Isolation and characterization of a 30-kDa endometrial glycoprotein synthesized during the estrous cycle and early pregnancy of the pig.

Endometrial polypeptide synthesis, which is regulated through ovarian steroid secretion and steroid production by the developing conceptus, not only provides the necessary secretory components vital to conceptus development but also presents the adhesive changes in the epithelial surface essential for conceptus attachment. In the present study, a 30-kDa, basic endometrial glycoprotein (pGP30) was isolated and characterized during the estrous cycle and early pregnancy of the pig. Uterine flushings and endometrial culture media were obtained from gilts on Days 0, 5, 10, 12, 15, and 18 of the estrous cycle and Days 10, 12, 15, and 18 of pregnancy. A polyclonal antibody was generated to pGP30 after isolation of medium from Day 15 pregnant endometrial cultures separated by gel filtration and PAGE. Western blot analysis indicated that the antiserum reacted with isoforms of pGP30 and cross-reacted with a 90-kDa component in serum that was not removed after cleavage of the oligosaccharide chains from the 90-kDa glycoprotein. Antiserum did not detect a 30-kDa band in media from cultures of kidney, fat, heart, muscle, liver, or serum; however, heart and muscle did contain bands of different molecular masses that cross-reacted with the antiserum. Multiple bands of higher molecular mass (35-40 kDa) were detected in the endometrial cultures from gilts on Days 0 through 10 of the estrous cycle. Treatment of ovariectomized gilts with estradiol-17 beta stimulated a similar response. During the mid- to late luteal phase of the estrous cycle (Days 12-18), the 30-kDa band as well as an additional 32-kDa band was present on Western blots. Administration of progesterone for 14 days stimulated the synthesis of both the 30- and 32-kDa products in ovariectomized gilts. However, only the pGP30 was detected on Days 12-18 of pregnancy. Immunocytochemical localization with antiserum to pGP30 indicated that the glycoprotein is present in the endometrial epithelium, with the surface epithelium demonstrating the strongest reaction product. Discrete changes in staining and cellular localization were observed during the early stages of the estrous cycle (Days 0-5) and the midluteal (Day 10) phase. A similar response was achieved with administration of steroids to ovariectomized gilts. Data indicate that discrete changes in epithelial synthesis of the endometrial glycoprotein occur at the time of conceptus trophoblastic elongation and placental attachment in the pig.

Effects of phenylbutazone on thiamylal disposition and anaesthesia in ponies.

Phenylbutazone given during the perisurgical period has been reported to increase the intensity and duration of thiamylal anaesthesia in horses. A possible mechanism of competitive plasma protein binding has been suggested. The purpose of the present study was to experimentally reproduce the phenomenon of increased intensity and/or duration of thiamylal anaesthesia and to determine if there is competitive displacement of plasma protein bound thiamylal by phenylbutazone. Six ponies each received one of three treatments, 11 mg/kg intravenous (i.v.) thiamylal; 8.8 mg/kg i.v. phenylbutazone; and 11 mg/kg i.v. thiamylal with 8.8 mg/kg i.v. phenylbutazone given 9 min later. Thirteen blood samples were collected from 0 time through 600 min following drug administration and plasma drug concentrations quantified by high performance liquid chromatography. The pharmacokinetics of thiamylal and phenylbutazone were best described by three- and two-compartment models, respectively. There were no significant differences in pharmacokinetic parameters for thiamylal in the presence of phenylbutazone. However, there were differences in phenylbutazone pharmacokinetics when preceded by thiamylal administration. Unbound phenylbutazone concentrations were increased at 171, 231 and 351 min when given with thiamylal, accompanied by decreases in per cent bound phenylbutazone (P < 0.05). There were also significant (P < 0.05) changes in per cent plasma protein binding of thiamylal and phenylbutazone between 120 and 360 min, when in combination. No changes in intensity or duration of anaesthesia were observed.

Effects of localized Pasteurella haemolytica infection on erythromycin-binding properties of bovine alpha-1-acid glycoprotein, albumin, serum, and tissue chamber fluids.

