galbeta-1,4GlcNAcbeta-1,6]Galbeta+ ++-1,4Glcbeta-1, 1'Ceramide (+info)
Electrospray mass spectra from protein electroeluted from sodium dodecylsulfate polyacrylamide gel electrophoresis gels.
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Electrospray ionization/tandem mass spectrometry of proteins separated on sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels is severely limited by the requirement that the protein be completely separated from the SDS. As shown here, the gaseous noncovalent SDS adducts of protonated proteins thus formed can be dissociated by infrared irradiation. ESI spectra from myoglobin in SDS-containing solutions show molecular ions adducted with up to 15 dodecyl sulfates, but ir irradiation of these ions causes complete dissociation to expel the SDS. (+info)
SFS, a novel fibronectin-binding protein from Streptococcus equi, inhibits the binding between fibronectin and collagen.
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The obligate parasitic bacterium Streptococcus equi subsp. equi is the causative agent of strangles, a serious disease of the upper respiratory tract in horses. In this study we have, using shotgun phage display, cloned from S. equi subsp. equi and characterized a gene, called sfs, encoding a protein termed SFS, representing a new type of fibronectin (Fn)-binding protein. The sfs gene was found to be present in all 50 isolates of S. equi subsp. equi tested and in 41 of 48 S. equi subsp. zooepidemicus isolates tested. The sfs gene is down-regulated during growth in vitro compared to fnz, a previously characterized gene encoding an Fn-binding protein from S. equi subsp. zooepidemicus. Sequence comparisons revealed no similarities to previously characterized Fn-binding proteins, but high scores were obtained against collagen. Besides similarity due to the high content of glycine, serine, and proline residues present in both proteins, there was a nine-residue motif present both in collagen and in the Fn-binding domain of SFS. By searching the Oklahoma S. pyogenes database, we found that this motif is also present in a potential cell surface protein from S. pyogenes. Protein SFS was found to inhibit the binding between Fn and collagen in a concentration-dependent way. (+info)
The influence of betaine on untrained and trained horses exercising to fatigue.
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Because exercise fatigue has been associated with the accumulation of lactic acid, factors that influence lactate metabolism during exercise can potentially enhance performance. The objective of this study was to examine the effects of supplemental betaine on eight mature Thoroughbred horses before and after 8 wk of conditioning. The effects of betaine were tested in two cross-over design experiments, allowing each horse to receive both the control and betaine treatments at each fitness level. Ingestion of 80 mg of betaine/kg of BW for 14 d before exercise testing did not alter plasma lactate, glucose, free fatty acids (FFA), or triglyceride concentrations during exercise in the untrained or trained horses. A time x treatment interaction (P < .05) was observed for plasma lactate in untrained horses during recovery from exercise, and plasma lactate concentrations were lower (P < .05) at 60 min after exercise when untrained horses received betaine. Plasma FFA concentrations were lower (P < .05) before exercise and at 720 min after exercise when untrained horses received betaine. These data indicate that betaine may influence lactate metabolism following exercise in untrained horses; however, betaine does not seem beneficial for trained horses. (+info)
Binding of peripheral proteins to mixed lipid membranes: effect of lipid demixing upon binding.
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Binding isotherms have been determined for the association of horse heart cytochrome c with dioleoyl phosphatidylglycerol (DOPG)/dioleoyl phosphatidylcholine (DOPC) bilayer membranes over a range of lipid compositions and ionic strengths. In the absence of protein, the DOPG and DOPC lipids mix nearly ideally. The binding isotherms have been analyzed using double layer theory to account for the electrostatics, either the Van der Waals or scaled particle theory equation of state to describe the protein surface distribution, and a statistical thermodynamic formulation consistent with the mass-action law to describe the lipid distribution. Basic parameters governing the electrostatics and intrinsic binding are established from the binding to membranes composed of anionic lipid (DOPG) alone. Both the Van der Waals and scaled particle equations of state can describe the effects of protein distribution on the DOPG binding isotherms equally well, but with different values of the maximum binding stoichiometry (13 lipids/protein for Van der Waals and 8 lipids/protein for scaled particle theory). With these parameters set, it is then possible to derive the association constant, Kr, of DOPG relative to DOPC for surface association with bound cytochrome c by using the binding isotherms obtained with the mixed lipid membranes. A value of Kr (DOPG:DOPC) = 3.3-4.8, depending on the lipid stoichiometry, is determined that consistently describes the binding at different lipid compositions and different ionic strengths. Using the value of Kr obtained it is possible to derive the average in-plane lipid distribution and the enhancement in protein binding induced by lipid redistribution using the statistical thermodynamic theory. (+info)
Dehydration and crystallization of trehalose and sucrose glasses containing carbonmonoxy-myoglobin.
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We report a study wherein we contemporarily measured 1) the dehydration process of trehalose or sucrose glasses embedding carbonmonoxy-myoglobin (MbCO) and 2) the evolution of the A substates in saccharide-coated MbCO. Our results indicate that microcrystallization processes, sizeably different in the two saccharides, take place during dehydration; moreover, the microcrystalline structure is maintained unless the dry samples are equilibrated with a humidity >/=75% (>/=60%) at 25 degrees C for the trehalose (sucrose) sample. The evolution of the parameters that characterize the A substates of MbCO indicates that 1) the effects of water withdrawal are analogous in samples dried in the presence or in the absence of sugars, although much larger effects are observed in the samples without sugar; 2) the distribution of A substates is determined by the overall matrix structure and not only by the sample water content; and 3) the population of A0 substate (i. e., the substate currently put in relation with MbCO molecules having the distal histidine out of the heme pocket) is largely enhanced during the dehydration process. However, after rehumidification its population is largely decreased with respect to the values obtained, at similar water content, during the first dehydration run. (+info)