Ultrastructural localization of plasma retinol-binding protein in rat liver

Immunocytochemical studies were carried out to examine the subcellular localization of plasma retinol-binding protein (RBP) in rat liver. The studies used normal, retinol-deficient, and retinol-repleted retinol-deficient rats with or without colchicine pretreatment. Affinity-purified monomeric Fab' fragments from the IgG fraction of rabbit anti-rat RBP were conjugated to horseradish peroxidase. This conjugate effectively penetrated into tissue sections and enabled RBP to be localized by high resolution immunoelectron microscopy. In the normal liver parenchymal cell, RBP was found to be localized in the synthetic and secretory structures including endoplasmic reticulum (ER), Golgi complex (GC), and secretory vesicles. With the method used, significant localization of RBP was not observed in hepatic cells other than parenchymal cells. The distribution of RBP-positive areas within parenchymal cells changed markedly with retinol depletion. Thus, a heavy accumulation of RBP in the ER, accompanied by a marked decrease of the RBP-positive GC and secretory vesicles, was demonstrated in liver parenchymal cells from retinol-deficient rats. After repletion of deficient rats with retinol, the RBP that accumulated in the ER appeared to move rapidly from the ER through GC and secretory vesicles to the cell surface. Pretreatment with colchicine led to marked increase in RBP-positive secretory vesicles in retinol-repleted rat liver parenchymal cells. The results reported here demonstrate that the specific block in hepatic RBP secretion seen in retinol deficiency involves an inhibition of the movement of RBP from the ER to the GC in the parenchymal cell.     

Localization of retinol-binding protein messenger RNA in the rat kidney and in perinephric fat tissue

The cellular localization of retinol-binding protein (RBP) messenger RNA (mRNA) in the kidney, and the developmental pattern of the renal expression of the RBP gene, were studied in the Sprague-Dawley rat. In situ hybridization studies were conducted with single-stranded cRNA probes, using sections of adult and young rat kidneys. These studies revealed specific localization of RBP mRNA in the outer stripe of the medulla, specifically localized in the S3 segment of the proximal tubules. Northern blot analysis demonstrated that RBP mRNA was not detectable in the kidney before birth or during the first week postpartum, but was clearly detected by the end of the second week of age. No RBP mRNA was observed in the kidney by in situ hybridization at 12 days of age. At 26 days of age, however, RBP mRNA was clearly detected by the in situ hybridization technique, localized in the same anatomic region as that observed in the adult kidney. Transthyretin mRNA was not detected in the adult kidney. Previous studies have shown that immunoreactive RBP is localized in the convoluted segment of the proximal tubules of the rat kidney. The present results demonstrate that RBP mRNA in the kidney is localized in an anatomic region (the S3 segment of the proximal tubules) different from that of immunoreactive RBP. In addition, an intense RBP mRNA hybridization signal was detected in the perinephric fat tissue of 26- and 40-day-old and adult rats. Further analysis of RNA from epididymal fat showed a level of RBP mRNA approximately 20% of that of liver. The function of RBP synthesized in the kidney and adipose tissue remains to be determined. We have previously hypothesized that RBP synthesized in extrahepatic tissue may function in the recycling of retinol back to the liver or to other target tissues.     

Cellular localization of ovarian proopiomelanocortin messenger RNA during follicular and luteal development in the rat

Opioid peptides are expressed in the reproductive system and have been reported to regulate reproductive function. The present study used in situ hybridization to selectively localize ovarian cells containing high levels of proopiomelanocortin (POMC) mRNA, an opioid precursor, during different stages of ovarian development. Prepubertal rats were primed with PMSG to stimulate follicular development, followed by hCG to induce ovulation. Treatment groups consisted of control (no treatment), PMSG (2 days post-PMSG), 1 day corpus luteum (CL; 1 day post-hCG), and 8 day CL (8 days post-hCG). POMC mRNA-containing cells were present in antral follicles, CL, and the interstitial compartment. With gonadotropin treatment, the percentage of follicles containing heavily labeled cells increased in the PMSG and 1 day CL groups. The number of POMC mRNA-containing cells per follicle also increased in the 1 day CL group. In the CL, no difference was observed in the percentage of CL exhibiting labeled cells between the 1 day CL and 8 day CL groups; however, more labeled luteal cells per CL were present in the 1 day CL group. A marked increase in POMC mRNA-containing cells was observed in the interstitial compartment of the 1 day CL group. These results indicate that the number of POMC mRNA-containing cells increases with follicular development and CL formation; however, the ovarian distribution suggests that the labeled cells could be nonendocrine cells, possibly white blood cells. The in situ hybridization findings are indicative of low total concentrations of ovarian POMC mRNA, suggesting mainly an autocrine or paracrine role for POMC or POMC-derived peptides.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential regulation of three sodium channel messenger RNAs in the rat central nervous system during development.

