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Biomedical subjects

T R Hansen

Publications and source records attributed to T R Hansen.

At least 19 recordsLinked to original sources
PubMed1997

Increased bax and interleukin-1beta-converting enzyme messenger ribonucleic acid levels coincide with apoptosis in the bovine corpus luteum during structural regression.

Recent reports have demonstrated that prostaglandin F2alpha-induced generation of reactive oxygen species or their intermediates inhibits progesterone synthesis and may also serve as a trigger for apoptosis in the corpus luteum (CL). BCL-2, an inhibitor of apoptosis in a wide variety of cell types, has been reported to prevent oxidative stress-induced cell death. Thus, the present studies were conducted to determine whether levels of mRNA encoding BCL-2 and related members of this gene family (BAX and BCL-Xshort, which induce apoptosis; BCL-Xlong, a BCL-2 homologue that prevents apoptosis) differed in functional (Day 21 of pregnancy) versus regressed (Day 21 of the estrous cycle) CL in the bovine ovary. Levels of mRNAs encoding p53, a transcriptional regulator of the bcl-2 and bax genes, and interleukin-1beta-converting enzyme (ICE), a protein recently implicated in the induction of apoptosis whose expression may be enhanced by oxidative stress, were also assessed. Partial cDNA clones encoding bovine bax, bcl-x, p53, and Ice were isolated using the reverse transcriptase-polymerase chain reaction (RT-PCR) technique with total RNA prepared from functional or regressed CL. A bovine bcl-2 cDNA could not be isolated from luteal tissue RNA despite the use of several primer pairs for amplification. Total RNA was then extracted from functional or regressed CL and analyzed by Northern blot analysis. The occurrence of apoptosis in regressed CL, as evidenced by the presence of internucleosomal DNA cleavage, was associated with a significant increase in both bax and Ice mRNA levels as compared with levels of bax and Ice expression in functional CL (p < 0.05, n = 3). There were no significant differences in bcl-x or p53 mRNA levels in functional versus regressed CL. Analysis of bcl-x mRNA by RT-PCR revealed that the long form was the primary, if not only, mRNA expressed in functional and regressed bovine luteal tissue. On the basis of data that increased expression of bax is associated with, and may be required for, apoptosis in ovarian granulosa cells and germ cells, we propose that BAX may play a similar role in apoptosis induction during luteal regression. Moreover, the increased Ice mRNA levels in regressed CL provides the first evidence that the ICE family of death proteases may be involved in luteolysis.

Animals↗
PubMed1996

Pepsin-inhibitory activity of the uterine serpins.

Among the major products secreted by the uteri of cattle, sheep, and pigs during pregnancy are glycoproteins with amino acid sequences that place them in the serpin (serine proteinase inhibitor) superfamily of proteins. The inferred amino acid sequences for bovine uterine serpin (boUS-1) and ovine uterine serpin (ovUS-1) exhibit about 72% sequence identity to each other but only about 50% and 56% identity, respectively, to two distinct porcine uterine serpins (poUS-1 and poUS-2). Despite these differences in primary structure, the uterine serpins possess well-conserved reactive center loop regions that contain several motifs present in the propeptide regions of pepsinogens. One such motif, VVVK, aligns with the first 4 amino acids of the aspartic proteinase inhibitor pepstatin. Although no inhibitory activity toward any serine proteinase has been found, at least one of the uterine serpins, ovUS-1, can bind specifically to immobilized pepsin A and can weakly inhibit the proteolytic activities of pepsin A and C (but not cathepsins D and E). OvUS-1 is the first specific inhibitor of aspartic proteinases to be identified in vertebrates and provides another example of a serpin with "crossover" activity. The pregnancy-associated glycoproteins (PAGs), which are secreted by the trophoblast layer of the placentas of ungulate species and are inactive members of the aspartic proteinase family, can also bind ovUS-1 and may be the natural target partners for the uterine serpins.

Amino Acid Sequence↗
PubMed1996

Ubiquitin cross-reactive protein is released by the bovine uterus in response to interferon during early pregnancy.

