Generation of recombinant bovine interferon tau in the human embryonic kidney cell line and its biological activity.

The objective of this study was to generate recombinant bovine interferon tau (rbIFNT) in mammalian hosts. The complementary DNA encoding bovine IFNT2 was cloned for the construction of pRcRSV-bIFNT2 expression vector. The expression vector was transfected to 293 cells. Transfected cells harboring expression vector were selected with G418. Highly expressing clonal line was adapted to serum-free suspension culture in a spinner flask. The recombinant protein had 24 kDa apparent molecular mass, suggesting being expressed as a glycoprotein, and was purified from serum-free conditioned medium by the combination of Diethylaminoethanol Sepharose ion exchange and Sephacryl S-200 HR gel filtration. A total of 7.3 mg rbIFNT was obtained from 13.5 L conditioned medium. Generated rbIFNT was biologically active in terms of antiviral activity measured by the plaque inhibition assay with Madin-Darby bovine kidney cells and the vesicular stomatitis virus. The recombinant protein was also utilized for immunization to raise antibodies in the rabbit. The generated antibody was capable of use in both Western blotting and the binding assay. The results in the present study suggest that a certain amount of rbIFNT is raised in mammalian hosts by using conventional plasmid vector and its antibody provides useful tools for studies in the biology of bovine IFNT.

Optimized protocol for soluble prokaryotic expression, purification and structural analysis of human placenta specific-1(PLAC1).

Placenta specific -1 (PLAC1) has been recently introduced as a small membrane-associated protein mainly involved in placental development. Expression of PLAC1 transcript has been documented in almost one hundred cancer cell lines standing for fourteen distinct cancer types. The presence of two disulfide bridges makes difficult to produce functional recombinant PLAC1 in soluble form with high yield. This limitation also complicates the structural studies of PLAC1, which is important for prediction of its physiological roles. To address this issue, we employed an expression matrix consisting of two expression vectors, five different E. coli hosts and five solubilization conditions to optimize production of full and truncated forms of human PLAC1. The recombinant proteins were then characterized using an anti-PLAC1-specific antibody in Western blotting (WB) and enzyme linked immunosorbent assay (ELISA). Structure of full length protein was also investigated using circular dichroism (CD). We demonstrated the combination of Origami™ and pCold expression vector to yield substantial amount of soluble truncated PLAC1 without further need for solubilization step. Full length PLAC1, however, expressed mostly as inclusion bodies with higher yield in Origami™ and Rosetta2. Among solubilization buffers examined, buffer containing Urea 2 M, pH 12 was found to be more effective. Recombinant proteins exhibited excellent reactivity as detected by ELISA and WB. The secondary structure of full length PLAC1 was considered by CD spectroscopy. Taken together, we introduced here a simple, affordable and efficient expression system for soluble PLAC1 production.

An effective placental cotyledons proteins extraction method for 2D gel electrophoresis.

Effective protein extraction is essential especially in producing a well-resolved proteome on 2D gels. A well-resolved placental cotyledon proteome, with good reproducibility, have allowed researchers to study the proteins underlying the physiology and pathophysiology of pregnancy. The aim of this study is to determine the best protein extraction protocol for the extraction of protein from placental cotyledons tissues for a two-dimensional gel electrophoresis (2D-GE). Based on widely used protein extraction strategies, 12 different extraction methodologies were carefully selected, which included one chemical extraction, two mechanical extraction coupled protein precipitations, and nine chemical extraction coupled protein precipitations. Extracted proteins were resolved in a one-dimensional gel electrophoresis and 2D-GE; then, it was compared with set criteria: extraction efficacy, protein resolution, reproducibility, and recovery efficiency. Our results revealed that a better profile was obtained by chemical extraction in comparison to mechanical extraction. We further compared chemical extraction coupled protein precipitation methodologies, where the DNase/lithium chloride-dense sucrose homogenization coupled dichloromethane-methanol precipitation (DNase/LiCl-DSH-D/MPE) method showed good protein extraction efficiency. This, however, was carried out with the best protein resolution and proteome reproducibility on 2D-gels. DNase/LiCl-DSH-D/MPE was efficient in the extraction of proteins from placental cotyledons tissues. In addition, this methodology could hypothetically allow the protein extraction of any tissue that contains highly abundant lipid and glycogen.

