Biomedical subjects

R M Roberts

Publications and source records attributed to R M Roberts.

At least 73 records · Page 4Linked to original sources

PubMed1996

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.

Amino Acid Sequence↗

PubMed1996

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.

Amino Acid Sequence↗

PubMed1996

Ontogeny and regulation of luteinizing hormone receptor messenger ribonucleic acid within the ovine corpus luteum.

Although LH regulates corpus luteum (CL) function and LH receptor (LHR) binding has been characterized within ovine CL, changes in LHR mRNA expression have not been documented. The objectives here have been to assess relative amounts of LHR mRNA in ovine CL throughout the estrous cycle and during luteolysis by ribonuclease protection analysis. In experiment 1, LHR mRNA was quantified within CL collected on Days 3, 7, 10, 13, and 16 post-estrus (n = 3, 4, 4, 3, and 4, respectively). LHR mRNA differed throughout the luteal phase (p < 0.01), with higher concentrations found during the midluteal phase (Days 10 and 13) than during the early (Day 3) or late (Day 16) luteal phase (p < or = 0.05). In experiment 2, LHR mRNA content was determined within pools of Day 10 small (n = 4) or large (n = 4) luteal cells. Relative amounts of LHR mRNA were not different (p = 0.1) in small (2.23 +/- 0.22 relative units) vs. large (1.62 +/- 0.22 relative units) luteal cells. In experiment 3, CL were collected at 0, 6, 12, or 24 h after injection of 15 mg prostaglandin F2 alpha) on Day 10 post-estrus (n = 4, 4, 3, and 4, respectively). LHR mRNA content fell during PGF2 alpha-induced luteolysis (p < 0.002), decreasing significantly by 6 h (1.76 vs. 1.10 relative units; p < or = 0.05). By 24 h, levels were approximately 60% of those detected within 0-h controls (1.76 vs. 0.79 relative units; p < 0.05). These results indicate that changes in LHR mRNA during the luteal phase correlates well with previously reported changes in LHR binding. After PGF2 alpha administration, LHR mRNA falls earlier than the reported drop in LHR binding sites occurs.

Animals↗

PubMed1996

Maternal recognition of pregnancy.

Maternal recognition of pregnancy reflects the various ways in which the mother responds to the presence of a conceptus within her reproductive tract. A part of the biochemical information she senses may be irrelevant to pregnancy outcome, but some reflects the attempts by the conceptus to gain some measure of control over corpus luteum function, uterine blood supply, the mother's immune system, and other aspects of maternal physiology. Most probably as a result of ongoing genetic conflict between the mother and the conceptus, a bewildering range of placental structures and trophoblast signaling mechanisms are encountered in eutherian mammals despite the fact that the uterus and conceptus share a common interest, which is the successful outcome of the pregnancy. Here we review some of the ways that such mammals maintain luteal function in early pregnancy and briefly discuss the related topics of embryonic loss and maternal monitoring of conceptus fitness. We next address the view that the conceptus is an intruder, recognized as foreign by the mother, that likely survives by using strategies analogous to those employed by successful parasites. In this context, we describe the pregnancy-associated glycoproteins, multiple isoforms of which are released at the trophoblast-endometrial interface during pregnancy of ungulate species. These molecules, which are structurally related to pepsin, are proposed to bind and sequester antigenic peptides, thereby serving an immunoprotective role.

Animals↗

PubMed1996

Differential gene expression within the ovine corpus luteum: identification of secreted protein acidic and rich in cysteine as a major secretory product of small but not large luteal cells.