A 16-kDa protein has been identified that is secreted by the bovine endometrium in response to conceptus-derived interferon (IFN)-tau during early pregnancy. Because this uterine protein was similar in size to a human ubiquitin cross-reactive protein (hUCRP) that also was regulated by IFN, we suspected that they might be related. To test this hypothesis, uterine flushings, medium from cultured endometrium, and endometrial tissues were examined for the presence of ubiquitin-immunoreactive proteins. Immunoreacting proteins were detected through use of one-dimensional (1D)-PAGE and Western blotting with ubiquitin and hUCRP antiserum (1:500). A 16-kDa protein that cross-reacted with ubiquitin and hUCRP antisera was released by the endometrium and was present in uterine flushings from all Day 18 pregnant females examined (n = 12). The immunoreacting 16-kDa protein was absent in all nonpregnant females examined (n = 23). Regulation of this uterine protein by recombinant type I IFNs (rbIFN-tau, rbIFN-alpha, and roIFN-tau), using 0, 0.5, 5, and 25 nm of each IFN, was evaluated in nonpregnant (Day 12) heifers (n = 5) using 1D-PAGE and Western blotting. Release of the 16-kDa protein into medium was negligible in controls (0 nm IFN). For each IFN, a dose-dependent increase (p < 0.05) in release of the immunoreacting 16-kDa protein was noted. We conclude that the 16-kDa protein that is produced by the endometrium in response to IFN-tau during early pregnancy also shares epitopes with hUCRP and ubiquitin. The 16-kDa protein has been named bovine UCRP.

Animals↗
PubMed1996

Polyubiquitin up-regulation in corpora lutea of prostaglandin-treated ewes.

Genes that encode mRNAs for ubiquitin are activated by cells in metabolic distress. Cytosolic proteins that consequently become conjugated to ubiquitin are targeted for degradation. We hypothesized that ubiquitin mediates the endocrine demise of the corpus luteum induced by prostaglandin (PG) F2 alpha. Indeed, polyubiquitin gene expression increased abruptly (within 2 h) in luteal tissues of ewes treated with PGF2 alpha-before the precipitous decline in glandular progesterone accumulation indicative of functional luteolysis. A corresponding elevation in ubiquitin immunostaining was localized to large (PG-sensitive) luteal cells. It is suggested that luteal progesterone biosynthesis is disrupted by ubiquitination of steroidogenic regulatory proteins-perhaps those involved in the mechanics of mitochondrial delivery and side-chain cleavage of cholesterol.

Animals↗
PubMed1996

Polyubiquitin up-regulation in corpora lutea of prostaglandin-treated ewes.

Genes that encode mRNAs for ubiquitin are activated by cells in metabolic distress. Cytosolic proteins that consequently become conjugated to ubiquitin are targeted for degradation. We hypothesized that ubiquitin mediates the endocrine demise of the corpus luteum induced by prostaglandin (PG) F2alpha. Indeed, polyubiquitin gene expression increased abruptly (within 2 h) in luteal tissues of ewes treated with PGF2alpha--before the precipitous decline in glandular progesterone accumulation indicative of functional luteolysis. A corresponding elevation in ubiquitin immunostaining was localized to large (PG-sensitive) luteal cells. It is suggested that luteal progesterone biosynthesis is disrupted by ubiquitination of steroidogenic regulatory proteins--perhaps those involved in the mechanics of mitochondrial delivery and side-chain cleavage of cholesterol.

Animals↗
PubMed1995

Baculovirus-insect cell production of bioactive choriogonadotropin-immunoglobulin G heavy-chain fusion proteins in sheep.

A hybrid cDNA encoding a fusion protein between the beta subunit of hCG (beta hCG) and constant domains of a mouse IgG heavy chain (CH1-3) was inserted into a baculovirus expression vector. Insect cells transfected with foreign DNA synthesized multimeric forms of fusion protein that inhibited hCG-induced steroid hormone secretion by mouse Leydig tumor cells. Leydig cells were lysed by beta hCG-CH1-3 in the presence of complement. Intravenous injection of beta hCG-CH1-3 in rams was associated with testicular mononuclear leukocyte infiltration, interstitial tissue damage, and a transient depression in circulatory testosterone (levels returned to normal within 2 wk). It appears that targeted cell-killing can be mediated by recombinant proteins composed of the receptor-binding moiety of hormones and truncated effector (Fc) regions of lethal antibodies.