Chitosan oligosaccharide affects antioxidant defense capacity and placental amino acids transport of sows.

BACKGROUND: Chitosan oligosaccharide (COS) is widely consumed as a functional food due to its multiple health effects, but few studies about COS supplement on placental antioxidant and nutrition transport capacity were reported. Taken pregnant sow as a model, we aimed to investigate the effects of dietary COS supplementation during late gestation on placental amino acids transport and antioxidant defense capacity of sows. From day (d) 85 of gestation to parturition, sixteen pregnant sows were divided into a control group (basal diet without COS supplementation) and a COS group (30 mg COS/kg basal diet). Plasma sample of sow was collected on d 110 of gestation, and placenta tissue was obtained during parturition. Then plasma antioxidant enzyme's activities, the relative level of oxidant stress related genes, amino acids transport related genes and mTOR pathway molecules in placenta were determined. RESULTS: Results showed that maternal dietary supplementation with COS increased (P < 0.05) plasma total SOD, caused a downtrend in plasma MDA (0.05 < P < 0.10) on d 110 of gestation. Interestingly, the mRNA expression of some antioxidant genes in the placenta were increased (P < 0.05) and pro-inflammatory cytokines were reduced (P < 0.05) by COS supplement, whereas no significant difference was observed in the activities of placental total SOD and CAT between two groups. Additionally, further study demonstrated that COS feeding stimulated mTOR signaling pathway, increased amino acids transporters expression in placenta. CONCLUSIONS: These observations suggested that COS supplement in sow's diet during late gestation enhanced antioxidant defense capacity of sows, promoted placental amino acids transport, which may contribute to the health of sows and development of fetus during gestation.

Extremely stable soluble high molecular mass multi-protein complex with DNase activity in human placental tissue.

Human placenta is an organ which protects, feeds, and regulates the grooving of the embryo. Therefore, identification and characterization of placental components including proteins and their multi-protein complexes is an important step to understanding the placenta function. We have obtained and analyzed for the first time an extremely stable multi-protein complex (SPC, ∼ 1000 kDa) from the soluble fraction of three human placentas. By gel filtration on Sepharose-4B, the SPC was well separated from other proteins of the placenta extract. Light scattering measurements and gel filtration showed that the SPC is stable in the presence of NaCl, MgCl2, acetonitrile, guanidinium chloride, and Triton in high concentrations, but dissociates efficiently in the presence of 8 M urea, 50 mM EDTA, and 0.5 M NaCl. Such a stable complex is unlikely to be a casual associate of different proteins. According to SDS-PAGE and MALDI mass spectrometry data, this complex contains many major glycosylated proteins with low and moderate molecular masses (MMs) 4-14 kDa and several moderately abundant (79.3, 68.5, 52.8, and 27.2 kDa) as well as minor proteins with higher MMs. The SPC treatment with dithiothreitol led to a disappearance of some protein bands and revealed proteins with lower MMs. The SPCs from three placentas efficiently hydrolyzed plasmid supercoiled DNA with comparable rates and possess at least two DNA-binding sites with different affinities for a 12-mer oligonucleotide. Progress in study of placental protein complexes can promote understanding of their biological functions.

Human endogenous retrovirus W activity in cartilage of osteoarthritis patients.

The etiology of viruses in osteoarthritis remains controversial because the prevalence of viral nucleic acid sequences in peripheral blood or synovial fluid from osteoarthritis patients and that in healthy control subjects are similar. Until now the presence of virus has not been analyzed in cartilage. We screened cartilage and chondrocytes from advanced and non-/early osteoarthritis patients for parvovirus B19, herpes simplex virus-1, Epstein Barr virus, cytomegalovirus, human herpes virus-6, hepatitis C virus, and human endogenous retroviruses transcripts. Endogenous retroviruses transcripts, but none of the other viruses, were detected in 15 out the 17 patients. Sequencing identified the virus as HERV-WE1 and E2. HERV-W activity was confirmed by high expression levels of syncytin, dsRNA, virus budding, and the presence of virus-like particles in all advanced osteoarthritis cartilages examined. Low levels of HERV-WE1, but not E2 envelope RNA, were observed in 3 out of 8 non-/early osteoarthritis patients, while only 3 out of 7 chondrocytes cultures displayed low levels of syncytin, and just one was positive for virus-like particles. This study demonstrates for the first time activation of HERV-W in cartilage of osteoarthritis patients; however, a causative role for HERV-W in development or deterioration of the disease remains to be proven.

