Changes in ovarian NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase activity in pregnant and pseudopregnant rabbits.

The specific activity of NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (PGDH) was found to increase in the ovaries of pregnant and pseudopregnant rabbits. The mean specific activity of cytosolic ovarian PGDH in 14- to 28-day pregnant rabbits was 24.3 +/- 8.1 nmol NADH formed/min/mg protein (n = 16) using PGE1 as substrate whereas in nonpregnant rabbits the specific activity was 1.5 +/- 0.8 nmol NADH formed/min/mg protein (n = 8). The reaction was dependent on NAD+; NADP+ did not support the reaction. In grouping the PGDH activities from pregnant rabbits into second (14-18 days) and third (2-28 days) trimester periods, no significant difference between values was found (26.1 +/- 8.9 vs 23.4 +/- 8.1 nmol NADH formed/min/mg protein, respectively). Western blot analysis of the ovarian cytosol using an antibody which was made to the purified lung PGDH of pregnant rabbits recognized an ovarian protein of identical molecular mass (30 kDa). Ovarian PGDH activities were also examined in rabbits treated with pregnant mare's serum gonadotrophin (PMSG) and human chorionic gonadotrophin (hCG) to induce a state of superovulatory/pseudopregnancy and only on day 11 following hCG treatment was an increase in PGDH specific activity observed. On day 11, the specific activity was 14.8 +/- 4.3 nmol NADH formed/min/mg protein whereas values on days 10 and 12 were only 1.1 +/- 1.1 and 1.0 +/- 0.8, respectively. PGDH activities on days 3, 7 and 16 were also low.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and properties of lung 15-hydroxyprostaglandin dehydrogenase from pregnant rabbits

A NAD-dependent 15-hydroxyprostaglandin dehydrogenase (PGDH) was purified to a specific activity of over 25,000 nmol NADH formed/min/mg protein with 50 microM prostaglandin E1 as substrate from the lungs of 28-day-old pregnant rabbits. This represented a 2600-fold purification of the enzyme with a recovery of 6% of the starting enzyme activity. The lungs of pregnant rabbits were used because a 42- to 55-fold induction of the PGDH activity was observed after 20 days of gestation. The enzyme was purified by CM-cellulose, DEAE-cellulose, Sephadex G-75, octylamino-agarose, and hydroxylapatite chromatography. The enzyme could not be purified by affinity chromatography using NAD- or blue dextran-bound resins. The purified enzyme was specific for NAD and had a subunit molecular weight of 29,000. The optimal pH range for the oxidation of prostaglandin E1 was between 10.0 and 10.4 using 3-(cyclohexylamino)propanesulfonic acid as the buffer. The Km and Vmax values for prostaglandin E1 were 33 microM and 40,260 nmol/min/mg protein, respectively, while the Km and Vmax values for prostaglandin E2 were 59 microM and 43,319 nmol/min/mg protein, respectively. The Km for prostaglandin F2 alpha was four times the value for prostaglandin E1. The PGDH activity was inhibited by p-chloromercuriphenylsulfonic acid but the enzymatic activity was restored by the addition of dithiothreitol. n-Ethylmaleimide also produced a rapid decline in enzymatic activity but when NAD was included in the incubation system, no inhibition was observed.     

Identification of ethanol-inducible P-450 isozyme 3a as the acetone and acetol monooxygenase of rabbit microsomes

