Interaction of the retinol/cellular retinol-binding protein complex with isolated nuclei and nuclear components.

Retinol (vitamin A alcohol) is involved in the proper differentiation of epithelia. The mechanism of this involvement is unknown. We have previously reported that purified cellular retinol-binding (CRBP) will mediate specific binding of retinol to nuclei isolated from rat liver. We now report that pure CRBP delivers retinol to the specific nuclear binding sites without itself remaining bound. Triton X-100-treated nuclei retain the majority of these binding sites. CRBP is also capable of delivering retinol specifically to isolated chromatin with no apparent loss of binding sites, as compared to whole nuclei. CRBP again does not remain bound after transferring retinol to the chromatin binding sites. When isolated nuclei are incubated with [3H]retinol-CRBP, sectioned, and autoradiographed, specifically bound retinol is found distributed throughout the nuclei. Thus, CRBP delivers retinol to the interior of the nucleus, to specific binding sites which are primarily, if not solely, on the chromatin. The binding of retinol to these sites may affect gene expression.

Comparison of the level of cellular retinoid-binding proteins and susceptibility to retinoid-induced growth inhibition of various neoplastic cell lines.

The presence and level of cellular retinol-binding protein (CRBP) and cellular retinoic acid-binding protein (CRABP) were determined in several neoplastic cell lines. These cells exhibited different degrees of susceptibility to growth inhibition in culture by two retinoids, retinyl acetate and retinoic acid. CRABP was detected in 10 and CRBP in 3 of the 11 tested cell lines. The levels of CRBP and CRABP were in the ranges 15-3,400 and 4-1,290 pmol per 10(9) cells, respectively, as determined by sucrose gradient centrifugation. Cell lines that contained CRABP included S91 and B16 melanomas; Mm5mT and DMBA No. 8 mammary adenocarcinomas; BW5147, BW5147.RicR, and P3 neoplastic lymphoid cells; F361.2 (a hybrid cell line obtained by fusion of MSV3T3 and BW5147); MSV3T3 sarcoma; and RAW8 lymphosarcoma. All but the last two cell lines were inhibited by retinoic acid in culture. CRBP was detected in extracts of S91, Mm5mT, and RAW8. Retinyl acetate inhibited the growth of all cell lines with the exception of RAW8, MSV3T3, and F361.2. No correlation was found between the level of either binding protein and the extent of growth inhibition by either retinyl acetate or retinoic acid. Neither of the binding proteins was detected in L1210-A5 leukemia cells, whose proliferation can be inhibited by both retinyl acetate and retinoic acid. These data indicated that screening cell lines for the presence and level of CRBP and CRABP is not sufficient to predict the susceptibility of cultured cells to growth inhibition by retinoids.

Water-soluble, dextran-linked retinal: preparation, vitamin A-like activity in rats, and effects on melanoma cells.

A new, water-soluble, polymer-linked form of retinal was synthesized and tested for its ability to support the growth of vitamin A-deficient noninbred Holtzman rats and to inhibit the proliferation of melanoma cells in culture. Retinal was conjugated to the hydrazide of carboxymethyldextran in the presence of alpha-and beta-cyclodextrins. The aqueous solutions of the product contained between 200 and 1,000 micrograms retinal/ml as opposed to the low water solubility (< 0.01 micrograms/ml) of retinal itself. The retinal-dextran complex, although barely resorbed from the gastrointestinal tract, supported the growth of rats fed a vitamin A-deficient diet when administered ip at 2.3 mumol of retinal equivalent/kg body weight. Retinal and the retinal-dextran complex exhibited differential cytotoxicity toward S91 melanoma cells and caused cell lysis at 10 and 500 microM (retinal residue), respectively. At noncytotoxic doses both free retinal and its dextran-linked derivative reduced the cell proliferation rate in a time- and dose-dependent fashion with median inhibitory doses of 1 and 4 microM (retinal residue), respectively. These data demonstrated that the water-soluble retinal-dextran complex retained certain biologic activities of retinal and was less cytotoxic.