Limited information is available regarding secretory proteins of the corpus luteum (CL), and the potential local role these proteins may play in control of luteal function. An ovine small luteal cell complementary DNA library was immunologically screened with a polyclonal antiserum generated against small cell secretory proteins. A relatively abundant complementary DNA (approximately 2.1 kb) encoding a calcium binding glycoprotein Secreted Protein Acidic and Rich in Cysteine (SPARC) was isolated. Production of SPARC protein by ovine luteal cells was confirmed by immunoprecipitating it from labeled culture medium. Expression of SPARC messenger RNA (mRNA) was examined within CL collected on days 3, 7, 10, 13, and 16 post estrus (n = 4, 4, 4, 3, and 4 respectively), and within pools of purified small (n = 3) and large (n = 4) luteal cells by Northern and dot blot analysis. Amounts of SPARC mRNA increased during the early luteal phase, peaked by day 7 (P < 0.05) and subsequently declined on days 10 and 13 (P < 0.05). SPARC mRNA content was significantly higher in the small than in the large cells (P < 0.003). In situ hybridization showed that SPARC mRNA was localized to the thecal layer of Graafian follicles and to day 3 and day 10 CL. Within CL, immunohistochemistry indicated that SPARC protein was associated with small luteal cells (spindle shaped, avg = 17 microM in diameter) but not with large cells. This specific localization to small cells was confirmed by colocalization of SPARC with 3 beta-hydroxysteroid dehydrogenase. We conclude that SPARC is a major secretory product of small steroidogenic luteal cells of the ovine CL. As SPARC is known to modulate many aspects of tissue reorganization, expression by small luteal cells may play a role in regulation of CL maturation.

3-Hydroxysteroid Dehydrogenases↗

PubMed1996

Treatment with recombinant bovine interferon-tau in utero attenuates secretion of prostaglandin F from cultured endometrial epithelial cells.

Endometrial oxytocin receptors and total production of PGF by endometrial epithelial cells were measured in 10 cyclic cows after intrauterine injections of recombinant bovine interferon-tau plus BSA or BSA alone. Cows received twice daily injections (via intrauterine catheters) of 200 micrograms of recombinant bovine interferon-tau plus 1.3 mg of BSA (n = 5) or 1.5 mg of BSA (n = 5) from d 14 to 17 after estrus. On d 17, the reproductive tracts of each cow was removed at slaughter, and endometrial epithelial cells were cultured with 0, 2, or 50 ng/ml of recombinant bovine interferon-tau. After 24 h, oxytocin (2 x 10(-7) M) was added to one-half of the culture wells, and the medium was sampled at 0, 30, and 90 min for analysis of total PGF (PGF plus 13, 14-dihydro-15-keto-PGF2 alpha). In vivo treatment with recombinant bovine interferon-tau + BSA reduced total secretion of PGF in culture (1.49 +/- 0.06 vs. 2.80 +/- 0.07 ng/micrograms of DNA), but did not block the oxytocin-induced stimulation in total secretion of PGF. In vitro treatment of cells with recombinant-bovine interferon-tau did not decrease basal secretion of total PGF. Oxytocin receptor binding at d 17 was low in both treatments but slightly attenuated in the group treated with recombinant bovine interferon-tau.

Animals↗

PubMed1995

The gene encoding bovine pregnancy-associated glycoprotein-1, an inactive member of the aspartic proteinase family.

Bovine pregnancy-associated glycoprotein 1 (bPAG1) is a member of the aspartic proteinase family. It becomes detectable in maternal serum soon after implantation and is produced specifically in the invasive binucleate cells of the placenta. As a result of a key mutation within its catalytic center, bPAG1 appears to be proteolytically inactive. Its gene consists of nine exons (size range 99-281 bp) and eight introns (87-1800 bp) organized in a manner very similar to those of proteolytically active mammalian aspartic proteinases. The transcription start point (tsp) is located 53 or 54 bp upstream from the start codon (ATG) and 19 bp downstream from a 5'-TATATAA sequence. Southern blot analyses have indicated the presence of two bPAG1 genes. By screening with an antiserum raised against bPAG1, a less common cDNA with 91% sequence identity to the bPAG1 transcript has been isolated from a placental cDNA library and presumably represents the second gene. At least eight other genes with sequences that hybridize relatively weakly to the bPAG1 probe are present in the bovine genome. Despite the similarities in the transcribed portion of the genes encoding PAG1, pepsinogen and other mammalian aspartic proteinases, the sequences upstream from the tsp of bPAG1 are unique.

