Purification and immunolocalization of ovine placental retinol-binding protein.

A retinol-binding protein (RBP), synthesized and secreted by ovine allantois in vitro, was purified from culture medium. The protein consisted of three isoelectric variants (pI 5.3-6.1) of identical molecular masses of about 23,000 Da as determined by two-dimensional PAGE under reducing conditions. Thirty-one of the first 34 N-terminal amino acids of the purified protein were sequenced and shown to have complete homology with bovine placental and bovine plasma RBP. The ultraviolet absorption spectrum and fluorescence excitation and emission spectra of the purified ovine placental RBP indicated the presence of bound retinol. Metabolic labeling studies demonstrated that the protein was synthesized by placental membranes. Using antiserum to bovine placental RBP, ovine placental RBP was immunolocalized in trophectoderm of 13-day-old blastocysts and trophectodermal cells of the chorion, endodermal cells lining the allantois, and ectodermal cells lining the amnion of 23-, 45-, and 53-day-old conceptuses. Results from this study suggest that ovine placental membrane epithelia synthesize and secrete RBP. Transport, storage, and metabolism of retinol mediated by placental RBP may be essential for normal embryonic development during pregnancy.     

Early gestational expression of retinol-binding protein mRNA by the ovine conceptus and endometrium

Communication between the mother and the early developing embryo is mediated by a variety of signals secreted by either the uterus or the embryo to elicit a response from the other. These signals include prostaglandins, proteins, and steroids. Recently, retinol-binding protein (RBP) has been described as a product of both the conceptus and endometrium in several species. Utilizing a cDNA clone to bovine RBP, we have described RBP mRNA expression in the endometrium, early conceptus, and extraembryonic membranes of sheep. Endometrial RBP mRNA expression did not differ between samples collected on day 13 of the estrous cycle and early pregnancy. In cyclic animals, RBP mRNA expression decreased two-fold between days 13 and 16, presumably a result of luteal regression and the consequential withdrawal of progesterone. In pregnant animals, endometrial RBP mRNA expression likewise decreased between days 13 and 16 and remained at this reduced level through day 30, despite the presence of a functional corpus luteum. Initiation of embryonic RBP expression appeared to coincide with early stages of blastocyst elongation at day 13. Levels of expression increased dramatically with conceptus development, peaked on day 23, and declined afterwards. Results from restriction enzyme analysis of genomic DNA indicated that RBP was encoded by a single gene per haploid genome. Differences in the temporal and tissue-specific expression of the protein, despite the apparent utilization of a single gene, suggest complex regulation of RBP gene expression. © 1994 Wiley-Liss, Inc.     

Expression of retinol-binding protein and cellular retinol-binding protein in the bovine ovary

Retinol (vitamin A) is essential for reproduction, and retinoids have been suggested to play a role in ovarian steroidogenesis, oocyte maturation, and early embryonic development. Retinol is transported systemically and intercellularly by retinol-binding protein (RBP). Within the cell, cellular retinol-binding protein (CRBP) functions in retinol accumulation and metabolism. Since the actions of retinoids are mediated, in part, by retinoid-binding proteins, the objective of this study was to investigate cell-specific expression of RBP and CRBP in the bovine ovary. Immunocytochemical analysis (ICC) localized RBP to the thecal and granulosa cell layers of antral and preantral follicles with the most intense staining in the cells of large, healthy follicles. The tunica adventitia of arterial blood vessels also exhibited RBP staining. Immunostaining of CRBP was most intense in the granulosa cells of preantral follicles and present, but diminished, in thecal and granulosa cells of antral follicles. Within the corpus luteum, both proteins were observed in large luteal cells, but only RBP was observed in small luteal cells. Northern blot analysis demonstrated that thecal and granulosa cells from antral follicles and luteal tissue expressed RBP and CRBP mRNA. Synthesis and secretion of RBP by thecal cells, granulosa cells, and luteal cells were demonstrated by immune-complex precipitation of radiolabeled RBP from the medium of cultured cells or explants, followed by SDS-PAGE and fluorography. Follicular fluid was collected from small (<5 mm) and large (8-14 mm) follicles, pooled according to follicular size, and analyzed for retinol, RBP, estradiol-17beta, and progesterone. Concentrations of retinol, RBP, and estradiol were greater in the fluid of large follicles. Results demonstrate retinoid-binding protein expression by bovine ovaries and provide physical evidence that supports the concept that retinoids play a role in ovarian function.     