The levels of the mRNAs encoding sodium channels I, II and III in various regions of the developing rat central nervous system (from embryonal day 10 to postnatal day 90) have been examined by blot hybridization analysis with specific probes. The three sodium channel mRNAs exhibit different temporal and regional expression patterns. The expression of sodium channel I mRNA rises after a lag phase to adult levels during the second and third postnatal weeks with stronger increases in caudal regions of the brain and in spinal cord. Sodium channel II mRNA increases steadily until the first postnatal week, keeping high adult levels in rostral regions of the brain or reaching low adult levels after the second postnatal week in most caudal regions of the brain and in spinal cord; cerebellum shows low levels during the first two postnatal weeks but high adult levels. In all regions, sodium channel III mRNA attains maximum levels around birth and decreases during the first and second postnatal weeks to reach variable low adult levels. These results suggest that sodium channel III is expressed predominantly at fetal and early postnatal stages and sodium channel I predominantly at late postnatal stages, whereas sodium channel II is expressed throughout the developmental stages studied with greater regional variability.     

Immunohistochemical studies on the localization of cellular retinol-binding protein in rat testis and epididymis

The immunohistochemical localization of cellular retinol-binding protein (CRBP) was studied in rat testis and epididymis. Parallel studies were also carried out on the localization of plasma retinol-binding protein (RBP) and transthyretin (TTR) in testis. The studies employed antibodies purified by immunosorbent affinity chromatography, permitting the specific staining and localization of each antigen by the unlabeled peroxidase-antiperoxidase method. For RBP and TTR, specific immune staining was found in the interstitial spaces between the seminiferous tubules, and not in the tubules themselves. In contrast, strong specific immune staining for CRBP was found in the seminiferous tubules, with a striking localization within Sertoli cells. Moreover, a distinct cyclic variation of specific staining for CRBP within Sertoli cells was observed during the spermatogenic cycle. This cyclic variation was seen with regard to both the intensity of staining and to the anatomic distribution of CRBP within the Sertoli cells. Within the epididymis CRBP was selectively localized to the proximal portion of the caput epididymidis, with variations in intensity of the staining of the epithelium of the ducts in different histological zones. Specific immune staining for CRBP was very weak or absent in the other portions of the epididymis. These results were confirmed by radioimmunoassay. Vitamin A-deficient rats showed markedly reduced specific immune staining for CRBP in both testes and epididymides, and greatly reduced levels of CRBP in these tissues on radioimmunoassay. These studies on the localization of CRBP provide information concerning the specific cells and anatomic loci within the testis and epididymis where retinol may be playing an important role in sperm formation and maturation.     

Detection of cellular retinol-binding protein messenger RNA in the somatic cells of the rat seminiferous tubules

A cDNA clone coding for Cellular Retinol-Binding Protein (CRBP) was used as a probe to study the expression of the gene in the somatic cells of the seminiferous tubules (Sertoli and peritubular cells). In this paper we demonstrate that these cells are actively involved in the synthesis of the specific mRNA. In Sertoli cells the gene is modulated by the hormones effective in spermatogenesis, such as FSH and testosterone. Moreover, peritubular cells revealed an approximately two times higher concentration of CRBP steady-state mRNA levels when compared with Sertoli cells.     