Amino Acid Sequence↗

PubMed1995

A bifunctional vector suitable for both site-directed mutagenesis and recombinant expression of interferon-tau in Escherichia coli.

In order to produce workable quantities of a large number of mutant forms of recombinant ovine and bovine interferon-t (IFN-t), a bifunctional vector, pME-2, was developed, which combines a mutant selection system and a strong promoter providing controlled expression. An EcoRI/KpnI fragment containing the complete Trp promoter, a Shine-Dalgarno sequence, and an AT-rich region from the pTrp-2 expression vector was inserted into the large fragment of EcoRI/KpnI-digested pALTER-1 plasmid, which had been modified by eliminating a ClaI site. The pALTER-1 phagemid provides a highly efficient, antibiotic-dependent system for selection of mutant plaques. The existing T7 promoter was then eliminated from the recombinant phagemid to create the pME-2 vector. Ovine and bovine IFN-t genes lacking the coding region for the signal peptide, but with an ATG codon ahead of the open reading frame, were inserted into the multicloning site of pME-2. Following site-directed mutagenesis designed to produce elongations, truncations, and single and multiple amino acid replacements in the protein products, mutant genes were selected in Escherichia coli BMH 71-18 and efficiently expressed in E. coli JM-101 in response to the inducer of the Trp promoter indole acetic acid. The recombinant IFN were solubilized from washed inclusion bodies in guanidinium-HCl and 2-mercaptoethanol and allowed to refold in aerated buffer. The procedure provides high yields of fully active, homogeneous IFN-t and can be accomplished within 1 week.

Amino Acid Sequence↗

PubMed1995

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.

Amino Acid Sequence↗

PubMed1995

Pronuclear visibility, development and transgene expression in IVM/IVF-derived porcine embryos.

A total of 1550 zygotes was used to assess the timing of pronuclear visibility, embryo development following DNA microinjection, and transgene expression in IVM/IVF-generated porcine embryos. After centrifugation, pronuclei could be seen in 61.6% of zygotes. In 55.3% of these only 1 pronucleus was visible. Pronuclear visibility was highest at 20 h post-insemination. Zygotes were microinjected with 1 of 2 LacZ gene constructs driven by either the SV40 early promoter (pSVON) or the human cytoplasmic beta actin promoter (pbActinLacZ). Development and transgene expression were assessed after either 48 h or 7 d in culture. After 48 h, significantly more zygotes with a single visible pronucleus developed to the 8-cell stage than zygotes in which no pronucleus had been seen (43.0 vs 24.8%), while those with 2 pronuclei were intermediate (31.4%). After 7 d, no difference in development to the morula stage was observed between noninjected control embryos (25.5%) and embryos with 1 (21.0%) or 2 pronuclei (22.5%); however, the proportion of embryos reaching the morula stage in the nonpronuclear group was significantly reduced (9.1%). After 48 h in culture, transgene expression was significantly higher in embryos with 2 pronuclei at the time of injection than in those with 1 (36.4 vs 17.9%). After 7 d in culture, 41.5% of morulae derived from zygotes with 2 pronuclei and 29.97% of thsoe derived from zygotes with 1 pronucleus showed signs of transgene expression. At this stage, significantly more morulae expressed the pbActinLacZ than the pSVON transgene (43.8 vs 25.8%). More than 80% of putative transgenic morulae or blastocysts showed evidence of mosaicism. These results demonstrate that IVM/IVF porcine embryos are able to develop in culture and express a microinjected transgene.

Journal Article↗

PubMed1995

A three-dimensional model of interferon-tau.