Treatment of rabbits with 1% (v/v) acetone for 1 week resulted in the appearance in blood serum of 88 +/- 14 14 nmol/ml 1-hydroxyacetone (acetol) and 70 +/- 9 nmol/ml 1,2-propanediol. Untreated rabbits had no detectable 1,2-propanediol or acetol. Hepatic microsomes from control, ethanol-, and acetone-treated rabbits catalyzed the hydroxylation of acetone at rates of 0.32 +/- 0.01, 2.01 +/- 0.43, and 3.64 +/- 0.23 nmol/min/mg of protein, respectively. The same microsomal preparations catalyzed the hydroxylation of acetol at rates of 0.33 +/- 0.04, 0.94 +/- 0.20, and 1.08 +/- 0.12 nmol/min/ mg of microsomal protein, respectively. Isozyme 3a purified from acetone- or ethanol-treated rabbits was identical as judged by comparison of the high performance liquid chromatographic profiles of tryptic digests of the two proteins. Antibody to isozyme 3a inhibited greater than 90% of the acetone monooxygenase activity from untreated, acetone-, or ethanol-treated rabbits. In contrast, the antibody only inhibited 30% of the acetol monooxygenase activity of microsomes from untreated rabbits. The inhibition was increased to about 70% after acetone or ethanol treatment. Although the activities were inhibited to different extents, a comparison of the rates attributable to isozyme 3a from antibody inhibition experiments indicated that both activities were induced to a similar extent by ethanol. Similarly, acetone also increased both activities to the same extent but was more effective than ethanol. In a reconstituted system, isozyme 3a was the only isozyme of six forms from rabbit liver to exhibit acetone monooxygenase activity. Isozyme 3a was the most active enzyme in the hydroxylation of acetol, but isozymes 2, 3b, and 4 also were able to catalyze the reaction. Antibody to isozyme 3a also inhibited greater than 90% of the acetone hydroxylase activity and 70% of the acetol hydroxylase activity of microsomes from acetone-treated rats. Two proteins were immunochemically stained on Western blots of microsomes from untreated and acetone-treated rats, one of which was increased by acetone treatment. These results suggest that isozyme 3a in rabbit and an immunochemically homologous enzyme in rat are responsible for acetone and acetol hydroxylation, the initial steps in the proposed gluconeogenic pathways for acetone.     

前列腺素脱氢酶在大肠杆菌中的表达纯化及鉴定

15-羟基前列腺素脱氢酶（PGDH）属于抑癌基因,在多种肿瘤中表达缺失,在肿瘤的发生发展中起着重要作用。提取人正常大肠黏膜组织总RNA,利用RT-PCR方法扩增得到PGDH基因的编码序列,克隆入原核表达载体pBV220,测序鉴定正确后转化E.coli DH5α,经温控诱导表达,表达产物进行SDS-PAGE和Western blot,证实为相对分子质量约为29000的PGDH-His6蛋白,表达产物以包涵体形式存在,3h诱导表达量最高,约占菌体总蛋白的30%。经Ni2+配体亲和层析纯化得到纯度大于95%的目的蛋白。重组PGDH简单复性后具有一定的生物活性,约为3.7×104U/mg,为下一步研究其在肿瘤中的作用奠定了基础。     

Ovine osteopontin: II. Osteopontin and alpha(v)beta(3) integrin expression in the uterus and conceptus during the periimplantation period.

Osteopontin (OPN) is an acidic 70-kDa glycoprotein that is cleaved by proteases to yield 45-kDa and 24-kDa fragments. The 70-kDa and 45-kDa proteins contain a Gly-Arg-Gly-Asp-Ser (GRGDS) sequence that binds to cell surface integrins (primarily alpha(v)beta(3) heterodimer) to promote cell-cell attachment and cell spreading. A 70-kDa acidic protein was previously detected by two-dimensional (2D) PAGE in Day 17 pregnant endometrial cytosolic extracts using Stainsall and identified as immunoreactive OPN using Western blotting. Three forms of immunoreactive OPN proteins (70, 45, and 24 kDa) were detected by 1D PAGE and Western blot analysis of endometrial extracts. OPN protein in endometrial extracts did not differ between cyclic and pregnant ewes. However, the amount of 45-kDa OPN increased in uterine flushings from pregnant ewes between Days 11 and 17. Immunoreactive OPN was localized to luminal and glandular epithelia of both cyclic and pregnant ewes, and to trophectoderm of Day 19 conceptuses. The alpha(v) and beta(3) integrins were detected on Day 19 endometrium and conceptuses by immunofluorescence. It was reported that OPN mRNA increases in the uterine glands of pregnant ewes and secretion of OPN protein into the uterine lumen increases during early pregnancy. The present results demonstrate accumulation of OPN protein on endometrial LE and conceptus trophectoderm. Therefore, it is hypothesized that progesterone and/or interferon-tau induce expression, secretion and/or proteolytic cleavage of OPN by uterine epithelium. Secreted OPN is then available as ligand for alpha(v)beta(3) integrin heterodimer on trophectoderm and uterus to 1) stimulate changes in morphology of conceptus trophectoderm and 2) induce adhesion between luminal epithelium and trophectoderm essential for implantation and placentation.     