The in vitro erythromycin-binding properties of bovine alpha-1-acid glycoprotein (AAG) and albumin were studied by using equilibrium dialysis. In addition, the proportions of free erythromycin in bovine serum and tissue chamber fluid before and 4 days after inoculation of subcutaneous tissue chambers with Pasteurella haemolytica were measured. At a concentration of 5 micrograms/ml, erythromycin was moderately bound to AAG (39% +/- 4% free) and was only slightly bound to albumin (86% +/- 2% free). Scatchard analysis of the data describing binding to pure bovine AAG indicated that erythromycin was bound to a single high-affinity (6.45 x 10(4) M-1) site on the protein. At lower total concentrations of erythromycin, the free concentrations of the antibiotic were lower in serum samples collected after infection (49% +/- 3% at 5 micrograms of erythromycin per ml) than in those collected before inoculation (55% +/- 3% at 5 micrograms of erythromycin per ml). Inoculation had no effect on binding to macromolecules in chamber fluids. Inoculated tissue chambers served as a convenient model for studying the effect of infection on drug-macromolecule interactions in interstitial fluid.

Effect of Pasteurella haemolytica infection on alpha 1-acid glycoprotein and albumin concentrations in serum and subcutaneous tissue chamber fluid of calves.

Thermoplastic tissue chambers were implanted subcutaneously in the paralumbar fossae of 10 calves. Concentrations of alpha 1-acid glycoprotein (AAG) and albumin in serum and subcutaneous tissue chamber fluid were measured before and after inoculation of Pasteurella haemolytica into tissue chambers. Two months after implantation, serum and tissue chamber fluid samples were collected and all tissue chambers were then inoculated with P haemolytica. Additional serum and chamber fluid samples were collected two, four, six and 10 days after inoculation. The concentrations of AAG and albumin in the samples were measured by radial immunodiffusion assay and the bromcresol method, respectively. P haemolytica inoculation resulted in an increase in the serum and chamber fluid AAG concentrations and an increase in chamber fluid albumin concentrations, suggesting that the proportion of drugs bound to serum and interstitial proteins may be affected by P haemolytica infection.

Purification and characterization of Pz-peptidase B, a neutral metalloendopeptidase from bovine spermatozoa.

We have demonstrated previously that the Pz-peptide synthetic substrate is cleaved by two distinct spermatozoal peptidases, Pz-peptidases A and B. To facilitate further investigations, Pz-peptidase B was purified from bovine spermatozoa. The soluble extract from 81 grams of washed epididymal spermatozoa was fractionated by a five-step procedure consisting of anion-exchange, lectin affinity, hydrophobic interaction, chromatofocusing, and gel filtration chromatography. This method yielded 1 mg of 170-fold purified Pz-peptidase B with a 26% recovery. The purified Pz-peptidase B was electrophoretically homogeneous and possessed a monomeric molecular weight of 90,700. Isoelectric focusing revealed microheterogeneity with pIs ranging from 5.02 to 5.09. Pz-peptidase B was irreversibly inactivated at pH 3.5 or below, and activity was reduced at moderate ionic strengths. Hydrolysis of the Pz-peptide was maximal at pH 7.5. Pz-peptidase B was strongly inhibited by a metal chelator and phosphoramidon. Reactivation of metal-depleted enzyme by various metal ions suggested that Pz-peptidase B was a zinc metallopeptidase. Pz-peptidase B hydrolyzed a wide variety of synthetic substrates and physiologically activity peptides at the amino side of hydrophobic amino acids. These results established that Pz-peptidase B should be classified as a neutral metalloendopeptidase. Overall, the properties of Pz-peptidase B were very similar to those of previously described neutral metalloendopeptidases implicated in degradation of regulatory peptides.

Chromatographic separation and characterization of Pasteurella haemolytica cytotoxin.