Probing the structure of human protein disulfide isomerase by chemical cross-linking combined with mass spectrometry.

Protein disulfide-isomerase (PDI) is a four-domain flexible protein that catalyzes the formation of disulfide bonds in the endoplasmic reticulum. Here we have analyzed native PDI purified from human placenta by chemical cross-linking followed by mass spectrometry (CXMS). In addition to PDI the sample contained soluble calnexin and ERp72. Extensive cross-linking was observed within the PDI molecule, both intra- and inter-domain, as well as between the different components in the mixture. The high sensitivity of the analysis in the current experiments, combined with a likely promiscuous interaction pattern of the involved proteins, revealed relatively densely populated cross-link heat maps. The established X-ray structure of the monomeric PDI could be confirmed; however, the dimer as presented in the existing models does not seem to be prevalent in solution as modeling on the observed cross-links revealed new models of dimeric PDI. The observed inter-protein cross-links confirmed the existence of a peptide binding area on calnexin that binds strongly both PDI and ERp72. On the other hand, interaction sites on PDI and ERp72 could not be uniquely identified, indicating a more non-specific interaction pattern. BIOLOGICAL SIGNIFICANCE: The present work demonstrates the use of chemical cross-linking and mass spectrometry (CXMS) for the determination of a solution structure of natural human PDI and its interaction with the chaperones ERp72 and calnexin. The data shows that the dimeric structure of PDI may be more diverse than indicated by present models. We further observe that the temperature influences the cross-linking pattern of PDI, but this does not influence the overall folding pattern of the molecule.

Identification of pregnancy-associated glycoproteins and alpha-fetoprotein in fallow deer (Dama dama) placenta.

BACKGROUND: This paper describes the isolation and characterization of pregnancy-associated glycoproteins (PAG) from fetal cotyledonary tissue (FCT) and maternal caruncular tissue (MCT) collected from fallow deer (Dama dama) pregnant females. Proteins issued from FCT and MCT were submitted to affinity chromatographies by using Vicia villosa agarose (VVA) or anti-bovine PAG-2 (R#438) coupled to Sepharose 4B gel. Finally, they were characterized by SDS-PAGE and N-terminal microsequencing. RESULTS: Four distinct fallow deer PAG (fdPAG) sequences were identified and submitted to Swiss-Prot database. Comparison of fdPAG with PAG sequences identified in other ruminant species exhibited 64 to 83% identity. Additionally, alpha-fetoprotein was identified in fetal and maternal tissues. CONCLUSION: Our results demonstrate the efficacy of VVA and bovine PAG-2 affinity chromatographies for the isolation of PAG molecules expressed in deer placenta. This is the first report giving four specific amino acid sequences of PAG isolated from feto-maternal junction (FCT and MCT) in the Cervidae family.

Presence of auto-antibody against two placental proteins, annexin A1 and vitamin D binding protein, in sera of women with pre-eclampsia.

Pre-eclampsia (PE) is one of the most complex and life-threatening pregnancy disorders. PE is characterized by maternal hypertension and proteinuria. There is much evidence to support an immunological etiology for PE and auto-immunity is considered a predisposing factor for PE. The aim of the present study was the investigation of placental proteins as targets for auto-antibodies in PE patients. 2D-PAGE technique was used for separation of the total human placental proteins. After separation, protein spots were transferred to the PVDF membranes and blotted with sera from 20 PE patients and compared with membranes blotted with 20 sera from normal women. MALDI TOF/TOF mass spectrometry technique was used for identification of differentially blotted spots. Moreover, the results of mass analysis were confirmed using western blot with commercial mAbs and RT-PCR technique. The results indicated that two placental proteins, annexin A1 and vitamin D binding protein (DBP), might be targeted by PE sera. The expression of annexin A1 and DBP was also confirmed at RNA level using the RT-PCR technique. Furthermore, the mass results were confirmed by western blotting with commercial mAbs against two targeted proteins. The data of the present study suggest two new placental proteins, annexin A1 and DBP, as placental immune targets. Considering the relation among vitamin D deficiency, increased risk of PE, and the role of annexin A1 in the resolution of inflammation, production of antibody against annexin A1 and DBP may be considered a new auto-immune hypothesis in pre-eclampsia that calls for further investigation in future work.