A prostaglandin omega-hydroxylase cytochrome P-450 (P-450PG-omega) purified from lungs of pregnant rabbits

Cytochrome P-450-dependent prostaglandin omega-hydroxylation is induced over 100-fold during late gestation in rabbit pulmonary microsomes (Powell, W.S. (1978) J. Biol. Chem. 253, 6711-6716). Purification of cytochromes P-450 from lung microsomes of pregnant rabbits yielded three fractions. Two of these fractions correspond to rabbit lung P-450I (LM2) and P-450II (LM5), which together constitute 70-97% of total cytochrome P-450 in lung microsomes from nonpregnant rabbits. The third form, which we designate rabbit cytochrome P-450PG-omega, regioselectively hydroxylates prostaglandins at the omega-position in reconstituted systems with a turnover of 1-5 min-1. Titration with purified pig liver cytochrome b5, demonstrated a 4-fold maximum stimulation at a cytochrome b5 to a P-450 molar ratio of 1-2. Rabbit lung P-450PG-omega formed a typical type I binding spectrum upon the addition of prostaglandin E1 with a calculated K8 of 1 microM, which agreed reasonably well with the kinetically calculated Km of 3 microM. Cytochrome P-450PG-omega was isolated as a low-spin isozyme with a lambda max (450 nm) in the CO-difference spectrum distinguishable from P-450I (451 nm) and P-450II (449 nm). Sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis demonstrated that although purified P-450PG-omega had a relatively low specific content (12.1 nmol mg-1), it appeared homogeneous with a calculated minimum Mr of 56,000, intermediate between rabbit LM4 and LM6. When lung microsomes from pregnant and nonpregnant rabbit were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a protein band, with a Mr identical to P-450PG-omega, was observed in the pregnant rabbit, whereas this band appeared to be very faint or absent in microsomes from the nonpregnant rabbit. Purification of cytochromes P-450 from nonpregnant rabbit lung yielded only P-450I and P-450II. P-450PG-omega appears to be a novel rabbit P-450, possessing high activity towards omega-hydroxylation of prostaglandins, and is greatly induced during pregnancy in rabbit lung.     

Characterization of a bovine pregnancy-associated protein using two-dimensional gel electrophoresis, N-terminal sequencing and mass spectrometry

Bovine pregnancy-associated protein (bPAP) isolated from pregnant bovine urines by two-dimensional electrophoresis (2-DE) was characterized by N-terminal sequencing, internal sequencing, and mass spectrometric analyses using matrix-assisted laser desorption/ionization-time of flight mass spectrometry and tandem mass spectrometry. Database search using the amino acid sequences and the peptide mass profiles showed that the protein is a novel bovine pregnancy-associated protein of which the N-terminus has a high similarity to collagen alpha. The protein has a molecular mass of 21 kDa and a pI of 6.1. The expression profiles of the protein from the urine of 30 pregnant and 20 nonpregnant cows characterized by 2-DE indicated that the expression of bPAP during pregnancy increased to over nmol from the pmol level basal expression of bPAP at the nonpregnant state with < 3% of false negatives and < 10% of false positives. Using the peptide sequence information, polyclonal antibodies against the bPAP protein were generated. The purified polyclonal antibodies against the peptide sequences of bPAP detected the 21 kDa protein on the blots of pregnant cow urine by Western blot analysis. In addition, analysis showed that the expression of bPAP in the urine is associated with pregnancy, but that the urine concentration of bPAP is not correlated with the duration of the pregnancy.     