The interferon-tau (IFN-tau) are type I IFN whose expression is restricted to the embryonic trophectoderm of the developing placenta of ruminant ungulate species, where they act as hormones of pregnancy. Here computer modeling has been used to generate homology models of bovine and ovine IFN-tau based on the refined crystal structure of murine IFN-beta. The IFN-tau structure, like that of MuIFN-beta, is based on five long alpha helices (A-E), one short helix in the middle of the loop connecting helices C and D and a long loop between helices A and B. BoIFN-tau differs from MuIFN-beta in three important respects. First, as in all IFN-tau, there is a carboxyl tail of nine amino acids that cannot be accurately modeled but that would have a length of approximately 30 A when fully extended. Second, like the IFN-alpha subtype, all IFN-tau have a three-amino acid insertion in loop AB and a likely disulfide bridge between Cys29 and Cys139 that lead to marked conformational differences between them and MuIFN-beta in a region (Leu22 to Arg33 in IFN-tau) believed to interact with the receptor. Third, all IFN-tau, as well as the related IFN-omega, possess a Gly at position 126 (rather than the equivalent Arg on MuIFN-beta and IFN-alpha) that will impair an extensive hydrogen bonding interaction between helix D and loop AB. As a result, the polypeptide segment around this region (Phe36 to Gln40) of loop AB is likely to be considerably more flexible than in other type I IFN.

Amino Acid Sequence↗

PubMed1995

Porcine pregnancy-associated glycoproteins: new members of the aspartic proteinase gene family expressed in trophectoderm.

Pregnancy-associated glycoproteins (PAG) are members of the aspartyl proteinase gene family that were initially identified in cattle (bPAG) and sheep (oPAG) as placenta-specific antigens in maternal blood. The objective of this study was to determine whether PAG are expressed in pig trophoblast. A porcine conceptus cDNA library was screened with 32P-labeled ovine and bovine PAG cDNA. Of the approximately 10(4) plaques that were initially screened, a very high number (approximately 5.3%) were positive. Two distinct types were identified, and full-length clones representing each type (1371 bp, pPAG1; 1378 bp, pPAG2) were fully sequenced in both directions. Their open reading frames coded polypeptides of 389 and 387 amino acids, respectively, including 15 amino acid signal peptides. Each had several potentials sites for N-glycosylation. Both were members of the aspartic proteinase gene family, with approximately 50% amino acid sequence identity to porcine pepsinogen and 64% to each other. They were only distantly related to PAG of ruminant species (53% and 49% identify in amino acid sequence to oPAG1 and bPAG1, respectively). Interestingly, pPAG1 had amino acid substitutions within its catalytic center (Gly-->Ala81, domain 1; Thr-->Ser263, Thr-->Ser265, Ser-->Ala266, domain 2) that together were likely to render it enzymatically inactive, whereas pPAG2 retained sequences identical to pepsin in these regions. Western blotting of secretory products of porcine trophoblast with anti-oPAG1 and anti-bPAG1 antisera indicated that pPAG, like PAG from ruminants, had an unexpectedly high M(r)(approximately 70,000).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1995

Expression of two transgenes in in vitro matured and fertilized bovine zygotes after DNA microinjection.

Bovine zygotes produced by in vitro maturation-in vitro fertilization (IVM-IVF) were examined for their potential to serve as recipients of transgenes. Pronuclei, which were maximally visible at about 22 h after IVF, were injected with a SV40-LacZ construct (pSVON). Injected zygotes had lower cleavage rates (49.1%, n = 1162, P < 0.01) than did either noninjected controls (87.4%, n = 1420) or noninjected zygotes in which pronuclei were not visible (67.6%, n = 803). Zygotes that were injected into their pronuclei cleaved as well as zygotes injected cytoplasmically. At 48 h after injection, when most embryos had reached the four- and eight-cell stages, more zygotes in the pronuclear group (22.7%, n = 125) stained positively for LacZ than did zygotes in the cytoplasmic group (8.0%, n = 125). A group of zygotes injected into the pronucleus with pSVON was cultured for 9 days. More morulae (10.8%, n = 134) than blastocysts (3.2%, n = 31) expressed the LacZ gene, indicating that silencing of expression occurred as development progressed. Another group of zygotes was injected with a beta-actin-LacZ gene construct (pbActinLacZ) and, of the embryos assayed at 48 h, 10.6% (n = 255) stained positively. At 9 days, 36.3% of morulae (n = 91) and 21% of blastocysts (n = 33) expressed the transgene. Almost all putative transgenic embryos injected with either construct showed a mosaic pattern of LacZ expression, with an average of only 2-3 cells staining at the eight-cell stage and the majority of cells in positive blastocysts showing no evidence of expression.