Biochemical and immunologic properties of the cytotoxin (leukotoxin) produced by Pasteurella haemolytica were examined. Crude, bacteria-free supernatants from logarithmic phase P haemolytica were fractionated, using a series of column chromatographic techniques. Sequential anion exchange chromatography, gel-filtration chromatography, and chromatofocusing resulted in a cytotoxic substance (cytotoxin-C) of approximately 160 kilodaltons (kD), as determined by use of gel-filtration chromatography. Polyacrylamide-gel electrophoresis of cytotoxin-C yielded 3 protein bands with relative mobilities of 0.37, 0.42, and 0.63. On the basis of immunoblotting with a cytotoxin-neutralizing bovine immunoglobulin for antigen detection, the 2 low-mobility bands shared a strong region of immunogenicity. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, principal protein constituents of cytotoxin-C were found at 160, 66, 57, and 23 kD. Using immunoblotting with cytotoxin-neutralizing immunoglobulin, strong, distinct reactions with the 66- and 57-kD bands were detected. Immunization of rabbits and mice with cytotoxin-C resulted in sera that reacted strongly with cytotoxin-C in enzyme-linked immunosorbent assays and immunodiffusion assays. The major immunogenic proteins also were detected by use of immunoblotting with anticytotoxin-C sera from rabbits and mice. Postinoculation rabbit sera neutralized crude cytotoxin.

Comparison of the antigens associated with saline solution, potassium thiocyanate, and sodium salicylate extracts of Pasteurella haemolytica serotype 1.

Pasteurella haemolytica antigenic extracts were made, using saline solution, potassium thiocyanate (KSCN), and sodium salicylate (SS) extraction procedures. Of the 3 techniques, saline solution extraction resulted in the lowest protein concentration and lowest ribonucleic acid-to-protein ratio. The extracts varied in protein:carbohydrate ratios, with the KSCN extract being highest and the saline solution extract the lowest. Each extract contained lipopolysaccharide, as determined by detectable quantities of 2-keto, 3-deoxyoctonate. The saline solution extract contained the fewest protein bands by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis, but contained the highest molecular weight proteins. All 3 extracts were reasonably similar antigenically, as detected by immunoblotting. Many of the protein bands present in the KSCN or SS extracts did not seem to be antigenic. Each extract was subjected to chromatofocusing, and the greatest antigenic peak, for each extract, failed to bind to the exchanger. These highly antigenic peaks, designated as saline solution, KSCN, or SS antigens, were similarly high in carbohydrate content, had similar antigenic-profiles, and contained high molecular weight (greater than 200,000) antigenic material, most likely carbohydrate in nature, as detected by immunoblotting. Inoculation of mice with 1 of the 3 extracts or the saline solution antigen resulted in marked antibody responses; however, protection against intraperitoneal challenge exposure to P haemolytica was minimal.

Serum antibodies to antigens derived from a saline extract of Pasteurella haemolytica: correlation with resistance to experimental bovine pneumonic pasteurellosis.

The serum antibody response was determined to 6 antigen groups (AG's) derived from a saline extract (SE) of Pasteurella haemolytica, serotype 1. Using an enzyme-linked immunosorbent assay, sera were analyzed from 65 calves that had been previously vaccinated with saline, the unfractionated SE, a bacterin, or live P. haemolytica. The serum antibody responses to the 6 AG's were correlated with resistance to an experimental transthoracic challenge with the organism. The antibody responses to AG's 1, 5, and 6 appeared to be potentially important in resistance to challenge. In the 3 experiments conducted, a significantly higher (p less than 0.05) increase in antibody was seen to AG's 1, 5, and 6 in calves vaccinated with live organisms compared to those vaccinated with the bacterin. A significant correlation (p less than 0.05) was seen between high antibody to AG 1 and resistance to challenge in all 3 experiments. In 2 of the 3 experiments, a significant correlation (p less than 0.05) was seen among high antibody titers to AG's 5 and 6 and resistance, whereas in 1 experiment the correlation was significant (p less than 0.05) between antibody to AG 4 and resistance. A rise in antibody to AG's 2 and 3 was seen only in calves vaccinated with SE. Because AG's 1, 5, and 6 are higher in carbohydrate than the other AG's, this suggests that antibody to polysaccharide antigens may be important to resistance. Other potentially protective antigens of P. haemolytica are discussed.

Saline-extracted antigens of Pasteurella haemolytica: separation by chromatofocusing, preliminary characterization, and evaluation of immunogenicity.