Relationship between the level of estrone sulfate in the plasma and the number of fetuses during pregnancy in the gilt

Estrone sulfate was measured in the plasma of pregnant and nonpregnant gilts between Days 10 and 32 after estrus. Estrone sulfate was found to rise sharply in pregnant gilts beginning at Day 18 and to decline at Day 30 to Day 32. Estrone levels were not related to litter size. The level of estrone sulfate on Days 20, 22, 24 and 26 was significantly correlated with litter size at slaughter on Day 32. Reduction of the number of live fetuses by crushing them in utero at Day 40 or between Days 30 to 60 did not cause a subsequent reduction in the level of estrone sulfate, whereas reduction at Day 24 did cause a decline in estrone sulfate. The level of estrone sulfate in plasma of gilts at 20 to 28 days after mating was higher in pregnant than in nonpregnant gilts. The relative level of estrone sulfate would enable one to estimate litter size at Days 20 to 28 days but not later. Because of the limitations of the assay in exact quantitation of the levels of estrone sulfate, the results can only be considered qualitative.     

Stimulation of 2',5'-oligoadenylate synthetase activity in sheep endometrium during pregnancy, by intrauterine infusion of ovine trophoblast protein-1, and by intramuscular administration of recombinant bovine interferon-alpha I1.

In Expt 1, activity of 2',5'-oligoadenylate (2',5'-A) synthetase in endometrium collected on Day 16 (oestrus is Day 0) from the uterine horn ipsilateral to the corpus luteum was greater (P less than 0.001) for pregnant (135.5 +/- 1.72 nmol/mg protein/h) than for cyclic ewes (58.5 +/- 0.99 nmol/mg protein/h). In pregnant ewes, activity of 2',5'-A synthetase in endometrium collected from the contralateral uterine horn (119.5 +/- 1.72 nmol/mg protein/h) did not differ from that of the ipsilateral horn. In Expt 2, three ovariectomized ewes were treated with progesterone for 10 days and then with oestrogen for 2 days. Activity of 2',5'-A synthetase on Day 13 was 18% greater (P less than 0.10) in endometrium collected from the uterine horn receiving infusions of 30 micrograms ovine trophoblast protein-1 (oTP-1) twice a day on Days 10, 11 and 12(57.7 +/- 0.22 nmol/mg protein/h) than from the uterine horn receiving control infusions of serum protein (SP; 48.8 +/- 0.22 nmol/mg protein/h). In Expt 3, activity of 2',5'-A synthetase on Day 15 was not significantly greater in endometrium collected from the uterine horn of cyclic ewes receiving infusions of 30 micrograms oTP-1 twice a day on Days 12, 13 and 14 (46.5 +/- 0.37 nmol/mg protein/h) than in endometrium from the uterine horn receiving infusions of SP (38.2 +/- 0.37 nmol/mg protein/h). When results of Expt 2 and Expt 3 were combined, intrauterine infusion of oTP-1 increased (P less than 0.05) activity of 2',5'-A synthetase in endometrium by 20%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of osteopontin at the maternal-placental interface in pigs