Animals↗
PubMed1995

Secretion of bovine uterine proteins in response to type I interferons.

Bovine interferon-tau (bIFN-tau) is secreted by the developing conceptus and initiates antiluteolytic events by interacting with uterine membrane receptors. We have identified three endometrial proteins (approximately 8, 16, and 28 kDa; P8, P16, and P28; respectively) that are secreted in response to recombinant (r) bIFN-tau. The objective of this study was to determine whether or not secretion of these proteins was a unique response to IFN-tau during early pregnancy. Three experiments were designed to examine secretion of endometrial proteins as a function of time in culture (0, 3, 6, 12, 18, 24 h), stage of the estrous cycle and pregnancy (Days 15, 18, 0/21), and dose of Type I IFN (0, 0.5, 5, and 25 nM; rbIFN-tau, rbIFN-alpha, and roIFN-tau). Endometrium was cultured for times specified with L-[3H]leusine to generate radiolabeled proteins. Secreted proteins were quantitated by using one-dimensional (1D)-PAGE, fluorography, and densitometry. Secretion of P8, P16, and P28 increased over time (p < 0.0001) in culture and in response to 25 nM rbIFN-tau (p < 0.05). Secretion of P8 in response to rbIFN-tau was higher (p < 0.05). Secretion of P8 in response to rbIFN-tau was higher (p < 0.0005) in endometrium collected from pregnant than nonpregnant heifers, but did not differ across the days examined. Although secretion of P8 was higher (p < 0.0001) in the presence than in the absence of rbIFN-tau, it was not affected by rbIFN-alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Recombinant interferon-tau regulates secretion of two bovine endometrial proteins.

Conceptus-derived interferon-tau (IFN-tau) has been implicated in the process of maternal recognition of pregnancy in the bovine. This type I IFN interacts with a uterine receptor complex to elicit secondary maternal responses, one of which is secretion of uterine proteins. We investigated the effects of pregnancy and recombinant (r) bovine (bo) IFN-tau (10(7) antiviral units/mg) on secreted proteins by cultured bovine endometrial explants. Explants were cultured for 24 h with [3H]leucine and dialyzed medium analyzed by one- and two-dimensional SDS polyacrylamide gel electrophoresis (PAGE) and fluorography. In one-dimensional PAGE experiments, endometrium representing early pregnancy (days 16-21) and the estrous cycle (days 16-19) was cultured in the presence of 5 nM rboIFN-tau and showed an increase (two- to five-fold) in secretion of 12- and 28-kD proteins. Further examination of these proteins by using two-dimensional PAGE indicated that the 12-kD protein was basic (pI > or = 7.5), whereas the 28-kD protein was acidic (pI approximately 5.0). Isoelectric focusing in the acidic range revealed that the 28-kD protein was composed of several isoelectric variants (pI 4.5-5.5). Although the functions of these secretory proteins are currently unknown, they serve as useful markers for IFN action and may act as secondary signals to protect the early developing conceptus.

Animals↗
PubMed1992

Nucleotide and amino acid sequence of pap-gene (pediocin AcH production) in Pediococcus acidilactici H.

N-terminal analysis of purified pediocin AcH produced a partial sequence of 23 amino acids. This sequence matched perfectly with a segment of 23 amino acids in a 62 amino acid molecule generated from the 186 nucleotide sequence open reading frame in a Hind III fragment in pSMB74 encoding pap-gene (pediocin AcH production). It is suggested that the molecule is translated as inactive prepediocin AcH of 62 amino acids. Then through enzymatic modifications the leader segment of 18 amino acids is removed from the NH2-terminal. The remaining segment of 44 amino acids is active pediocin AcH of 4628 M(r).