Intracellular localization of messenger RNAs for cytoskeletal proteins

We have analyzed intracellular distributions of mRNAs for the cytoskeletal proteins actin, vimentin, and tubulin by in situ hybridization. Although polyadenylated RNA was homogeneously distributed throughout the cell, actin mRNA demonstrated a nonhomogeneous distribution in 95% of randomly selected chicken embryonic myoblasts and fibroblasts, as detected by isotopic and nonisotopic techniques. Actin mRNA concentrations were highest at cell extremities, generally in lamellipodia, where grain densities were up to 16-fold higher than in areas near the nucleus. Vimentin mRNA, unlike actin mRNA, was distributed near the nucleus. Tubulin mRNA appeared most concentrated in the peripheral cytoplasm. These results demonstrate that cytoplasmic mRNAs are localized in specific, nonrandom cellular patterns and that localized concentrations of specific proteins may result from corresponding localization of their respective mRNAs. Hence, actin mRNA distribution may result in increased concentration of actin filaments in lamellipodia of motile cells.     

An extracellular retinol-binding glycoprotein in the rat eye—characterization,localization and biosynthesis

We have identified and partially purified interstitial retinol-binding protein (IRBP) from the subretinal space of the rat. It appeared to be glycosylated. Its apparent mol. wt was 270,000 by gel-filtration and 144,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Rat IRBP cross-reacted with anti-bovine IRBP sheep and rabbit sera, bound all-trans-[15-³H] retinol and was bound by concanavalin A. IRBP was not detected in the cytosols of the neural retina or retinal pigment epithelium and choroid. This distribution was confirmed by immunocytochemistry using a fluorescence-labeled second antibody. Immunospecific fluorescence was most intense in the interphotoreceptor matrix in a 6.5 μm band adjacent to the retinal pigment epithelium. It was less intense over the remainder of the rod outer segment layer and was comparatively faint over the inner segment region. Its occurrence in the interstitial space between the photoreceptors and retinal pigment epithelium coupled with the fact it bound all-trans-[15-³H] retinol supports the concept that it may be implicated in the transport of retinoids between the retina and the retinal pigment epithelium during the visual cycle. When incubated with [³H]leucine or [³H]glucosamine, isolated retinas (but not retinal pigment epithelium and choroid) secreted labeled IRBP into the medium. This suggests that the retina plays a role in regulating the amount of IRBP in the subretinal space.     

Accumulation of the early histone messenger RNAs during the development of Strongylocentrotus purpuratus

The developmental profile of amounts of the mRNAs for the early histones was determined. Sea urchin embryos (Strongylocentrotus purpuratus) were labeled with ³H nucleoside, and the RNA was extracted and fractionated by electrophoresis on polyacrylamide gels. Radioactivity in the resolved mRNA bands was determined and converted to molar quantities by knowledge of the precursor pool-specific activity. Between the 16- and the 200-cell stages 7–10 × 10⁶ molecules of each core histone mRNA, and 2.5 × 10⁵ molecules of H1 mRNA accumulate. During the subsequent few hours of cleavage the accumulated mRNAs disappear with a half-life of 1.5–2 hr. It is argued that mRNA half-life may be regulated during cleavage.     

Immunocytochemical studies on the localization of plasma and of cellular retinol-binding proteins and of transthyretin (prealbumin) in rat liver and kidney

The immunocytochemical localization of cellular retinol-binding protein (CRBP), of plasma retinol-binding protein (RBP), and of plasma transthyretin (TTR) was studied in rat liver and kidney. The studies employed normal rats, retinol-deficient rats, and rats fed excess retinol. Antisera were prepared in rabbits against purified rat CRBP, RBP, and TTR. The primary antibodies and goat anti-rabbit IgG were purified by immunosorbent affinity chromatography, using the respective pure antigen coupled to Sepharose as the immunosorbent. This procedure effectively removed cross-reactive and heterophile antibodies, which permitted the specific staining and localization of each antigen by the unlabeled peroxidase-antiperoxidase method. CRBP was found to be localized in two cell types in the liver, the parenchymal cells and the fat-storing cells. Diffuse cytoplasmic staining for CRBP was seen in all the parenchymal cells. Much more intense staining for CRBP was seen in the fat-storing cells. The prominence of the CRBP-positive fat- storing cells changed markedly with vitamin A status. Thus, these cells were most prominent, and appeared most numerous, in liver from rats fed excess retinol. Both RBP and TTR were localized within liver parenchymal cells. The intensity of RBP staining increased markedly in retinol-deficient rat liver, consistent with previous biochemical observations. With the methods employed, specific staining for RBP or TTR was not seen in cells other than the parenchymal cells. In the kidney, all three proteins (CRBP, RBP, and TTR) were localized in the proximal convoluted tubules of the renal cortex. Staining for RBP was much more intense in normal kidney than in kidney from retinol- deficient rats. These findings reflect the fact that RBP in the tubules represents filtered and reabsorbed RBP. The pattern of specific staining for CRBP among the various tubules was very similar to that seen for RBP on adjacent, serial sections of kidney. The function of CRBP in the kidney is not known.     