Noninvasive, epitheliochorial placentation in the pig follows a prolonged preimplantation period characterized by migration, spacing and elongation of conceptuses, and secretion of estrogen for maternal recognition of pregnancy. Osteopontin (OPN) is an extracellular matrix protein that binds integrins to promote cell-cell attachment and communication. OPN appears to play a key role in conceptus implantation and maintenance of pregnancy in sheep; however, a role for OPN in the porcine uterus has not been established. Therefore, this study examined OPN expression and function in the porcine uterus and conceptus (embryo/fetus and associated extraembryonic membranes). Northern and slot blot hybridization detected an increase in endometrial OPN expression between Days 25 and 30, and levels remained elevated through Day 85 of pregnancy. In situ hybridization localized OPN mRNA to discrete regions of the uterine luminal epithelium (LE) on Day 15 of pregnancy and to the entire LE thereafter. Glandular epithelial (GE) expression of OPN mRNA was first detected on Day 35 of pregnancy and increased through Day 85. Both 70- and 45-kDa forms of OPN protein were detected in cyclic and pregnant endometrium by Western blotting. OPN protein was localized to the LE and GE by immunofluorescence; however, only the 70-kDa OPN was detected in uterine flushings. OPN protein was present along the entire uterine-placental interface after Day 30 of pregnancy. In addition, OPN mRNA and protein were localized to immune-like cells within the stratum compactum of the endometrium in both Day 9 cyclic and pregnant gilts. Incubation of OPN-coated microbeads with porcine trophectoderm and uterine luminal epithelial cells induced Arg-Gly-Asp (RGD)-dependent integrin activation and transmembrane accumulation of cytoskeletal molecules at the apical cell surface as assessed by immunofluorescence detection of talin or alpha-actinin as markers for focal adhesions. These results suggest that OPN, expressed by uterine epithelium and immune cells, may interact with receptors (i.e., integrins) on conceptus and uterus to promote conceptus development and signaling between these tissues as key contributors to attachment and placentation in the pig.     

Myometrial adenylyl cyclase protein decreases on the last day of pregnancy in the rat

To determine whether gestation-related changes in responsiveness of the rat uterus to beta-adrenergic agonists are mediated at the level of adenylyl cyclase, we measured myometrial adenylyl cyclase activity and protein quantities during pregnancy and labor. In rat myometrial membranes, basal adenylyl cyclase activity increased from the nonpregnant state to mid (Days 12-14) and then late (Days 18-20) gestation and then decreased intrapartum (Day 22). Stimulated adenylyl cyclase activity, at the level of the beta-adrenergic receptor (isoproterenol, 10(-4) M), the G protein (GTP, 10(-5) M), or the adenylyl cyclase enzyme (MnCl(2), 20 mM), was similarly altered during gestation. Total adenylyl cyclase protein was quantified by [(3)H]forskolin binding assay in myometrial membranes from nonpregnant and pregnant (Day 14, Day 20, Day 21, and intrapartum Day 22) rats. Adenylyl cyclase protein increased progressively from nonpregnant rats to pregnant rats at mid (Day 14) and late (Day 20) gestation, but it decreased abruptly to nonpregnant levels on Day 21, the day before parturition, and remained at similar levels on Day 22 (intrapartum). The gestation-related increase in expression of myometrial adenylyl cyclase protein may facilitate uterine quiescence during pregnancy, and the abrupt decrease of adenylyl cyclase protein on the last day of pregnancy may be a contributing mechanism for the initiation of labor.     

Molecular and biochemical characterization of D-phosphoglycerate dehydrogenase from Entamoeba histolytica. A unique enteric protozoan parasite that possesses both phosphorylated and nonphosphorylated serine metabolic pathways.