Purification of pregnancy-associated glycoproteins from late-pregnancy Bubalus bubalis placentas and development of a radioimmunoassay for pregnancy diagnosis in water buffalo females.

BACKGROUND: Pregnancy-associated glycoproteins (PAGs) were first described as placental antigens present in the blood serum of the mother soon after implantation. Here, we describe the purification of several pregnancy-associated glycoproteins from water buffalo placenta (wbPAGs). A specific radioimmunoassay (RIA) was developed for early pregnancy diagnosis in buffalo species. RESULTS: Amino-terminal microsequencing of immunoreactive placental proteins allowed the identification of eleven wbPAGs sequences [Swiss-Prot accession numbers: P86369 to P86379]. Three polyclonal antisera (AS#858, AS#859 and AS#860) were raised in rabbits against distinct wbPAG fractions. A new RIA (RIA-860) was developed and used to distinguish between pregnant (n=33) and non-pregnant (n=26) water buffalo females. CONCLUSIONS: Our results confirmed the multiplicity of PAG expression in buffalo placenta. In addition, the RIA-860 system was shown to be sensitive, linear, reproducible, accurate and specific in measuring PAG concentrations in buffalo plasma samples from Day 37 of gestation onwards.

Expanded protein expression profile of human placenta using two-dimensional gel electrophoresis.

Studying proteins expressed in placenta is important to understand its function in pregnancy and fetal growth. Here, we present protein expression profiling from normal human placenta by 2-D gel - MS/MS approach that resulted in identification of 117 unique proteins. Integration with earlier analyses resulted in a profile of 423 non-redundant proteins, 75 of them being new identifications unique to this study including their isoforms. We present a compilation of placental protein expressions identified by proteomic approaches, their functions and known clinical implications. We believe that our dataset would be a useful resource for studies related to placental dysfunction.

Glycodelin-A stimulates interleukin-6 secretion by human monocytes and macrophages through L-selectin and the extracellular signal-regulated kinase pathway.

Macrophages represent the second major type of decidual leukocytes at the fetomaternal interface. Changes in macrophage number and activity are associated with fetal loss and pregnancy complications. Glycodelin-A (GdA) is an abundant glycoprotein in the first-trimester decidua. It is involved in fetomaternal defense and early placental development through its regulatory activities in various immune cells. The N-glycosylation of GdA mediates the binding and therefore the activities of the molecule. In this study, we studied the biological activities of GdA in the functions of human monocytes/macrophages. GdA was purified from amniotic fluid by affinity chromatography. GdA treatment did not affect the viability, cell death, or phagocytic activity of the monocytes/macrophages. GdA, but not recombinant glycodelin without glycosylation, induced IL-6 production as demonstrated by cytokine array, intracellular staining, and ELISA. GdA also induced phosphorylation of ERK in monocytes/macrophages. The involvement of ERKs in IL-6 induction was confirmed using pharmacological inhibitors. Co-immunoprecipitation showed that L-selectin on the monocytes/macrophages was the binding protein of GdA. Treatment with anti-L-selectin antibody reduced GdA binding and GdA-induced IL-6 production. GdA-treated macrophages suppressed IFN-γ expression by co-cultured T-helper cells in an IL-6-dependent manner. These results show that GdA interacts with L-selectin to induce IL-6 production in monocytes/macrophages by activating the ERK signaling pathway. In turn, the increased IL-6 production suppresses IFN-γ expression in T-helper cells, which may play an important role in inducing a Th-2-polarized cytokine environment that flavors the immunotolerance of the fetoplacental unit.