基质Gla蛋白在大鼠附睾发育过程中的表达及定位

目的明确基质Gla蛋白(matrix Gla protein,MGP)在大鼠附睾发育过程中的表达特征。方法采用实时定量PCR和免疫荧光染色方法,对MGP在大鼠附睾不同发育阶段的表达及定位进行检测。结果实时定量PCR结果显示,MGP mRNA在6d、10d、3w、5w、7w、8w、10w和12w的大鼠附睾中均有表达,其表达量在3w达到最高峰,3w至8w表达量逐渐降低,成年大鼠(10～12w)MGP的表达量逐渐升高并稳定在较高水平。免疫荧光染色显示MGP在10d、3w的大鼠附睾各个节段均有表达,在7w、12w的表达主要集中于大鼠附睾体部和尾部,且MGP定位于附睾上皮主细胞和亮细胞。结论MGP在大鼠附睾发育的关键分化期高表达,成年后主要定位于附睾体部和尾部的主、亮细胞,可能对附睾的形态发育和管腔钙稳态的维持起重要作用。     

Identification and cytogenetic localization of vitelline membrane messenger RNAs in Drosophila

During stages 9 and 10 of oogenesis in Drosophila the major proteins involved in vitelline membrane (VM) formation are synthesized and secreted by the somatic follicle cells surrounding the oocyte. To identify potential mRNAs involved in VM protein synthesis, newly synthesized poly(A)-containing RNA from egg chambers of different developmental stages was studied. Urea-agarose gel electrophoresis revealed two RNA bands in stage 10 egg chambers in the size range expected for those which encode the smaller VM proteins. These RNA bands, T₁ and T₂, are specifically enriched in stage 10 follicle cell preparations. In vitro translations in reticulocyte lysates in the absence and presence of microsomal membranes showed both RNA bands code for products that are synthesized in precursor forms which are processed to species that comigrate with VM proteins. T₂ directed the synthesis of processed species that comigrated with the 23- to 24-kDa and 17.5-kDa VM proteins (J. Fargnoli and G. L. Waring, 1982, Dev. Biol. 92, 306–314) while the T₁ translation product comigrated with the 14-kDa protein. To determine the cytogenetic location of the genes encoding T₁ and T₂ RNAs, radiolabeled T₁ and T₂ RNAs were hybridized in situ to salivary gland chromosomes. The results suggest that the structural genes coding for the small vitelline membrane proteins are localized at two sites on the second chromosome: 39DE and 42A.     

Cellular retinol-binding protein messenger RNA levels in normal and retinoid-deficient rats