A putative phosphoglycerate dehydrogenase (PGDH), which catalyzes the oxidation of d-phosphoglycerate to 3-phosphohydroxypyruvate in the so-called phosphorylated serine metabolic pathway, from the enteric protozoan parasite Entamoeba histolytica was characterized. The E. histolytica PGDH gene (EhPGDH) encodes a protein of 299 amino acids with a calculated molecular mass of 33.5 kDa and an isoelectric point of 8.11. EhPGDH showed high homology to PGDH from bacteroides and another enteric protozoan ciliate, Entodinium caudatum. EhPGDH lacks both the carboxyl-terminal serine binding domain and the 13-14 amino acid regions containing the conserved Trp139 (of Escherichia coli PGDH) in the nucleotide binding domain shown to be crucial for tetramerization, which are present in other organisms including higher eukaryotes. EhPGDH catalyzed reduction of phosphohydroxypyruvate to phosphoglycerate utilizing NADH and, less efficiently, NADPH; EhPGDH did not utilize 2-oxoglutarate. Kinetic parameters of EhPGDH were similar to those of mammalian PGDH, for example the preference of NADH cofactor, substrate specificities and salt-reversible substrate inhibition. In contrast to PGDH from bacteria, plants and mammals, the EhPGDH protein is present as a homodimer as demonstrated by gel filtration chromatography. The E. histolytica lysate contained PGDH activity of 26 nmol NADH utilized per min per mg of lysate protein in the reverse direction, which consisted 0.2-0.4% of a total soluble protein. Altogether, this parasite represents a unique unicellular protist that possesses both phosphorylated and nonphosphorylated serine metabolic pathways, reinforcing the biological importance of serine metabolism in this organism. Amino acid sequence comparison and phylogenetic analysis of various PGDH sequences showed that E. histolytica forms a highly supported monophyletic group with another enteric protozoa, cilliate E. caudatum, and bacteroides.     

Validation of a new pregnancy-associated glycoprotein radioimmunoassay method for the detection of early pregnancy in ewes

The aim of the current study was to describe the use of a pool of different antisera raised against pregnancy-associated glycoproteins (PAGs; purified from both ovine and caprine placentas) for early pregnancy diagnosis in ovine species. Sixty-three pluriparous Sarda ewes (Ovis aries) were synchronized. Blood samples were withdrawn on Days 18, 24, 26, 28, 30, and 50 after mating. These samples were assayed for progesterone (radioimmunoassay [RIA] including an extraction step) and for pregnancy-associated glycoproteins (RIA-706 and RIA-srPool). Progesterone concentrations were under 1.0 ng/mL in all nonpregnant Sarda ewes. In pregnant ewes, mean progesterone concentrations ranged from 2.4 ng/mL (Day 24, single pregnancies) to 4.4 ng/mL (Day 28, multiple pregnancies). During all periods of examination, PAGs remained lower than 0.8 ng/mL in nonpregnant ewes. On Day 18 of pregnancy, PAG concentrations could be detected in 26 of 43 (60.5%) and in 41 of 43 (95.3%) pregnant ewes using the RIA-706 and RIA-srPool methods, respectively. From Day 24 to Day 50, using both RIA methods, PAGs could be detected in all pregnant ewes. On Day 24, the best threshold for pregnancy diagnosis was obtained by use of RIA-srPool, maximal concentration in nonpregnant ewes being 0.3 ng/mL and minimal concentration in pregnant ewes being 4.8 ng/mL. In general, progesterone and PAG concentrations were higher in multiple pregnancies than in single pregnancies. However, because of large individual variations, single pregnancies could not be differentiated from multiple pregnancies.     

Monoclonal antibodies recognize a cell surface marker of epithelial differentiation in the rabbit reproductive tract

Monoclonal antibodies against the cell surface were produced by immunizing mice with endometrial scrapings prepared from 6-day pregnant rabbits. Spleen cells from an immune mouse were fused with myeloma cells and cultured by standard hybridoma technology methods. Hybridoma supernatants were screened for reaction with the apical epithelial surface by immunohistochemistry on frozen sections of uterus from 6-day pregnant rabbits, and positive colonies were cloned by limiting dilution. Ascites fluid was produced in mice from hybridoma clones that gave a consistent pattern of apical epithelial surface staining through 6 sub-clonings. Antibodies in the ascites fluid were tested by immunohistochemistry on frozen sections of uterus, oviduct, lung, liver and kidney from nonpregnant or 6-day pregnant rabbits. At a dilution of 1:5000, the antibodies recognized an antigen that was specific to the apical surface of luminal but not glandular epithelium of the 6-day pregnant uterus and could not be detected in the nonpregnant uterine epithelium. At higher concentrations of antibody (1:100 to 1:1000), crossreaction was seen with antigens in stromal and myometrial cells of pregnant and nonpregnant uterus. At a dilution of 1:5000, the antibody also crossreacted with some components of lung, liver and kidney but without discriminating between the two reproductive states. In the oviduct, staining of the surface epithelium was specific to the pregnant state. We conclude that this monoclonal antibody has a high affinity for a luminal epithelial cell surface antigen in the reproductive tract of the pregnant rabbit and shows multiple organ reactivity with other tissues that is not affected by pregnancy. This antigen will provide a useful cell surface marker of epithelial differentiation in the progestational reproductive tract.     

