Prolactin receptor heterogeneity in bovine fetal and maternal tissues.

Study of diverse PRL actions at a variety of fetal and maternal targets during pregnancy is complicated by receptor heterogeneity and multiple ligands circulating at this time. In the present studies, we have examined PRL receptors at a variety of potential targets by reverse transcription-PCR and Western analysis. Bovine tissues contain two different transcripts for the PRL receptor; the one that encodes a short form includes an additional 39 bases at a position identical to the deviation from the long form found in rodents and sheep. Western analyses of PRL receptors in microsomal fractions from various maternal and fetal tissues revealed considerable size heterogeneity. Collectively, the larger immunoreactive moieties (apparent Mr 100 kDa or greater) and the smaller species (47-55 kDa) correlated well with the relative abundance of the transcripts for the different forms of the receptor and varied considerably among tissues. N-Glycosylation was shown to be the major, but not the only, modification of both receptor forms when transiently transfected into COS-7 and END-6.2 cells. Much of the short form could be reduced to the mobility predicted from the complementary DNA by culture with tunicamycin; this was not true of the long form, suggesting modifications specific for its cytoplasmic domain. Differences in the pattern of immunoreactive species in the COS-7 and END-6.2 cells are consistent with cell-specific modifications. The ability of these receptor forms to mediate a transcriptional response to PRL and its placental relative, placental lactogen, was evaluated with a PRL response element inserted upstream from a thymidine kinase promoter/reporter gene construct transiently transfected into CHO-K1 cells. Both hormones were able to stimulate reporter gene expression through the long form, but not the short form, of the receptor. These studies will facilitate examination of tissue-specific actions of PRL and related hormones during pregnancy.

Glycoproteins of the aspartyl proteinase gene family secreted by the developing placenta.

Pregnancy in cattle and sheep can be diagnosed by the presence of placentally-derived antigens (pregnancy-associated glycoproteins or PAG-1) in maternal serum soon after implantation begins at about Day 20 following conception. Molecular cloning of their cDNA has revealed that PAG-1 belong to the aspartic proteinase gene family and have about 50% amino acid sequence identity to pepsin. However, critical amino acid substitutions at the active site regions suggest that both bovine and ovine PAG-1 are enzymatically inactive. PAG-1 expression has been shown by in situ hybridization and immunocytochemistry to be localized to the trophoblast binucleate cells, which invade maternal uterine endometrium during implantation. The glycoproteins are concentrated in dense cytoplasmic granules that are discharged after the binucleate cells have migrated to the maternal side of the placental barrier. We suggest, therefore, that the PAG-1 might have an endocrine function either as carriers of other bioactive peptides or by acting as hormones themselves. Recently screening of placental libraries with nucleic acid probes has identified additional cDNA that are very abundant and code for polypeptides (PAG-2 and PAG-3) related to, but antigenically and structurally distinct from PAG-1 described above. These molecules have sequences of amino acids at their catalytic centers that are consistent with their being potentially functional proteinases but their role during pregnancy, like that of PAG-1, is unclear.

Instrument flight performance under the influence of certain combinations of antiemetic drugs.

Two different combinations of antiemetic drugs were evaluated using a digital flight simulator. Drug treatments consisted of a lactose placebo, a combination of thiethylperazine (10 mg) and cimetidine (300 mg), and a combination which added promethazine (25 mg) to the two-drug combination. The performance effects of these combinations were evaluated on both a dual task (instrument flight task with the Sternberg Memory Scanning task) and a single task condition (Sternberg task only) for 3 h post drug ingestion. Analysis indicated a significant treatment effect on three of the six flight performance variables and that the three-drug combination, containing promethazine, was primarily responsible for the decrease in performance. Implications for operation in a radiation environment are that thiethylperazine and cimetidine will not cause significant performance decrements, but the addition of promethazine to those two drugs will significantly impair performance. The Sternberg task was sensitive to changes in workload.

Specific binding of an exonic splicing enhancer by the pre-mRNA splicing factor SRp55.

Purine-rich exonic splicing enhancers (ESEs) have been identified in many alternatively spliced exons. Alternative splicing of several ESE-containing exons has been shown to depend on subsets of the SR protein family of pre-mRNA splicing factors. In this report, we show that purified SR protein family member SRp55 by itself binds a 30-nt ESE-containing exon, the alternatively spliced exon 5 of avian cardiac troponin T. We show that purified SRp55 binds specifically to this RNA sequence with an apparent Kd of 60 nM as assayed by gel mobility retardation experiments. Mutations in the exon 5 sequence that increase or decrease exon 5 inclusion in vivo and in vitro have correspondingly different affinities for SRp55 in our assays. The exon 5 sequence contains two purine-rich motifs, common to many ESEs, and both are required for SRp55 binding. Hill plot analysis of binding titration reactions indicates that there is a cooperative binding of at least two SRp55 proteins to the exon sequence. Chemical modification interference studies using kethoxal show that SRp55 binding to exon 5 requires the N1 and/or the N2 of almost every G residue in the exon. Dimethylsulfate modification interference studies indicate that none of the N1 positions of A residues in the exon are important for binding. We postulate that SRp55 may recognize both primary sequence and RNA secondary structural elements within pre-mRNA.

Trophoblast-specific processing and phosphorylation of pregnancy-associated glycoprotein-1 in day 15 to 25 sheep placenta.