Human amniotic glycodelin actively regulates changes in ß-catenin immunoreactivity in cultured human umbilical vein endothelial cells (HUVEC).

OBJECTIVE: Assessment of the capacity of Glycodelin A (GdA) to modulate the aggregation of cultured human umbilical vein endothelial cells. METHODS: Highly purified Glycodelin A (GdA) from late first trimester amniotic fluid has been added to cultured cells and its biological activity has been observed with immunofluorescent staining of ß-catenin molecules. RESULTS: GdA induces translocation of ß-catenin molecules promoting cell-to-cell adhesion and formation of adherents junctions through cytoskeletal reorganization. CONCLUSION: These data provide further mechanistic insight into the specificity of cell-to-cell adhesion, thus corroborating the role of GdA in promoting angiogenesis.

Differential actions of glycodelin-A on Th-1 and Th-2 cells: a paracrine mechanism that could produce the Th-2 dominant environment during pregnancy.

BACKGROUND: The maternal-fetal interface has a unique immunological response towards the implanting placenta. It is generally accepted that a T-helper type-2 (Th-2) cytokine prevailing environment is important in pregnancy. The proportion of Th-2 cells in the peripheral blood and decidua is significantly higher in pregnant women in the first trimester than in non-pregnant women. Glycodelin-A (GdA) is a major endocrine-regulated decidual glycoprotein thought to be related to fetomaternal defence. Yet the relationship between its immunoregulatory activities and the shift towards Th-2 cytokine profile during pregnancy is unclear. METHODS: GdA was immunoaffinity purified from human amniotic fluid. T-helper, T-helper type-1 (Th-1) and Th-2 cells were isolated from the peripheral blood. The viability of these cells was studied by XTT assay. Immunophenotyping of CD4/CD294, cell death and GdA-binding were determined by flow cytometry. The mRNA expression, surface expression and secretion of Fas/Fas ligand (FasL) were determined by quantitative polymerase chain reaction, flow cytometry and ELISA, respectively. The activities of caspase-3, -8 and -9 were measured. The phosphorylation of extracellular signal-regulated kinases (ERK), p38 and, c-Jun N-terminal kinase was determined by western blotting. RESULTS: Although GdA bound to both Th-1 and Th-2 cells, it had differential actions on the two cell-types. GdA induced cell death of the Th-1 cells but not the Th-2 cells. The cell death was mediated through activation of caspase -3, -8 and -9 activities. GdA up-regulated the expression of Fas and inhibited ERK activation in the Th-1 cells, which might enhance the vulnerability of the cells to cell death caused by a trophoblast-derived FasL. CONCLUSIONS: The data suggest that GdA could be an endometrial factor that contributes to the Th-2/Th-1 shift during pregnancy.

Identification of pregnancy-associated CA125-reactive protein as a carbohydrate-binding immunoglobulin G.

Cancer antigen 125 (CA125), also referred to as mucin 16, is expressed under both normal and pathological conditions and the complexity of its structure indicates multifunctionality, i.e. both the protein and carbohydrate parts may be involved in diverse interactions at different levels of cell and tissue organization. Its biological role is not understood, but involvement in immune response modulation and influence on cell adhesion have been speculated. This study aimed at isolation and characterization of endogenous ligands for CA125 as an initial step in gaining insight into its activity. A CA125-reactive fraction was separated from human placental extract by affinity chromatography. The isolated preparation was characterized by SDS-PAGE, immunoblotting, peptide mass fingerprinting and binding assay. The CA125-reactive fraction from placental extract was identified as carbohydrate-binding IgG. The glycan composition of inhibitors of carbohydrate-binding pointed to sialic acid as one determinant for recognition but indicated that sialylation was not alone and that glycotopes containing galactose, N-acetylgalactosamine and N-acetylglucosamine were also important. CA125-reactive IgG could be selectively enriched using fetuin as the ligand and represents a distinct IgG subfraction differing from abundant natural carbohydrate-binding antibodies. Taking advantage of the particular properties of ligands for CA125 may have biomedical potential for use as biological modifiers or delivery agents and have an impact beyond pregnancy, since many immunoregulatory molecular pathways are common to embryonic development and malignant transformation.