Saline extracts of logarithmic-phase Pasteurella haemolytica, serotype 1, were separated by chromatofocusing. The resulting fractions were analyzed by immunodiffusion and an enzyme-linked immunosorbant assay, and six antigen groups (AG's) were identified. AG 1 did not bind to the column, AG's 2, 3 and 4 were eluted with a decreasing pH gradient, and AG's 5 and 6 were eluted with an increasing NaC1 gradient. Fractions containing each AG were pooled and further purified by gel filtration. The AG's were subsequently characterized as to protein, carbohydrate and 2-keto-3-deoxyoctanate (KDO) content. AG's 1, 5, and 6 had higher carbohydrate contents than AG's 2, 3 and 4. Only AG 5 contained detectable levels of KDO. The AG's were also analyzed by SDS-PAGE and immunoblotting. Each AG produced a characteristic pattern of proteins and antigens, although two antigenic proteins were common to all AG's. AG 1 contained the greatest number of antigenic proteins. Immunization of mice with each AG in Freund's incomplete adjuvant resulted in a strong antibody response to the homologous AG for four of the six AG's. Limited protection against a P. haemolytica challenge was observed in mice that were immunized with AG 2 or 4.

Identification and distribution of Pz-peptidases A and B in human semen.

Human semen was fractionated into fluid, particle and spermatozoal constituents using Percoll density gradient centrifugation followed by additional separation steps. All of the fractions isolated possessed both Pz-peptidase A and Pz-peptidase B activity. The effects of inhibitors on the Pz-peptidase A and B activities of all seminal fractions were similar, suggesting that hydrolysis of the Pz-peptidase was attributable solely to these two enzymes. Estimates of the activities in intact spermatozoa indicated that 1.6 +/- 0.5 mU of Pz-peptidase A and 1.6 +/- 0.7 mU of Pz-peptidase B were present per billion spermatozoa. The predominant source of Pz-peptidase B activity in semen was the ultra-low density particle fraction (110,000 X g pellet from seminal plasma), which contained 86% of the recoverable Pz-peptidase B activity. Pz-peptidase A and B activities of fluid and particle fractions isolated from azoospermic ejaculates from vasectomized donors were similar to the activities of the corresponding fractions from normal semen. This suggested that much of the Pz-peptidase A and B activities of semen originated in accessory gland secretions. The effects of EDTA, Zn2+ and Cu2+ on soluble Pz-peptidase A and B activities of particle-free seminal plasma suggested that neither was involved in the liquefaction of semen.

Identification and preliminary characterization of two distinct bovine seminal Pz-peptidases.

Two peptidases hydrolyzing the Pz-peptide substrate were identified in bovine semen. Each Pz-peptidase was strongly inhibited by chelating agents, suggesting both were metallopeptidases. However, these peptidases could be distinguished by other properties and were designated Pz-peptidases A and B. Pz-peptidase A hydrolyzed the Pz-peptide at the Leu-Gly bond, was inhibited by tosylphenylethylchloromethylketone (TPCK) but not by phosphoramidon and had a pH optimum near 6, whereas Pz-peptidase B cleaved the Pro-Leu bond, was inhibited by phosphoramidon but not by TPCK and had a pH optimum near 7. Seminal plasma, light particulates and cytoplasmic droplets contained almost exclusively Pz-peptidase A, and Pz-peptidase A predominated in sperm extracts. Pz-peptidase B was found primarily in sperm extracts, but Pz-peptidase B activity was also present in ultralight particulates. Pz-peptidase A of spermatozoa required Triton X-100 for complete extraction, but Pz-peptidase B was solubilized from spermatozoa by nitrogen decompression without detergents. Pz-peptidase B was inhibited by several detergents. In particular, addition of 0.1% Hyamine 2389 to sperm extracts inhibited 99% of the Pz-peptidase B activity. Thus, Pz-peptidase B may have been overlooked in previous studies employing extraction of spermatozoa with Hyamine 2389. The properties of both seminal PZ-peptidases were different from those of purified bovine testicular PZ-peptidase, suggesting that PZ-peptidases from these sources were not identical.

Immunocytochemical localization of acrosin on both acrosomal membranes and in the acrosomal matrix of porcine spermatozoa.