Steroid hormones induce cell proliferation and specific protein synthesis in primary chick oviduct cultures

A rapid method to obtain large amounts of tubular gland cells from chick oviduct was developed. Combined collagenase and trypsin treatment allowed within 1.5 h complete dissociation of the magnum portion of the oviduct. By differential attachment of cells, fibroblasts were separated from tubular gland- and ciliated cells. Tubular gland cells attached within 18 h to plastic Petri dishes, had large secretory granules and grew very actively. The responsiveness of cells to hormones and/or antihormone was tested by measurement of cell proliferation and specific protein synthesis. After 7 days of culture in the presence of estradiol (50 nM) or progesterone (100 nM), cell growth was increased by approximately 50 and 35% respectively. Tamoxifen (100 nM) inhibited the estradiol induced growth stimulation, but had also negative effects of its own. The anti-progesterone (in mammals) RU 486, inactive per se, did not antagonize progesterone induced growth. Ovalbumin- and conalbumin synthesis after 4-5 days of cultures under different hormonal conditions was assessed after immunoprecipitation of newly synthesized [35S]methionine labelled proteins. In the presence of estradiol (50 and 100 nM), progesterone (50 nM), and both estradiol and progesterone together (50 nM of each), ovalbumin and conalbumin synthesis was increased, when compared to control cultures without hormones, or to oviduct fibroblasts. Hormonal stimulation of ovalbumin synthesis was also shown in cell supernatant and culture medium after gel electrophoresis.     

Ligand-dependent regulation of intracellular protein transport: effect of vitamin a on the secretion of the retinol-binding protein

As a model of ligand-dependent protein secretion the biosynthesis, intracellular transport, and release of the retinol-binding protein (RBP) were studied in primary cultures of rat hepatocytes pulse-labeled with [35S]methionine. After various periods of chase RBP was isolated by immunoprecipitation and identified by SDS PAGE. Both normal and vitamin A-deficient hepatocytes synthesized RBP. The normal cells secreted the pulse-labeled RBP within 2 h. RBP synthesized by deficient cells was not secreted, and intracellular degradation of the protein appeared to be slow. Deficient cells could be induced to secrete RBP on the addition of retinol to the culture medium. This occurred also after protein synthesis had been blocked by cycloheximide. Since retinol induces the secretion of RBP, accumulated in the endoplasmic reticulum (ER), it seems reasonable to conclude that the transport of RBP from the ER to the Golgi complex is regulated by retinol.     

视黄醇结合蛋白的分离纯化

在变性条件下,应用Sephacryl-100凝胶过滤和Source-30Q阴离子交换两步分离,实现了分离纯化性质不稳定、易于降解的视黄醇结合蛋白(RBP)之目的。最后经过分步缓慢复性,获得具有生物活性的RBP,为其单克隆抗体制备及最终应用于临床营养评价和相关疾病的诊断创造了条件。     

视黄醇结合蛋白的分子生物学

视黄醇结合蛋白 (RBP)是维生素A(VitA)的运载蛋白 ,结构上为疏水小分子结合蛋白家族的成员。视黄醇结合蛋白在肝脏中合成并释放入血液后 ,通过与视黄醇 (ROH)、甲状腺素运载蛋白 (TTR)及细胞表面受体相互作用 ,在VitA的储存、代谢、转运到周围靶器官中发挥重要功能。本文从基因结构与表达、构效关系及临床意义等方面介绍了视黄醇结合蛋白的分子生物学。     

