The synthesis and secretion of proteins by the boar genital tract were studied in vitro by incubating epididymal tissues with [35S]methionine and cysteine. Characterization of the major neosynthesized proteins was performed electrophoretically by one- and two-dimensional PAGE analysis, and an epididymal protein cartography was established. Some of the proteins secreted were found to be unregionalized. Polarization studies of the secretions in the epididymal tubule were carried out by in vitro incubation of isolated tubules, and most of these unregionalized proteins were found not to be secreted in the epididymal lumen. Inside the epididymal lumen, protein secretion was highly regionalized, and electrophoresis analysis detected few proteins secreted at all points along the organ. A total of 146 epididymal proteins, covering 220 spots, were found to be secreted by the epididymis. The distal caput showed the highest number of spots, the lowest number of proteins secreted being found in the proximal caput and cauda. Most of the epididymal proteins analyzed are highly polymorphic in terms of both isoelectric point and molecular mass. The presence and importance of the different compounds in the various regions of the epididymis were established. Several distinct secretory regions of the epididymis can be determined by the presence of major characteristic proteins. The concentrations of a given protein in the fluids of various regions were not related to the respective secretion intensity of that protein. Identification of some major epididymal proteins was accomplished by N-terminal amino microsequencing and by the use of specific antisera. Of the various major proteins, clusterin, glutathione peroxidase, retinol-binding protein, lactoferrin, EP4, beta-N-acetyl-hexosaminidase, alpha-mannosidase, and procathepsin L were identified and localized along the organ. Several polypeptides found in this study remain unidentified.
Background and purpose:The adhesion molecule mucosal addressin cell adhesion molecule (MAdCAM) plays an essential role in the recruitment of lymphocytes to specialized high endothelial venules of the gastrointestinal tract and in their excessive tissue extravasation observed in inflammatory conditions, such as Crohn's disease. We have characterized the in vitro pharmacological properties of two monoclonal antibodies blocking MAdCAM, MECA-367 and PF-00547659, and determined their pharmacokinetic/pharmacodynamic profiles in vivo. Experimental approach: Functional adhesion assays and surface plasmon resonance were used to characterize, in vitro, the pharmacological properties of MECA-367 and PF-00547659. The in vivo effects of MECA-367 and PF-00547659 on restriction of b7 + memory T lymphocytes were determined in mice and macaques, respectively, over the pharmacological dose range to confirm pharmacokinetic/pharmacodynamic relationships. Key results: MECA-367 and PF-00547659 bound with high affinity to mouse and human MAdCAM with Kd values of 5.1 and 16.1 pmol·L -1 respectively and blocked the adhesion of a4b7 + leukocytes to MAdCAM with similar potency. MECA-367 and PF-00547659 induced a similar, dose-dependent two-to threefold increase in circulating populations of b7 + memory T-cells in the mouse and macaque; without affecting the b7 -populations. Conclusions and implications: PF-00547659 has potential utility in the treatment of inflammatory conditions by blocking tissue homing of activated a4b7 + leukocytes. The characterization of a rodent cross-reacting antibody as a surrogate for PF-00547659 in the search for potential pharmacological biomarkers and the determination of efficacious doses was effective in addressing the restricted orthologous cross-reactivity of PF-00547659 and the challenges this poses with respect to efficacy and safety testing.
The number of proteins secreted by the boar epididymis increased progressively from 1 mo of age to the adult period. The first specific secretory activity was revealed at 2 mo in the distal caput (hexosaminidase, clusterin, and lactoferrin) and in the corpus (train O/HE1). Train A and glutathione peroxidase specific to the proximal caput, and trains E and M specific to the corpus, appeared at 4 mo. At 5 mo, secretion of procathepsin L occurred in the middle caput and that of mannosidase and E-RABP in the distal caput. Approximately 48% of all the proteins secreted in the adult boar epididymis were dependent on the presence of androgens, either stimulated (33.6%) or repressed (14.4%); 47% were modulated by other factors, and 5% were unregulated. In the proximal caput, 50% of the specific secreted proteins were controlled essentially by factors emanating from the testis. In more distal regions, two proteins secreted in the corpus were regulated by factors from the anterior regions. The regionalization of the secretory activity of the epididymal epithelium resulted in a specific regulation for each protein, which was modulated according to the region of expression and influenced by either testicular or epididymal factors that remain to be identified.
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