Ovarian cancer-derived glycodelin impairs in vitro dendritic cell maturation.

Local immunosuppressive mechanisms shape the tumor microenvironment and contribute to carcinogenesis. In ovarian cancer such mechanisms have been shown to influence survival. Dendritic cells (DCs) are central immunity regulators and induce potent cytotoxic T-cell responses as well as peripheral tolerance depending on modulatory stimuli. Here, we show that ovarian cancer-derived glycodelin (Gd), a glycoprotein that physiologically modulates local immunity in early pregnancy, induces a tolerogenic DC phenotype. Gd was isolated with high performance liquid chromatography from the malignant ascites of ovarian cancer patients. DCs were generated from monocytes of healthy donors and exposed to Gd with or without an inflammatory stimulus (tumor necrosis factor-alpha and interleukin 1-beta). We investigated the effect of Gd on DC surface marker expression, endopinocytotic activity, cytokine profile, and lymphoproliferative activity. DCs that were exposed to Gd altered their phenotype as seen by a differential expression of costimulatory molecules, whereas expression of DC-specific intercellular adhesion molecule 3-grabbing nonintegrin, a marker of an immature phenotype, was increased. Functional data provided further evidence for the immature/tolerogenic properties of Gd-pretreated DCs. Antigen uptake was retained, production of interleukin-10 was increased, and lymphoproliferative activity was reduced. This effect was reversible by adding Gd-blocking antibodies. Gd, which is found in the malignant ascites of ovarian cancer patients, induces a tolerogenic phenotype in DC, thereby shaping an immunodeficient tumor micromilieu.

Effects of differential glycosylation of glycodelins on lymphocyte survival.

Glycodelin is a human glycoprotein with four reported glycoforms, namely glycodelin-A (GdA), glycodelin-F (GdF), glycodelin-C (GdC), and glycodelin-S (GdS). These glycoforms have the same protein core and appear to differ in their N-glycosylation. The glycosylation of GdA is completely different from that of GdS. GdA inhibits proliferation and induces cell death of T cells. However, the glycosylation and immunomodulating activities of GdF and GdC are not known. This study aimed to use ultra-high sensitivity mass spectrometry to compare the glycomes of GdA, GdC, and GdF and to study the relationship between the immunological activity and glycosylation pattern among glycodelin glycoforms. Using MALDI-TOF strategies, the glycoforms were shown to contain an enormous diversity of bi-, tri-, and tetra-antennary complex-type glycans carrying Galbeta1-4GlcNAc (lacNAc) and/or GalNAcbeta1-4GlcNAc (lacdiNAc) antennae backbones with varying levels of fucose and sialic acid substitution. Interestingly, they all carried a family of Sda (NeuAcalpha2-3(GalNAcbeta1-4)Gal)-containing glycans, which were not identified in the earlier study because of less sensitive methodologies used. Among the three glycodelins, GdA is the most heavily sialylated. Virtually all the sialic acid on GdC is located on the Sda antennae. With the exception of the Sda epitope, the GdC N-glycome appears to be the asialylated counterpart of the GdA/GdF glycomes. Sialidase activity, which may be responsible for transforming GdA/GdF to GdC, was detected in cumulus cells. Both GdA and GdF inhibited the proliferation, induced cell death, and suppressed interleukin-2 secretion of Jurkat cells and peripheral blood mononuclear cells. In contrast, no immunosuppressive effect was observed for GdS and GdC.

Detection, purification and identification of an endogenous inhibitor of L-Dopa decarboxylase activity from human placenta.

An endogenous inhibitor of L-Dopa decarboxylase activity was identified and purified from human placenta. The endogenous inhibitor of L-Dopa decarboxylase (Ddc) was localized in the membrane fraction of placental tissue. Treatment of membranes with phosphatidylinositol-specific phospholipase C or proteinase K did not affect membrane-associated Ddc inhibitory activity, suggesting that a population of the inhibitor is embedded within membranes. Purification was achieved by extraction from a nondenaturing polyacrylamide gel. The purification scheme resulted in the isolation of a single 35 kDa band, bearing L-Dopa decarboxylase inhibitory activity. The purified inhibitor was identified as Annexin V. The elucidation of the biological importance of the presence of an L-Dopa decarboxylase activity inhibitor in normal human tissues could provide us with new information leading to the better understanding of the biological pathways that Ddc is involved in.