Mitogen-activated protein kinase activation and regulation in the pressure-loaded fetal ovine heart

The mitogen-activated protein (MAP) kinase signaling pathways help to mediate the hypertrophic response of the pressure-loaded adult heart, although their importance in fetal myocardium is less known. The goal of this study was to determine the role the MAP kinase signaling pathways play in regulating the response of the fetal heart to a pressure load. Aortic (Ao) and pulmonary artery (PA) bands were placed in 132-day fetal sheep for 7 days. Protein levels of the total and active (phosphorylated) terminal MAP kinases extracellular signal-regulated kinase (ERK/P-ERK), c-Jun NH(2)-terminal kinase (JNK/P-JNK), and p38/P-p38 and the MAP kinase phosphatases MKP-1, MKP-2, and MKP-3 were made in the right and left ventricular (RV and LV) free walls. In both Ao- and PA-banded animals, total heart weight normalized to body weight was significantly increased, largely due to an increase in RV free wall mass in the Ao-banded animals and an increase in septal mass in the PA-banded fetuses. Total protein levels of the three terminal kinases and of P-ERK and P-JNK remained stable in both groups of banded animals. However, P-p38 was significantly increased in RV and LV of Ao- and PA-banded fetuses. Whereas MKP-1 and MKP-2 protein levels were unchanged following Ao- and PA-banding, MKP-3 protein levels were significantly increased in the RV of the PA-banded animals. These findings indicate that the MAP kinase signaling pathways are active in the fetal heart and help to modulate the response of prenatal myocardium to a pressure load.     

Binding specificities of cellular retinol-binding protein and cellular retinol-binding protein, type II

Cellular retinol-binding protein (CRBP) and cellular retinol-binding protein, type ii (CRBP(II] are cytoplasmic proteins that bind trans-retinol as an endogenous ligand. These proteins are structurally similar having greater than 50% sequence homology. Employing fluorescence, absorbance, and competition studies, the ability of pure preparations of CRBP(II) and CRBP to bind various members of the vitamin A family has been examined. In addition to trans-retinol, CRBP(II) was able to form high affinity complexes (K'd less than 5 X 10(-8) M) with 13-cis-retinol, 3-dehydroretinol, and all-trans-retinaldehyde. CRBP bound those retinol isomers with similar affinities, but did not bind trans-retinaldehyde. Neither protein bound retinoic acid nor 9-cis- and 11-cis-retinol. The spectra of 13-cis-retinol and 3-dehydroretinol, when bound, were shifted and displayed fine structure compared to their spectra in organic solution. However, the lambda max and fluorescent yield of a particular ligand were different when bound to CRBP(II) versus CRBP. It appears that CRBP(II) and CRBP bind trans-retinol, 13-cis-retinol, and 3-dehydroretinol in a planar configuration. However, the binding sites of CRBP(II) and CRBP are clearly distinct based on the observed spectral differences of the bound ligands and the observations that only CRBP(II) could bind trans-retinaldehyde. The ability of CRBP(II) to bind trans-retinaldehyde suggests a physiological role for the protein in accepting retinaldehyde generated from the cleavage of beta-carotene in the absorptive cell.     

Time-dependent physiological regulation of ovine placental GLUT-3 glucose transporter protein

