We hypothesize that the mechanisms governing bone formation and remodeling involve the assembly of some of the components of the extracellular matrix into supramolecular complexes. We have examined the associations of osteopontin (OPN) with other proteins isolated from demineralized rat long bones. Three ligand binding techniques were used to demonstrate the formation of complexes between osteopontin and osteocalcin (OCN). Using gel overlay assays, the binding between soluble 125I-OPN and OCN immobilized in acrylamide gels was visualized. Competition for 125I-OPN-OCN complexes was demonstrated when unlabeled OCN-enriched bone extract was included in gel overlay solutions. Also, gel overlay assays showed 125I-OCN binding to OPN. Saturable binding was shown in solid-phase filter binding assays, which yielded an equilibrium binding constant of moderately high affinity (approximately 10(-8) M). Specificity of OPN-OCN complex formation was confirmed by measuring binding in the presence of unlabeled OPN and OCN versus a bone-localized serum protein, alpha 2HS-glycoprotein. Finally, the formation of soluble complexes were demonstrated in a modified Hummel-Dreyer gel filtration assay. These results indicate that OPN and OCN form complexes in vitro. The possible functions of OPN-OCN complexes in osteoclast recruitment and attachment are discussed.

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During parturition, uterine-derived prostaglandins (PG) play an outstanding role regarding the functional elimination of the corpus luteum and the promotion of uterine contraction. The rate-limiting enzyme cyclooxygenase-2 (COX-2), highly regulated in a cell-type and localization specific manner throughout pregnancy, is involved in uterine prostanoid production. Prostaglandins exert their effects via G-protein-coupled receptors. Distribution and cellular localization of these receptors are decisive factors for prostaglandin-mediated actions. Since both COX-2 and PG receptors have only been assessed during pregnancy in the cow, these parameters were localized immunohistochemically near term to evaluate their specific role at parturition. Thus, during two periods, segments of the intercaruncular uterine wall were collected from cows at slaughter being eight and nine months pregnant, from cattle during caesarean section, and after spontaneous calving. Results reveal that COX-2 was mainly localized in the cytoplasm of surface epithelial cells with a high expression in animals with induced parturition. The enzyme could also be found in lower concentrations within the glandular epithelium without any effect of gestational time or labour. In contrast to relaxant prostaglandin E receptor type 2 (EP2), not showing any change in all tissue layers observed, contractile prostaglandin F(2alpha) receptor (FP) was modulated during the peripartal period revealing a peak expression in animals with induced parturition. FP was localized in surface and glandular epithelial cells as well as in endometrial stroma and myometrial smooth muscle cells. Our study indicates that labour and induction of parturition may have an effect on amounts of immunohistochemically detectable COX-2 and FP. EP2 remains rather unchanged during the peripartal period. COX-2 and FP thus contribute via changes in amount and distribution to mechanisms associated with parturition. During parturition uterine derived prostaglandins (PG) play an outstanding role 2 regarding the functional elimination of the corpus luteum and the promotion of uterine 3 contraction. The rate-limiting enzyme Cyclooxygenase-2 (COX-2), highly regulated in 4 a cell-type and localization specific manner throughout pregnancy, is involved in 5 uterine prostanoid production. Prostaglandins exert their effects via G protein-6 coupled receptors. Distribution and cellular localization of these receptors are 7 decisive factors for prostaglandin-mediated actions. Since both, COX-2 and PG 8 receptors have only been assessed during pregnancy in the cow, these parameters 9were localized immunohistochemically near term to evaluate their specific role at 10 parturition. Thus, during two periods, segments of the intercaruncular uterine wall 11 were collected from cows at slaughter being eight and nine months pregnant, from 12 cattle during caesarean section, and after spontaneous calving.

Loss-of-function mutations in Kv7.1 often lead to long QT syndrome (LQTS), a cardiac repolarization disorder associated with arrhythmia and subsequent sudden cardiac death. The discovery of agonistic IKs modulators may offer a new potential strategy in pharmacological treatment of this disorder. The benzodiazepine derivative (R)-L3 potently activates Kv7.1 channels and shortens action potential duration, thus may represent a starting point for drug development. However, the molecular mechanisms underlying modulation by (R)-L3 are still unknown. By combining alanine scanning mutagenesis, non-canonical amino acid incorporation, voltage-clamp electrophysiology and fluorometry, and in silico protein modelling, we show that (R)-L3 not only stimulates currents by allosteric modulation of the pore domain but also alters the kinetics independently from the pore domain effects. We identify novel (R)-L3-interacting key residues in the lower S4-segment of Kv7.1 and observed an uncoupling of the outer S4 segment with the inner S5, S6 and selectivity filter segments.