Amino Acid Sequence↗
PubMed1991

The genes for the trophoblast interferons and the related interferon-alpha II possess distinct 5'-promoter and 3'-flanking sequences.

The genes for trophoblast interferons (IFN) bovine trophoblast protein-1 (bTP-1) and ovine trophoblast protein-1 (oTP-1) are expressed massively in the trophectoderm of preimplantation bovine and ovine concepti during the period of maternal recognition of pregnancy. These 172-amino acid IFN are closely related to the IFN-alpha II, a family of "long" IFN expressed in virus-induced leukocytes. Genomic Southern blotting with a full-length bTP-1 cDNA revealed about 15 genes that bind the probe with varying intensities. By using more specific probes for the 3'-ends of the cDNA, we have shown that only four to five of these represent bTP-1 genes, whereas no more than another four are IFN-alpha II. Genes for bTP-1 and IFN-alpha II, all of which are intronless, have been isolated from bovine genomic libraries, and their nucleotide sequences were compared. Additional bTP-1 genes and two distinct oTP-1 genes have been isolated from bovine and ovine genomic DNA by using the polymerase chain reaction procedure in conjunction with specific 3'- and 5'- primers (derived from the bTP-1 gene sequences determined above). The promoter region up to 100 bases upstream from the transcription start sites of the trophoblast IFN are almost completely conserved across different genes and across species, yet show only limited sequence identity with IFN-alpha II. Two putative binding regions for interferon regulatory factor-1 as well as several GAAANN motifs (where N is any nucleotide) that have been implicated in viral responsiveness of alpha 1- and beta-IFN genes are retained at identical positions in all of the trophoblast genes. Putative interferon regulatory factor-1-binding nucleotide hexamers and GAAANN motifs are also present in the IFN-alpha II genes, but these are organized very differently than in the trophoblast IFN genes. A GAAATG motif is present in IFN-alpha II promoters but is absent in trophoblast IFN genes. Based on the evidence presented, it is proposed that the trophoblast interferons constitute a separate subclass of IFN-alpha distinct from IFN-alpha II and IFN-alpha I.

Animals↗
PubMed1991

Preoperative laboratory screening in healthy Mayo patients: cost-effective elimination of tests and unchanged outcomes.

We reviewed the results of preoperative screening laboratory tests in asymptomatic healthy patients who underwent elective surgical procedures at our institution in 1988. Substantially abnormal results were found in 160 of 3,782 patients. All such abnormalities involved five tests: aspartate aminotransferase, glucose, potassium, platelet count, and hemoglobin. Thirty of the abnormal test results were predictable on the basis of the history or physical examination. The abnormal test result prompted further assessment in 47 patients. No surgical procedure was delayed, and no association was noted between adverse outcome and any preoperative laboratory abnormality. Because of our findings in this analysis and similar studies on specific tests from other institutions, we no longer require preoperative laboratory screening tests for healthy patients.

Adult↗
PubMed1991

Slowed transcription and rapid messenger RNA turnover contribute to a decline in synthesis of ovine trophoblast protein-1 during in vitro culture.

Ovine trophoblast protein-1 (oTP-1) is produced in massive amounts by conceptuses during the Day-12-20-period of early pregnancy. The rate of production of oTP-1 declines during culture of conceptuses, however, suggesting a role for critical intrauterine factors for continued production. The present study was conducted to define the mechanism responsible for the in vitro decline in oTP-1 synthesis. Over 24 h culture, synthesis of oTP-1 was initially rapid but then declined such that there was little increase in either the protein or its antiviral activity after 12 h. By contrast, the release of total protein into the medium continued at an approximately linear rate for the entire 24-h period of culture. By using in situ hybridization to tissue sections and dot-blot analysis of tissue extracts with labeled cDNA probes, it was shown that the quantity of oTP-1 mRNA fell 3- to 5-fold during culture, whereas the amount of actin mRNA remained relatively constant. To determine whether this selective fall in levels of oTP-1 mRNA resulted from decreased transcription rate and/or high turnover rate of existing mRNA, conceptuses were either provided continuously with [3H]uridine for 24 h or labeled for 9 h and then exposed to medium enriched in unlabeled uridine and cytidine for a further 15 h. Specific incorporation of 3H into oTP-1 mRNA was assessed by hybridization to excess oTP-1 cDNA immobilized on nitrocellulose membranes. In the continuous labeling study, 3H in total RNA increased at an approximately linear rate for at least 18 h, whereas the content of 3H in oTP-1 mRNA peaked at 9 h and declined about 10-fold by 24 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