Isolation and sequence of an interferon-tau-inducible, pregnancy- and bovine interferon-stimulated gene product 15 (ISG15)-specific, bovine ubiquitin-activating E1-like (UBE1L) enzyme

Bovine (bov) interferon-stimulated gene product 15 (ISG15) is produced in the endometrium in response to conceptus-secreted interferon (IFN)-tau. ISG15 conjugates to endometrial proteins through an enzymatic pathway that is similar to ubiquitinylation. Ubiquitin-activating enzyme 1-like protein (UBE1L) initiates enzymatic conjugation by forming a thioester bond with ISG15, thus preparing it for transfer to the next series of enzymes. The bovUBE1L has not been described. We hypothesized that bovUBE1L was induced by pregnancy and IFN-tau in the endometrium. A 110-kDa protein was purified from bovine endometrial (BEND) cells based on affinity with recombinant (r) glutathione S-transferase (GST)-ISG15. This protein was digested in gel with trypsin. Seven peptides were purified using HPLC, sequenced using liquid chromatography-mass spectroscopy-mass spectroscopy and found to share 43-100% identity with human UBE1L. The full-length bovUBE1L cDNA was isolated from a BEND cell cDNA library, sequenced, and found to share 83% identity with human UBE1L cDNA. Northern blot revealed two mRNAs that were detected in greater (P<0.05) concentrations in endometrium from Day 17-21 pregnant versus nonpregnant cows. Western blots using antihuman UBE1L antibody revealed a similar pattern of pregnancy-associated expression of UBE1L protein in the uterus. The bovUBE1L mRNA was localized, using in situ hybridization, primarily to glandular and luminal epithelium, with more diffuse localization to stroma of the endometrium from pregnant cows. Because bovUBE1L was purified through its interaction with rGST-ISG15 and shares significant amino acid and cDNA sequence identity with human UBE1L, it is concluded that it mediates conjugation of ISG15 to uterine proteins in response to the developing and attaching conceptus.     

中国白兔白介素-10基因的克隆、表达及其抗体的制备

目的克隆并表达中国白兔IL-10基因,制备抗IL-10多克隆抗体。方法运用RT-PCR对从ConA诱导后的中国白兔外周血单核细胞(PMBCr)总RNA中扩增IL-10基因,克隆后进行测序和遗传进化分析,同时将其亚克隆pET28a中,在大肠杆菌中诱导表达,并用纯化的表达产物制备多克隆抗体。结果该基因全长537 bp,编码178个氨基酸。与欧洲兔IL-10基因同源性很高,但与鼠、鸡、河豚和斑马鱼等不同物种IL-10基因差异较大。经IPTG诱导后,重组菌体裂解物经SDS-PAGE电泳可检测到相对分子质量为23.4×103的重组蛋白。Western blot分析表明,中国白兔IL-10基因已经表达。重组表达的蛋白量可占菌体蛋白的15.2%。结论成功实现了中国白兔IL-10的原核表达,制备了小鼠抗兔IL-10的多克隆抗体。     

Expression of cytochrome CYP2B1/2 in nonpregnant, pregnant and fetal rats exposed to tobacco smoke