Immunocytochemical techniques were employed to determine, at the ultrastructural level, the location of acrosin in porcine spermatozoa. Antisera to highly purified porcine acrosin was produced in rabbits. The (Fab')2 fragments of the immunoglobulins were purified and conjugated with horseradish peroxidase (HRP). Washed, formaldehyde-fixed spermatozoa were reacted with the labeled antiacrosin immunoglobulins, utilizing a direct staining technique. Electron microscopy revealed that the peroxidase reaction product of HRP-antiporcine acrosin was distributed evenly over the outer acrosomal membrane of spermatozoa with intact acrosomes. The labeled antibody was also distributed evenly over the inner acrosomal membrane of cells when the overlying acrosomal structures were absent. In some spermatozoa, labeling was noted throughout the acrosomal matrix. No significant labeling was observed in control specimens when spermatozoa were exposed to HRP-antiporcine acrosin immunoglobulins that had been adsorbed previously with excess purified acrosin or exposed to HRP-conjugated rabbit antiporcine immunoglobulins. This pattern of labeling is consistent with the hypothesis that acrosin may function as a zona lysin. The observation that the outer acrosomal membrane and acrosomal matrix are labeled suggests that acrosin is not exclusively located on the inner acrosomal membrane and, thus, could participate in physiologic events other than zona penetration.

Distribution of Pz-peptidase in bovine epididymal and ejaculated semen.

Bovine epididymal or ejaculated semen was fractionated by density gradient centrifugation in Percoll, and seminal components recovered from the gradients were subjected to additional separation and washing steps. This procedure resulted in isolation of four major seminal constituents: particle-free extracellular fluid, washed light particulates, washed cytoplasmic droplets, and washed spermatozoa. When assayed using the Pz-peptide substrate, all the isolated seminal fractions contained substantial Pz-peptidase activity. The extracellular fluid Pz-peptidase was present in soluble form, but Triton X-100 was required for complete extraction of the Pz-peptidase activity from the spermatozoa, cytoplasmic droplets, and light particulates. The greatest Pz-peptidase activities were observed in the cytoplasmic droplet and epididymal sperm extracts, whereas the activities in extracellular fluid, extracts of light particulates, and extracts of ejaculated spermatozoa were relatively low. Most of the Pz-peptidase activity in extracts of epididymal spermatozoa was attributable to cytoplasmic droplets. The specific Pz-peptidase activities found by regression analysis were 6.1 mU/billion attached cytoplasmic droplets and 1.1 mU/billion spermatozoa. These results established that in the bovine, cytoplasmic droplets were the major source of Pz-peptidase activity in semen and that Pz-peptidase was not primarily a spermatozoal enzyme.

Study of bovine pulmonary response to Pasteurella haemolytica: specificity of immunoglobulins isolated from the bovine lung.

A procedure is described for the isolation and purification of the immunoglobulins (Ig)A and IgG from pulmonary lavage fluid from calves. These Ig were isolated from calves experimentally exposed to Pasteurella haemolytica via aerosolization and from nonexposed calves. The specificity of these fractions toward P haemolytica was examined, using an indirect fluorescent antibody test and agglutination reactions. Specific antibody activity was detected in the IgA and IgG fractions from calves exposed to P haemolytica and IgG fractions from nonexposed calves. None of the isolated Ig agglutinated P haemolytica.

Failure of immunoperoxidase staining to detect acrosin in the equatorial segment of spermatozoa (1).

An immunoperoxidase staining procedure that readily demonstrated acrosin in the rostral portion of the acrosome failed to detect acrosin in the equatorial segment of spermatozoa representing the mammalian orders artiodactyla (bull and boar), lagomorpha (rabbit) and primate (human).

Activity of eight enzymes of chicken seminal plasma in the eluent from agarose gel chromatography.

Chromatography of seminal plasma from fresh, untreated chicken semen on Bio-Rad A1.5m agarose gel yielded five major peaks of ultraviolet absorbancy at 280 nm. Two peaks with 280/260 absorbancy ratios less than unity suggested the presence of free nucleotides. Enzyme assays on the eluent fractions resulted in substantial single peaks of lactic dehydrogenase, glutamic oxaloacetic transaminase, phosphohexose isomerase, acid phosphatase and alkaline phosphatase activity. Acetylcholinesterase and aminopeptidase assays produced multiple peaks of activity. No trypsin-like enzyme activity was detected, suggesting the presence of a seminal plasma trypsin-like enzyme inhibitor. Molecular weight estimates were obtained for all enzyme activity peaks.