The polypeptides and genes for ovine and bovine trophoblast protein-1.

Ovine and bovine trophoblast protein-1 (oTP-1 and bTP-1) have been strongly implicated as antiluteolytic agents and responsible for maternal recognition of pregnancy in sheep and cattle, respectively. Both are interferons (IFN) belonging to the IFN-alpha family, but their length (172 residues versus 166 for most IFN-alpha) places them in an unusual subclass (the IFN-alpha II). The various isoforms of oTP-1 and bTP-1 produced by trophoblast tissue appear to arise in part from translation of multiple mRNAs which are themselves the products of distinct genes. These genes, like those for other IFN-alpha, are without introns. However, the genes for oTP-1 and bTP-1 form a distinct subgroup within the IFN-alpha II on the basis of their overall primary sequences and the high conservation of the 3'-untranslated ends of their transcription units. The bTP-1 genes also differ from the bovine IFN-alpha II in the organization of the promoter regions upstream from the transcription start site. Nevertheless, computer-aided analysis of the primary polypeptide sequences of oTP-1 and bTP-1 indicates that the molecules are likely to have approximately the same shapes and dimensions as all other IFN-alpha molecules. It remains to be determined whether they have unique biological properties which distinguish them from other IFN-alpha molecules.

Amino Acid Sequence↗
PubMed1990

Expression of trophoblastic interferon genes in sheep and cattle.

The trophoblastic interferons ovine and bovine trophoblast protein-1 (oTP-1 and bTP-1, respectively) have been implicated as mediators of maternal recognition of pregnancy in sheep and cattle. The objective of this study was to describe the onset and duration of gene expression for oTP-1 and bTP-1 in preimplantation ovine and bovine conceptuses by in situ hybridization and Northern analysis. Sections from paraffin-embedded ovine conceptuses, collected on Days 10, 11, 12, 13, and 15 of gestation (n = 1, 3, 3, 2, 2), and bovine conceptuses, collected on Days 12/13, 15/16, and 19 (n = 2, 4, 5), were hybridized to specific [35S]-labeled cDNA probes. Two different probes, one encompassing bases 442-918 and representing both coding and 3'-untranslated regions, and a second 3'-specific probe (bases 650-912) were used to detect oTP-1 mRNA. At all stages examined, oTP-1 mRNA was confined to trophectoderm of ovine conceptuses. Consistent with earlier studies, expression increased markedly at Day 13. oTP-1 mRNA was detected at low levels in seven of seven ovine conceptuses prior to Day 13 when the longer probe was employed. With the 3'-specific probe, however, oTP-1 mRNA was detected in only one of the seven ovine conceptuses prior to Day 13. Thus, although low amounts of oTP-1 mRNA may be present in ovine conceptuses prior to Day 13, massive induction of this mRNA occurs on Day 13 coincident with the initiation of maternal recognition of pregnancy.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1990

Interferons at the placental interface.