We immunolocalized the GLUT-3 glucose transporter isoform versus GLUT-1 in the late-gestation epitheliochorial ovine placenta, and we examined the effect of chronic maternal hyperglycemia and hypoglycemia on placental GLUT-3 concentrations. GLUT-3 was limited to the apical surface of the trophoectoderm, whereas GLUT-1 was on the basolateral and apical surfaces of this cell layer and in the epithelial cells lining the placental uterine glands. GLUT-3 concentrations declined at 17-20 days of chronic hyperglycemia (P < 0.05), associated with increased uterine and uteroplacental net glucose uptake rate, but a normal fetal glucose uptake rate was observed. Chronic hypoglycemia did not change GLUT-3 concentrations, although uterine, uteroplacental, and fetal net glucose uptake rates were decreased. Thus maternal hyperglycemia causes a time-dependent decline in the entire placental glucose transporter pool (GLUT-1 and GLUT-3). In contrast, maternal hypoglycemia decreases GLUT-1 but not GLUT-3, resulting in a relatively increased GLUT-3 contribution to the placental glucose transporter pool, which could maintain glucose delivery to the placenta relative to the fetus when maternal glucose is low.     

Steroid hormonal regulation of calcium-binding protein regucalcin mRNA expression in the kidney cortex of rats

Ethylenediamine tetraacetate ( EDTA ) inhibits lactoperoxidase (LPO)-catalyzed rate of iodide oxidation in concentration and pH-dependent manner. A plot of log Ki_app values against various pH yields a sigmoidal curve from which an ionisable group of pK_a value 6.0 could be ascertained for controlling the inhibition of catalytically active LPO by EDTA. Kinetic studies indicate that EDTA competitively inhibits iodide oxidation by acting as an electron donor. EDTA al so reduces LPO-compound-11 to the native ferric state by one-electron transfer as evidenced by the spectral shift from 428 to 412 nm. Optical difference spectroscopic studies indicate that EDTA binds to LPO with the apparent equilibrium dissociation constant (KD) of 12 ± 2 mM at pH 6.5. A plot of log K_D values against various pH produces a sigmoidal curve from which an ionisable group of LPO having pk_a = 5.47 could be calculated, deprotonation of which favours EDTA binding. EDTA also binds to LPO-CN- complex indicating its binding site away from heme iron centre. The K_D of LPO-EDTA complex is significantly increased (62 ± 5 mM) by iodide suggesting that EDTA binds close to the iodide binding site. EDTA also increases the K_D value of LPO-hydroquinone complex from 62 ± 5 mM to 200 ± 21 mM indicating that EDTA and aromatic donor binding sites are also close. We suggest that EDTA inhibits iodide oxidation competitively as an electron donor by interacting at or near the iodide binding site and these sites are close to the aromatic donor binding site.     

Involvement of tyrosine and lysine residues of retinol-binding protein in the interaction between retinol and retinol-binding protein and between retinol-binding protein and prealbumin. Acetylation with N-acetylimidazole and alkaline titration.

The behavior of holo-retinol-binding protein (RBP) from human plasma at alkaline pH was examined by absorption and circular dichroism measurements. Between pH 7.5 and 11.7 the ionization of the phenolic hydroxyl groups is reversible. However, there is a gradual irreversible loss of retinol as the pH is raised. After 4 hours at pH 11.7, 13 percent of retinol is lost from retinol-RBP. Alkaline titration of apo-RBP was time-independent and reversible between pH 7.5 and 11.7. The titration data of the phenolic hydroxyl groups in apo-RBP could be fitted with a single theoretical ionization curve of 8.6 phenolic groups having an apparent pK of 11. Acetylation of retinol-RBP with 10-fold molar excess of N-acetylimidazole over tyrosine resulted in the acetylation of all lysine residues and in the acetylation of 0.9 to 1.3 tyrosyl residues per molecule (out of eight). Acetylation of retinol-RBP, APO-RBP, and retinol-RBP-prealbumin complex with 50-fold molar excess of N-acetylimidazole resulted, again, with all of the lysine residues being acetylated and between 1.8 and 2.8 tyrosyl residues per molecule being acetylated. The acetylation did not affect the interaction between retinol and RBP. However, acetylation disrupted the normal binding between retinol-RBP and prealbumin. Deacetylation of tyrosyl residues with hydroxylamine failed to restore the normal binding of retinol-RBP to prealbumin. This excludes the acetylated tyrosyl-residues from being involved in the binding between the two proteins.