Four-month-old female Wistar rats were exposed for 20 days to tobacco smoke obtained from non-filter cigarettes. During the exposure, concentration of tobacco smoke was monitored indirectly by measuring the CO level (1500 mg/m3 air). The efficacy of exposure was assessed by measuring urine nicotine and cotinine levels. Cigarette smoke did not change total cytochrome P450 and b5 protein levels in any of the organs studied, and most of these organs did not show any changes in the activity of reductases associated with these cytochromes. Following exposure to tobacco smoke, fetal rat liver expressed CYP2B1/2 protein; in newborns (day 1) both liver and lung showed CYP2B1/2 protein expression and very low pentoxyresorufin O-dealkylase activity. Western blot analysis of adult liver, lung, heart, but not of brain microsomes, showed that tobacco smoke induced CYP2B1/2 in both nonpregnant and pregnant rats, though its expression was lower in the livers and hearts of pregnant females. In the rat and human placenta, neither rat CYP2B1/2 nor human CYP2B6 showed basal or tobacco smoke-induced expression at the protein level. This study shows clearly that the expression of CYP2B1/2, which metabolizes nicotine and some drugs and activates carcinogens, is controlled in rats by age-, pregnancy-, and tissue-specific regulatory mechanisms.     

Omega-hydroxylation of prostaglandin E1 in the isolated perfused lungs of pregnant rabbits

Cytochrome P450PG omega is induced in the rabbit lung in a gestational age-dependent manner and hydroxylates certain eicosanoids at their terminal, or omega (omega), carbon. This enzyme has been isolated from microsomal fractions and its activity has been characterized (Williams, D.E., et al., J. Biol. Chem. 259; 14600-14608, 1984). The experiments presented here examine the omega-hydroxylation activity of the intact lung during presentation of an eicosanoid substrate, prostaglandin E1 (PGE1), to the lung vasculature. Isolated, perfused lungs from three pregnant and four nonpregnant rabbits were injected with [3H]-PGE1. One-second fractions were collected from the perfusion effluent and were analyzed for metabolism of PGE1. Lungs isolated from pregnant rabbits metabolized PGE1 mainly to two polar derivatives, 20-hydroxy-PGE1 and 13,14-dihydro-15-keto-20-hydroxy-PGE1, whereas lungs from nonpregnant rabbits yielded mainly a relatively nonpolar metabolite, 13,14-dihydro-15-keto-PGE1. These metabolites were identified by coelution with standards that were generated enzymatically in vitro and whose structures were confirmed by gas chromatography/mass spectrometry (GC/MS).     

Rabbit relaxin: the influence of pregnancy and ovariectomy during pregnancy on the plasma profile.

A porcine relaxin radioimmunoassay was developed to evaluate the profile of immunoreactive relaxin in rabbit plasma. Relaxin was nondetectable in pseudopregnant (Days 1, 4, 5-8, 12, and 16), nonpregnant, and male rabbits. However, in pregnant rabbits, relaxin was detected during the peri-implantation period (Days 4-9). Peak concentrations were reached on Day 15 and were maintained until parturition (Day 32). Relaxin concentrations abruptly decreased on Day 1 postpartum to low but detectable concentrations that were unchanged during the first week postpartum. In contrast, progesterone concentrations peaked earlier (Day 13), decreased after Day 25, and were not detectable on Day 1 postpartum. The effect of ovariectomy on the profile of plasma relaxin was evaluated. Four pregnant rabbits were ovariectomized (Day 13) and treated with medroxyprogesterone acetate to maintain pregnancy. As in normal pregnant rabbits, relaxin was observed initially during the peri-implantation period (Days 4-9) and increased to peak concentrations by Day 16. These concentrations were maintained until parturition and abruptly decreased on Day 1 postpartum to low yet detectable concentrations during the first week postpartum. The concentrations of relaxin in the plasma of ovariectomized medroxyprogesterone-treated rabbits were not different from those in three sham controls. These results indicate that the ovary is not a significant source of relaxin in pregnant rabbits. The unique observation of the presence of relaxin during the peri-implantation period suggests that this hormone has a role in preparing the rabbit uterus for implantation. The continued presence of relaxin during the first week postpartum may represent residual hormone, or it may suggest a physiological role during the early postpartum period.