A study was conducted to determine the levels of cellular retinol-binding protein (CRBP) mRNA and protein in various tissues of the rat, to explore relationship between CRBP mRNA and protein levels in different tissues, and to examine the effects of changes in retinol nutritional status on the tissue distribution and levels of CRBP mRNA. Previous studies have shown that tissue CRBP protein levels are reduced in totally retinoid-deficient rats, but are otherwise minimally affected by changes in retinoid status. Three groups of male rats were compared: normal controls, retinoid-deficient, and retinol-repleted deficient rats. CRBP mRNA levels were measured by RNase protection assay and CRBP protein levels by radioimmunoassay in seven tissues. High levels of both CRBP mRNA and CRBP protein were found in the proximal epididymis, kidney, and liver; lower levels were seen in lung, testis, spleen, and small intestine. Tissue CRBP mRNA and protein levels were highly correlated (P less than 0.01) with each other. Retinoid deficiency did not alter the levels of CRBP mRNA found in the proximal epididymis, kidney, and liver. In contrast, CRBP mRNA levels in the lung, testis, spleen, and small intestine were reduced substantially in retinoid-deficient rats, to values that were only 23% to 50% of the corresponding values in the tissues of control rats. After oral repletion with retinol (4-18 h earlier), CRBP mRNA levels for these latter four tissues were found to have risen to control or near-control levels. The suggestion is raised that retinol repletion may have directly induced the expression of the CRBP gene in these particular tissues.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stem cell factor and c-kit gene expression and protein localization in the sheep ovary during fetal development

The aim of this study was to investigate stem cell factor and c-kit gene expression and protein localization in the mesonephros and ovary of sheep fetuses at different days of gestation, using RNA in situ hybridization and immunohistochemical procedures. At days 24 and 26 of gestation, stem cell factor mRNA and protein were present in cells throughout the developing gonad and mesonephros. From day 28 to day 40 of gestation, stem cell factor mRNA and protein became increasingly localized to the cortical region of the ovary, where most germ cells were present as actively proliferating oogonia. From day 40 to day 90 of gestation, stem cell factor mRNA and protein localization were confined mainly to the ovarian cortex. Moreover, within the cortical region, stem cell factor mRNA was low or absent where follicles were first forming and highest in the outer ovarian cortex, where germ cells were undergoing mitosis or the early stages of meiosis. In contrast, stem cell factor protein was present in newly forming follicles, as well as in mitotic and meiotic germ cells, which is consistent with the presence of both membrane-bound and soluble forms of this ligand. However, by day 90 of gestation, both stem cell factor mRNA and protein were observed in the granulosa cells of most (> 90%) primordial follicles. C-kit mRNA and protein were observed from day 24 of gestation in both germ cells and somatic cells but, with increasing gestational age, preferentially in germ cells (for example, pre-meiotic germ cells and both isolated oocytes and follicle-enclosed oocytes). C-kit protein, but not mRNA, was also observed in germ cells that were undergoing meiosis. The results indicate that the cells containing stem cell factor mRNA within the ovary up to day 90 of gestation originated from the gonadal blastema and from cells that migrated from the mesonephros before day 28 of gestation. Since stem cell factor mRNA was absent in both mesonephric cells migrating after day 28 of gestation and in regions where follicles were first forming, it is suggested that these later migrating mesonephric cells are the progenitors of the granulosa cells in the first forming follicles. In conclusion, during follicle formation, c-kit mRNA is localized to germ cells whereas c-kit, together with stem cell factor protein, is localized to both germ cells and somatic cells, consistent with the hypothesis that the presence of this receptor-ligand pair is essential to prevent apoptosis.     

2-D gel membrane protein patterns during fetal rat hepatocyte development

Fetal hepatocytes from rats of 13-20 days of gestation were 125I/surface-labelled. Autoradiographs of the plasma membrane proteins, analyzed by 2-D gel electrophoresis, were compared with those from adult hepatocytes and two hepatoma lines. The autoradiographic patterns of membrane proteins from 13 and 15 day fetal hepatocytes were similar. Between days 13 and 20 of gestation, at least 24 new iodinatable proteins appeared in the plasma membrane of fetal hepatocytes. Whilst between day 20 of gestation and adult life, at least 21 new membrane proteins appeared. Two membrane proteins, one acidic (MW 25,000) and a more basic protein (MW 45,000) were present in fetal hepatocytes but absent from adult hepatocytes. The membrane protein patterns from two hepatoma lines were less complex than those of adult hepatocytes.     