Identification of multiple pregnancy-associated glycoproteins (PAGs) purified from the European bison (Eb; Bison bonasus L.) placentas.

This paper describes the first identified chorionic PAGs in the European bison (Eb), named EbPAGs, predominantly expressed during early and mid-pregnancy (45-120 day post-coitum; dpc). Many EbPAGs were extracted from various cotyledonary tissues, precipitated, chromatographed (DEAE and VVA: Vicia villosa agglutinin), electrophoresed (1D- and 2D-PAGE), analysed by heterologous (cross-species) Western blotting and then micro-sequenced by Edman degradation. Finally, twelve selected VVA-purified isoforms (Ip 3.7-7.4) were entirely characterised. Nine identified NH(2)-terminal micro-sequences were found to be PAGs. On 45 dpc, three identified forms were named: EbPAG(67AkDa) (RGSNLTHPLRNIGDLFYVGN), EbPAG(55BkDa) (RGSNLTHPL) and EbPAG(50CkDa) (SQISLRGSNLTI). On 60 dpc, the next three forms were named: EbPAG(71DkDa) (RGSNLTIHPLRNIIDLFYVG), EbPAG(55EkDa) (RGSNLTHPLRNI) and EbPAG(50FkDa) (SQISLRGS). On 120 dpc, three other forms were named: EbPAG(71GkDa) (RGSNLTHPLRNIRDLFYVG), EbPAG(60HkDa) (RGSNLTTHPLRNIKDLVVYM) and EbPAG(50IkDa) (SGSNLTTV). These EbPAG ((A-I)) sequences are unique, as they are not identical to any other PAGs purified previously in related species of the Bovidae family. However, the EbPAGs (A-I forms) have some sequence resemblance to internal sequences of various full-length polypeptide PAG precursors (in silico translated from cloned cDNAs) identified in domestic cattle. Three other novel native isoforms (J1, J2 and K): EbUPG(45kDa) J1 (SKDNYKNYIPLIVPFAT), EbUPG(45kDa) J2 (SKDNQKNYIPLIVPFAT) and EbUPG(76kDa) K (SPEFTV), were temporarily named 'unknown placental glycoproteins' (UPGs), due to their efficient VVA-purification (specific for glycoproteins only) and a lack of considerable consensus to previously sequenced placental glycoproteins in the Bovidae family. This is the first study identifying NH(2)-terminals of multiple/diverse EbPAGs and some EbUPGs purified from the synepitheliochorial cotyledonary placenta of the endangered Bison bonasus (Red List).

Glycodelin A, an immunomodulatory protein in the endometrium, inhibits proliferation and induces apoptosis in monocytic cells.

Glycodelin A (GdA), is a lipocalin with an immunomodulatory role, secreted by the endometrium under progesterone regulation and proposed to play a role in protecting the fetus from maternal immune attack. Glycodelin A has an inhibitory effect on the proliferation of T cells and B cells and also on the activity of natural killer cells. We have earlier demonstrated that the inhibitory effect of glycodelin A on T cell proliferation is due to apoptosis induced in these cells through the caspase-dependent intrinsic mitochondrial pathway. Studies reported until now have shown that glycodelin modulates the adaptive immune responses. We, therefore, decided to look at its effect, if any, on the innate immune system. The effect of glycodelin on monocytes was studied using human monocytic cell lines, THP1 and U937, and primary human monocytes as model systems. We demonstrated that glycodelin inhibited the proliferation of THP1 and U937 and induced apoptosis in these cells as well as in primary monocytes. We found that this signaling was caspase-independent but followed the intrinsic mitochondrial pathway of apoptosis. No effect of glycodelin was seen on the phagocytic ability of monocytes post-differentiation into macrophages. These observations suggest that, at the fetomaternal interface, glycodelin plays a protective role by deleting the monocytes that could become pro-inflammatory. Importantly, leaving the macrophages untouched to carry on with efficient clearance of the apoptotic cells.