Animals↗

PubMed1995

Regulation and regulatory role of proteinase inhibitors.

Excess or ill-timed proteolytic events have, of necessity, to be restrained. Therefore, there are partner proteinase inhibitors to most of the known mammalian enzymes that cleave peptide bonds, a situation that implicates the inhibitors in a myriad of normal as well as pathological processes. Here, what is currently known about the regulatory functions, the mode of action, and the control of gene expression of the commoner extracellularly acting proteinase inhibitors is reviewed. The review covers four families of serine proteinase inhibitors: the serpins, the Kunitz, Kazal, and leukoproteinase inhibitors, which overlap considerably in the range of their specificities but differ considerably in their respective structures and likely functions. There then follows a description of the growing family of metalloproteinase inhibitors (usually known by the acronym TIMP), the structurally varied cysteine proteinase inhibitors, and finally alpha 2-macroglobulin, a large multisubunit inhibitor, that is relatively catholic in its range of targets. The review concludes with two short sections, the first an overview of the acute phase response, which features several proteinase inhibitors from the different families discussed earlier, and the second, our prediction of where this research field is headed over the next decade.

Animals↗

PubMed1995

Effect of bovine interferon-tau on body temperature and plasma progesterone concentrations in cyclic dairy cows.

We examined the effect of intramuscular injections of recombinant bovine interferon-tau on basal body temperature and plasma concentrations of progesterone in cows. Six mature, nonlactating Holstein cows were synchronized for estrus and assigned randomly to a replicated 3 x 3 Latin square design to receive 0, 1, or 5 mg of recombinant bovine interferon-tau intramuscularly on d 9, 12, or 15 of the estrous cycle. Rectal temperatures were measured, and blood was collected just prior to injection, at hourly intervals for 8 h, and at 10 and 12 h after injection. Treatment with 1 and 5 mg of recombinant bovine interferon-tau caused a rise in body temperature that peaked at 6 h (39.5 +/- .13 degrees C) and 4 h (40.3 +/- .13 degrees C), respectively. Analysis of homogeneity of regression for response curves indicated that 5 mg of recombinant bovine interferon-tau decreased plasma progesterone concentrations. This effect was not detected for the 1-mg dose of recombinant bovine interferon-tau. Intramuscular administration of 5 mg of recombinant bovine interferon-tau caused a hyperthermic response that was temporally associated with the decrease in plasma progesterone. Intramuscular administration of 1 mg of recombinant bovine interferon-tau caused a slight rise in temperature occurring 4 h later than that caused by 5 mg of recombinant bovine interferon-tau, and no decrease in plasma progesterone was detected. Recombinant bovine interferon-tau, administered at a 1-mg dose, warrants further study to determine whether it will alter survival rate of cattle embryos.

Animals↗

PubMed1995

Extension of corpus luteum lifespan and reduction of uterine secretion of prostaglandin F2 alpha of cows in response to recombinant interferon-tau.