Effect of the N-terminal sequence on the binding affinity of transthyretin for human retinol-binding protein

During vertebrate evolution, the N-terminal region of transthyretin (TTR) subunit has undergone a change in both length and hydropathy. This was previously shown to change the binding affinity for thyroid hormones (THs). However, it was not known whether this change affects other functions of TTR. In the present study, the effect of these changes on the binding of TTR to retinol-binding protein (RBP) was determined. Two wild-type TTRs from human and Crocodylus porosus, and three chimeric TTRs, including a human chimeric TTR in which its N-terminal sequence was changed to that of C. porosus TTR (croc/huTTR) and two C. porosus chimeric TTRs (hu/crocTTR in which its N-terminal sequence was changed to that of human TTR and xeno/crocTTR in which its N-terminal sequence was changed to that of Xenopus laevis TTR), were analyzed for their binding to human RBP by native-PAGE followed by immunoblotting and a chemilluminescence assay. The K(d) of human TTR was 30.41 ± 2.03 μm, and was similar to that reported for the second binding site, whereas that of crocodile TTR was 2.19 ± 0.24 μm. The binding affinities increased in croc/huTTR (K(d) = 23.57 ± 3.54 μm) and xeno/crocTTR (K(d) = 0.61 ± 0.16 μm) in which their N-termini were longer and more hydrophobic, but decreased in hu/crocTTR (K(d) = 5.03 ± 0.68 μm) in which its N-terminal region was shorter and less hydrophobic. These results suggest an influence of the N-terminal primary structure of TTR on its function as a co-carrier for retinol with RBP.     

Localization of immunoreactive transthyretin (prealbumin) and of transthyretin mRNA in fetal and adult rat brain

We used a combination of immunohistochemical and molecular-biological techniques to investigate the localization of transthyretin (TTR) in the brains of adult and fetal rats. The immunohistochemical studies employed antibodies purified by immunosorbent affinity chromatography, permitting the specific staining and localization of TTR using the unlabeled peroxidase-antiperoxidase method. TTR mRNA levels were measured by Northern-blot analysis of poly (A+) RNA, followed by hybridization to 32P-labeled TTR cDNA; TTR mRNA was localized in brain tissue sections by in situ hybridization. Immunoreactive TTR was found to be specifically localized in the choroid plexus epithelial cells of adult rat brain. High levels of TTR mRNA were found in poly (A+) RNA samples obtained from the choroid plexus. In addition, the specific localization of TTR mRNA in the epithelial cells of the choroid plexus was demonstrated by in situ hybridization. Neither immunoreactive TTR nor TTR mRNA were found in other regions of adult rat brains. The levels of TTR mRNA in the choroid plexus were at least 30 times higher than those observed in the adult liver. Immunoreactive TTR was observed in the brains of fetal rats on as early as the 11th day of gestation. This immunoreactive TTR was localized in the tela choroidea, the developmental forerunner of the choroid plexus. Immunoreactive TTR was also observed in the fetal choroid plexus as it began to form (14th day of gestation) as well as in the more completely developed choroid plexus (18th day of gestation).(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions of retinol with binding proteins: studies with rat cellular retinol-binding protein and with rat retinol-binding protein

The interactions of retinol with rat cellular retinol-binding protein (CRBP) and with rat serum retinol-binding protein (RBP) were studied. The equilibrium dissociation constants of the two retinol-protein complexes (Kd) were found to be 13 x 10(-9) and 20 x 10(-9) M for CRBP and for RBP, respectively. The kinetic parameters governing the interactions of retinol with the two binding proteins were also studied. It was found that although the equilibrium dissociation constants of the two retinol-protein complexes were similar, retinol interacted with CRBP 3-5-fold faster than with RBP; the rate constants for dissociation of retinol from CRBP and from RBP (koff) were 0.57 and 0.18 min-1, respectively. The rate constants for association of retinol with the two proteins (kon) were calculated from the expression: Kd = koff/kon. The kon's for retinol associating with CRBP and with RBP were found to be 4.4 x 10(7) and 0.9 x 10(7) M-1 min-1, respectively. The data suggest that the initial events of uptake of retinol by cells are not rate-limiting for this process and that the rate of uptake is probably determined by the rate of metabolism of this ligand. The data indicate further that the distribution of retinol between RBP in blood and CRBP in cytosol is at equilibrium and that intracellular levels of retinol are regulated by the levels of CRBP.