The antiluteolytic factors secreted by sheep and cattle conceptuses are closely related structurally to alpha-interferons (IFN-alpha s). They are known as ovine and bovine trophoblast protein-1 (oTP-1 and bTP-1), respectively. The mRNAs for oTP-1 and bTP-1 are transcribed from multiple genes and are the major translatable messages of Day 13-17 sheep conceptuses and Day 15-20 cattle conceptuses. The proteins belong to the 172-amino acid IFN-alpha II (or IFN-omega) subfamily and have the typical antiviral and antiproliferative properties of the 166-residue IFN-alpha Is. These embryonic interferons also bind to the IFN-alpha receptor, which is present in uterine endometrium in high concentrations, and can influence the production of prostaglandin F-2 alpha and the pattern of protein secretion in that tissue. Through use of in-situ hybridization procedures on tissue sections and Northern and dot blot analyses of extracted conceptus RNA, ovine oTP-1 mRNA has been shown to increase markedly around Day 13 and to decrease after about Day 15 of pregnancy. The mRNA is confined entirely to cells of the trophectoderm. Significant induction of mRNA that hybridizes to an oTP-1 cDNA occurs in response to exposure to polyI:polyC in Day 11 sheep blastocysts which normally have low levels of oTP-1 expression. However, the basis for induction in the normal progression of embryonic development remains unclear. The fact that preimplantation conceptuses of other species, e.g. pig, release substances with antiviral activity suggests that IFNs may have an important role in pregnancy that extends beyond the domestic ruminants.

Amino Acid Sequence↗
PubMed1989

Interferon production by the preimplantation sheep embryo.

Ovine trophoblast protein-1 (oTP-1), the major product secreted by the trophectoderm of the sheep conceptus between days 13 and 21 of pregnancy, is considered to mediate maternal recognition of pregnancy by maintaining the function of the corpus luteum. Its amino acid sequence has 40-55% identity with various mammalian interferons-alpha (IFN-alpha), and it has been shown to have antiviral activity. The present results confirm that oTP-1, which at days 15-17 of pregnancy is produced by a single embryo at more than 100 micrograms (greater than 1 million antiviral units) per day, is a functional IFN. A preparation of purified oTP-1 was made. Its amino-terminal sequence suggested that it consisted of a single homogeneous protein, so that its antiviral activity probably was not due to a contaminant. In a cytopathic effect inhibition assay with GBK-2 bovine cells challenged with vesicular stomatitis, its specific activity was 1.3 X 10(7) end point units/mg protein. It also protected GBK-2 cells against four other viruses, and A549 human cells against encephalomyocarditis virus. The antiviral activity was neutralized by an antiserum to human leukocyte IFN. Like human IFN-alpha, oTP-1 at concentrations as low as 10(-9) M inhibited the growth of GBK cells in culture and suppressed mitogen-stimulated incorporation of [3H]thymidine into ovine lymphocytes. Possible roles for oTP-1, functioning as an IFN-alpha during early pregnancy, are discussed.

Age Factors↗
PubMed1989

Complex binding of the embryonic interferon, ovine trophoblast protein-1, to endometrial receptors.

Ovine embryos produce an interferon (IFN)-alpha II in significant quantities during early pregnancy. This IFN, previously termed ovine trophoblast protein-1 (oTP-1), is a 172-amino-acid polypeptide which has been suggested to be the causal agent in maternal recognition of pregnancy in the ewe. Here we report the binding of oTP-1 and a recombinant bovine IFN-alpha I1 (rBoIFN-alpha I1; 165-166 amino acids long) to membrane preparations from ovine uterine endometrium. Both oTP-1 and rBoIFN-alpha I1 competed with each other for receptor binding. Based on Scatchard analysis, [125I]oTP-1 binding was determined to be complex and resolvable into a high-affinity (Kd = 3.8 x 10(-11) M, 30 fmoles/mg protein) and a low affinity (Kd = 1.7 x 10(-10) M; 96 fmoles/mg protein) component. Conversely [125I]rBoIFN-alpha I1 bound to only a single high-affinity receptor (Kd = 6.1 x 10(-11) M; 174 fmoles/mg protein). Cross-linking experiments using disuccinimidyl suberate revealed that [125I]oTP-1 associated with membrane polypeptides of two molecular weight classes (Mr 100,000 and 70,000), and could be displaced from both with rBoIFN-alpha I1. In contrast, [125I]rBoIFN-alpha I1 cross-linked to only the 100,000 Mr membrane polypeptide. These data provide evidence that the binding parameters of oTP-1 and rBoIFN-alpha I1 to endometrial receptors are different.

Animals↗