Two experiments tested the effect of recombinant ovine and bovine interferon-tau on corpus luteum lifespan, interestrous interval, and oxytocin-induced uterine secretion of prostaglandin F2 alpha. Cows received intrauterine injections of 100 micrograms of recombinant ovine interferon-tau plus 1.4 mg of BSA or of 1.5 mg of BSA alone in Experiment 1 and 200 micrograms of recombinant bovine interferon-tau plus 1.3 mg of BSA or 1.5 mg of BSA alone in Experiment 2. Twice daily injections (0700 and 1900 h) were split evenly between the uterine horns from d 14 to 24 of the experimental estrous cycle via an AI pipette in Experiment 1 and via intrauterine catheters in Experiment 2. On d 17, cows were injected with 100 IU of oxytocin, and plasma was collected for analysis of 13,14-dihydro-15-keto-prostaglandinF2 alpha. Recombinant ovine interferon-tau extended the lifespan of the corpus luteum (27.5 vs. 19.2 d) and interestrous interval (30.5 vs. 20.6 d) and abolished the oxytocin-induced increase in 13,14-dihydro-15-keto-prostaglandinF2 alpha, which peaked at 30 min for the BSA control group (210.8 pg/ml). Recombinant bovine interferon-tau also extended the lifespan of the corpus luteum (29.0 vs. 21.4 d) and interestrous interval (31.5 vs. 22.6 d) and abolished the oxytocin-induced increase in 13,14-dihydro-15-keto-prostaglandin F2 alpha, which peaked at 30 min for the BSA control group (205.6 pg/ml). In conclusion, recombinant ovine interferon-tau and recombinant bovine interferon-tau were effective antiluteolytic agents in cattle.

Animals↗

PubMed1994

Structure-function relationships in the interferon-tau (IFN-tau). Changes in receptor binding and in antiviral and antiproliferative activities resulting from site-directed mutagenesis performed near the carboxyl terminus.

The interferons-tau (IFN-tau) are Type I IFN, 172 amino acids in length, which are produced by the trophoblast of ruminant ungulate species and whose only known function is in triggering maternal responses to pregnancy. These IFN have the normal antiviral and antiproliferative activities exhibited by other Type I IFN, such as IFN-alpha and -beta, and are predicted to have similar structures based on five alpha-helices (A-E). A series of ovine IFN-tau variants was prepared with alterations in the carboxyl-terminal region in residues not expected to interact directly with the Type I receptor. Replacement of Ile143 with Thr in helix E significantly lowered receptor binding affinity to about 5% of control values, reduced antiviral activity to about 13% of control values, and abolished antiproliferative activity completely. Deletion of the terminal 11 residues from the carboxyl termini of these 172 amino acid IFN also reduced antiviral activities (approximately 5% of control) and antiproliferative proteins (1-2% of control), but had only slight negative influence on receptor binding. Deletion of Lys160, but not its replacement with Ala, had similar consequences to deleting the entire 11-amino acid carboxyl terminus. These studies show that mutations can be introduced into IFN-tau that influence antiviral and antiproliferative activities differentially and that the carboxyl tail of the molecule is important for signal transduction but not for primary receptor binding.

Animals↗

PubMed1994

Purification, characterization, and cDNA cloning of a Kunitz-type proteinase inhibitor secreted by the porcine uterus.

The porcine uterus synthesizes a proteinase inhibitor (M(r) 14,000) under the influence of progesterone that is relatively specific for plasmin and trypsin, but that also has weak affinity for chymotrypsin. Several isoforms of this uterine plasmin/trypsin inhibitor were purified by a procedure whose final two steps involved affinity chromatography on immobilized chymotrypsin and cation exchange chromatography. Amino-terminal sequencing showed that at least three of the isoforms were closely related. An oligonucleotide probe based on the protein sequence was used to identify a cDNA that contained an open reading frame coding for a mature protein (M(r) 10,295) of 93 amino acids. The inhibitor had a well defined, but unique, Kunitz domain of 64 residues at its amino terminus that shared 67% sequence identity to bovine pancreatic trypsin inhibitor. Its P1 residue was arginine rather than lysine. Northern analysis showed the presence of a single mRNA species (700 bases) that in adult female pigs appeared to be confined to the uterus. During pregnancy, UPTI mRNA expression was high until Day 30 and decreased significantly thereafter. By contrast, uteroferrin mRNA reached maximal concentrations in late pregnancy. These data are consistent with an earlier hypothesis that the inhibitor serves to neutralize the activities of one or more serine proteinases generated by the proliferating trophoblast during the formation of the noninvasive placenta of the pig.

Amino Acid Sequence↗