Bovine and ovine pregnancy-associated glycoproteins-1 (PAG-1) are products of binucleate trophoblast cells and belong to the aspartic proteinase gene family. Estimates of their relative molecular masses have varied considerably, from 47 to 90 kDa, even though the mature polypeptide has been inferred to be no more than 330 amino acids in length and that the glycosylated recombinant form synthesized in Chinese hamster ovary (CHO) or COS-1 cells had an apparent mass of 46 kDa. To establish the relationships among the various molecular forms, metabolic labeling, immunoprecipitation, and electrophoretic analysis were used to follow the biosynthesis of ovine PAG-1 (ovPAG-1) in placental explants. In time-course studies, ovPAG-1 could first be detected within 10 min as a 70-kDa form within the tissue. With time, forms of intermediate (53-61 kDa) and low (47 kDa) molecular mass began to accumulate. The latter predominated in medium after 6 h labeling. Pulse chase studies established that the 70-kDa forms were the precursors of the smaller species. Inhibition of glycosylation with tunicamycin or treatment with N-glycosidase F confirmed that ovPAG-1 contained N-linked oligosaccharide chains, but that this carbohydrate accounted for only a relatively small fraction (8-10 kDa) of the apparent mass. Consecutive treatment with neuraminidase and O-glycanase also reduced the apparent molecular mass of the precursor by approximately 11 kDa. OvPAG-1 incorporated 32P from [32P]orthophosphate into phosphoserine and phosphothreonine, but there was no incorporation of 35S from [35S]sulfate. The basis of the differences in molecular mass between the precursor and the final products remains to be elucidated, but the differences seem likely to be due to some unusual form of posttranslational modification introduced in the binucleate cell. The results of the study appear to explain the disparate size values that have been reported for these placenta-derived proteins.

A novel glycoprotein of the aspartic proteinase gene family expressed in bovine placental trophectoderm.

The pregnancy associated glycoproteins (PAG 1) that appear in the maternal serum of cattle and sheep soon after implantation are apparently inactive members of the aspartic proteinase family. Here we describe the isolation of a highly abundant cDNA (PAG 2 cDNA) that represents a second member of this gene family which is structurally related to bovine PAG 1, ovine PAG 1, and pepsin (58%, 58%, and 51% amino acid sequence identity, respectively). The bovine PAG 2 cDNA was identified in two ways. First, the bovine placental library was screened under relatively nonstringent conditions with an ovine PAG 1 cDNA. The second fortuitous approach employed immunoscreening with an antiserum raised against a partially purified factor that competed with bovine LH for binding to the LH receptor on the CL of the ovary. The full-length cDNA (1258 bp) codes for a polypeptide of 376 amino acids. Bovine PAG 2, unlike bovine PAG 1, has a catalytic center with a consensus sequence of amino acids. Its mRNA is expressed in fetal placenta but not in other fetal organs, and is localized to both the mononucleate and binucleate cells of the trophectoderm, whereas PAG 1 is expressed only in binucleate cells. PAG 2 is synthesized by placental explants as a 70-kDa glycoprotein that is processed to several smaller molecules. Western blot analysis of culture media developed with epitope-selected antibodies to PAG 2 reveals several bands ranging in apparent M(r) from 31,000-70,000, which correspond in size to the polypeptides present in the preparation used for immunization.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative modelling and analysis of amino acid substitutions suggests that the family of pregnancy-associated glycoproteins includes both active and inactive aspartic proteinases.

The pregnancy-associated glycoproteins (PAGs) are secretory products synthesized by the outer epithelial cell layer (chorion) of the placentas of various ungulate species. The amino acid sequences of eight PAGs have been inferred from cloned cDNA of cattle and sheep, as well as of the non-ruminant pig and horse. We compare the PAG sequences and present results of the three-dimensional models of boPAG-1 and ovPAG-1 that were constructed on the basis of the crystal structures of homologous porcine pepsin and bovine chymosin using a rule-based comparative modelling approach. Further, we compare peptide binding subsites defined by interactions with pepstatin and a decapeptide inhibitor (CH-66) modelled on the basis of crystal structures of other aspartic proteinases. We have extended our analysis of the peptide binding subsites to the other PAG molecules of known sequence by aligning the PAG sequences to the structural template derived from the pepsin family and by making use of the three-dimensional models of the boPAG-1 and ovPAG-1. The residues that are likely to affect peptide binding in the boPAG-1, ovPAG-1 and other PAG molecules have been identified. Sequence comparisons reveal that all PAG molecules may have evolved from a pepsin-like progenitor molecule with the equine PAG most closely related to the pepsins. The presence of substitutions at the S1 and other subsites relative to pepsin make it unlikely that either bovine, ovine or the porcine PAG-1 have catalytic activity. Only two of the eight PAGs examined (porcine PAG-2 and equine PAG-1) retain features of active aspartic proteinases with pepsin-like activity. Our results indicate that in the PAGs so far characterized the peptide binding specificities differ significantly from each other and from pepsin, despite their high sequence identities. Analysis of the various peptide binding subsites demonstrates why both bovine and ovine PAG-1 are capable of binding pepstatin. The strong negative charge in the binding cleft of boPAG-1 and ovPAG-1 indicates a preference for lysine- or arginine-rich peptides. PAGs represent a family where the possible peptide binding function may be retained through their binding specificities, but where the catalytic activity may be lost in some cases, such as the boPAG-1, ovPAG-1 and the poPAG-1.

Identification of the major pregnancy-specific antigens of cattle and sheep as inactive members of the aspartic proteinase family.

Pregnancy in cattle and sheep can be diagnosed by the presence of a conceptus-derived antigen in maternal serum that is secreted by trophoblast and placental tissue primarily as an acidic component of Mr 67,000. Molecular cloning of its cDNA reveals that the antigen belongs to the aspartic proteinase family and has greater than 50% amino acid sequence identity to pepsin, cathepsin D, and cathepsin E. The inferred sequences of the ovine and bovine polypeptides show approximately 73% identity to each other. Critical amino acid substitutions at the active site regions suggest that both proteins are enzymatically inactive. The antigen is a product of trophoblast binucleate cells that invade maternal endometrium at implantation sites.