The bovine intercaruncular uterine wall expresses steroid hormone receptors throughout pregnancy. Concentrations of specific hormones undergo massive changes during the peripartal period and modulate the synthesis of their own receptors. This is well documented for the placentome, but respective data concerning the intercaruncular uterine wall are completely lacking. Thus, intercaruncular uterine wall segments from cows (I) being 8 and 9 months pregnant (slaughtered cows) and (II) cows undergoing a premature caesarean section 269-282 days after artificial insemination (AI) with (IIa, b) or without (IIc) induction of birth with PGF(2alpha) agonist or (III) receiving a caesarean section during severe dystocia (n=6, 5, 5, 5, 6 and 4 animals, respectively) were studied. In four naturally calving cows (IV) endometrial biopsies were obtained within 30 min after the expulsion of the calf. All tissue probes were fixed for 24h in 4% formaldehyde, routinely embedded in paraffin, and cut at 4 microm. Progesterone receptors (PR), estrogen receptor alpha (ERalpha) and glucocorticoid receptors (GR) were assessed using specific antibodies and staining intensities were documented employing an immunoreactive score (IRS). PR, ERalpha and GR exhibited cell type- and location-specific distribution patterns. IRS for PR and ERalpha did not differ between groups. GR-IRS of endometrial stromal cells, however, were higher in animals undergoing premature caesarean section after induction of birth compared to animals slaughtered during month 8 or 9 of pregnancy or animals receiving caesarean section following dystocia. Results of the present study indicate that steroid hormone receptor amounts within the intercaruncular uterine wall do not (PR, ERalpha) - or in a tissue-specific manner (GR) only - change during the peripartal period, although respective hormones undergo massive changes during this period. This is in strict contrast to the placentome. Comparatively lower local tissue estrogen concentrations around term may be one cause for this difference.

Blood–brain barrier (BBB) integrity is necessary to maintain homeostasis of the central nervous system (CNS). NMDA receptor (NMDAR) function and expression have been implicated in BBB integrity. However, as evidenced in neuroinflammatory conditions, BBB disruption contributes to immune cell infiltration and propagation of inflammatory pathways. Currently, our understanding of the pathophysiological role of NMDAR signaling on endothelial cells remains incomplete. Thus, we investigated NMDAR function on primary mouse brain microvascular endothelial cells (MBMECs). We detected glycine-responsive NMDAR channels, composed of functional GluN1, GluN2A and GluN3A subunits. Importantly, application of glycine alone, but not glutamate, was sufficient to induce NMDAR-mediated currents and an increase in intracellular Ca2+ concentrations. Functionally, glycine-mediated NMDAR activation leads to loss of BBB integrity and changes in actin distribution. Treatment of oocytes that express NMDARs composed of different subunits, with GluN1 and GluN3A binding site inhibitors, resulted in abrogation of NMDAR signaling as measured by two-electrode voltage clamp (TEVC). This effect was only detected in the presence of the GluN2A subunits, suggesting the latter as prerequisite for pharmacological modulation of NMDARs on brain endothelial cells. Taken together, our findings argue for a novel role of glycine as NMDAR ligand on endothelial cells shaping BBB integrity.

Whole boar semen or seminal plasma has been demonstrated to advance the time of ovulation in gilts. As a means of clarifying this influence, the contribution of uterine lymphatics and their white cell populations has been examined. After duct visualisation with Evan's blue, lymph was sampled from a mesometrial vessel in eight pre-ovulatory gilts whose uterine lumen was infused simultaneously with whole semen in one ligated horn and saline in the contralateral ligated horn. Lymph was collected from cannulated vessels for periods of up to four hours under general anaesthesia. Thereafter, mesometrial lymph nodes, utero-tubal junction and uterine wall tissues were sampled. The proportion of nucleated cells in the sampled lymph increased towards the end of the collection period, but erythrocytes were found in all instances preventing a meaningful differentiation and identification of leukocytes. Prominent uterine lymph nodes were present in the mesometrium on both sides of the reproductive tract in 7 of 10 gilts. Differences in cellular contents were demonstrated between the side of the tract infused with semen and that infused with saline control. Two of 4 gilts had lower values for CD4 (Cluster Differentiation) and 3 of 6 gilts higher values for MHC II (Major Histocompatibility Complex) markers on the side challenged with semen. In contrast, values remained constant for CD8 but ranged widely for CD18. Immunohistochemical analysis of uterine tissue samples for MHC II+ cells revealed significant differences (P < 0.05) between the control and semen-treated ligated portions of the horns, as well as between the tissue sample of uterine wall and that from the utero-tubal junction, but there were no significant differences for CD4+ cells. It therefore remains plausible that semen-induced cytokines in the uterine lymph undergo counter-current transfer to the ipsilateral ovary and accelerate the final maturation of pre-ovulatory Graafian follicles.

Previous studies have shown that in vitro calcification of chick epiphyseal cartilage matrix vesicles is proteolipid-dependent. The purpose of this research is to examine the role of proteolipid in cartilage calcification in vivo by comparing the proteolipid concentration of normal and vitamin D-deficient chick epiphyseal cartilage, the relationship of proteolipid to other tissue lipids, and its ability to support in vitro apatite formation. Proteolipid was isolated from the upper growth centers (reserve cell zone, upper proliferative zone) and lower growth centers (lower proliferative, hypertrophic, and calcified cartilage zones) of long-bone epiphyses from 3-week-old normal and rachitic male white leghorn chicks by Sephadex LH-20 chromatography of the crude phospholipid component of the total lipid extract. In both normal and rachitic tissue the proteolipid/dry weight and proteolipid/total lipid ratios were greater in the lower growth center than in the upper zones. No statistically significant change in the proteolipid/total lipid ratio in rachitic tissues relative to comparable cell zones in normal cartilages was observed. However, there was an increase in the nonproteolipid phospholipid content of rachitic tissues, altering the relative proteolipid/phospholipid composition. Whereas proteolipids from normal tissue supported in vitro calcification, proteolipids from rachitic tissues did not, indicating a direct effect of vitamin D on proteolipid structure. These data support the hypothesis that failure of rachitic cartilage to calcify in vivo may be due in part to alterations in phospholipid and proteolipid metabolism.