Tissue-specific antibodies against human lung and breast carcinoma dehistonized chromatins.

Tissue-specific antisera against human lung and breast carcinoma dehistonized chromatins were obtained. The specificity of these antisera was determined by complement fixation. In the presence of antiserum against human lung carcinoma, only chromatins from lung carcinoma fixed complement significantly, whereas chromatins isolated from human breast carcinoma, HeLa cells, normal lung tissue, breast tissue, or term placenta were negative (i.e., inactive). In a similar assay with the use of antiserum against dehistonized breast carcinoma chromatins, only breast carcinoma chromatins fixed complement. Immunohistochemical localization of the antigens by the horseradish peroxidase bridge method demonstrated their presence in the nuclei.

Three-step purification of retinol-binding protein from rat serum.

Rat serum retinol-binding protein has been purified to apparent homogeneity in high yield by a new procedure utilizing three simple steps: DEAE-cellulose chromatography at pH 6.0, Sephadex G-75 gel filtration in the presence of 3.0 M urea, and finally DEAE-cellulose chromatography at pH 8.3. The second step accomplished the dissociation and separation of retinol-binding protein from its complex with prealbumin; this represents a substantial improvement over published procedures, in which sample recycling and preparative polyacrylamide gel electrophoresis were necessary. The purified retinol-binding protein was characterized by molecular weight measurement, fluorescence spectra and immunological properties.

Cellular retinol-binding protein.

1. A protein which binds retinol in vitro with high affinity and specificity was detected by sucrose gradient centrifugation or by gel filtration after preincubating rat tissue cytosols with all-trans-[3H]retinol. This protein sediments in the 2 S region of sucrose gradients. Molecular size determination by gel filtration indicates a molecular weight of 16 000. 2. Competition studies revealed that only all-trans-retinol, not retinal or retinoic acid, competes for binding. The binding of radioactive retinol is reversible. 3. This protein was detected in cytosols of rat liver, lung, spleen, brain, testis, ovaries, uterus and intestinal mucosa whereas heart or gastrocnemius muscle seem to lack this protein. 4. The cellular retinol binding protein was found in fetuses as early as day 12 of the gestation period and possessed the same specificity for the ligand as the one in adult tissues. 5. This binding component was not detected in cytosols prepared from Novikoff hepatoma, ascites hepatoma AS-30D, mouse Ehrlich ascites tumor and mouse pituitary tumor cell line AtT 20. 6. The cellular retinol binding protein seems to be different from that described to be present in the serum as suggested by difference in size and by the inability of the antisera against the serum retinol binding protein to remove the cellular binding protein from the cytosol preparations.

Purification and characterization of xanthine oxidase from livers of vitamin E deficient rabbits.

Xanthine oxidase which increases in activity during vitamin E deficiency was purified from livers of deficient rabbits. The procedure incorporates preparative sucrose gradient centrifugation and yields a homogeneous preparation on acrylamide gel electrophoresis. The purified enzyme exhibits a pH optimum of 8.1 and a Km value of 22 muM. Gel filtration chromatography gave the molecular weight of 280 000. Acrylamide gel electrophoresis in the presence of sodium dodecylsulphate reveals two types of subunits of molecular weights 52 000 and 99 000.

Evidence of a role for retinoic acid (vitamin A-acid) in the maintenance of testosterone production in male rats.

Animals maintained on retinol (vitamin A-alcohol)-deficient diets exhibit testicular atrophy and loss of the germinal epithelium. Retinoic acid (vitamin A-acid), when fed to retinol-deficient animals, does not prevent these lesions and had thus been thought not to play a role in the tests. Serum testosterone (T) levels, determined by RIA, in retinol-deficient rats were determined to be significantly lower than in control rats. In contrast, retinoic acid-fed, retinol-deficient rats exhibited serum T concentrations similar to those of control rats. No difference in immunoreactive serum LH levels was observed in the three groups. The response of serum T to ip administration of LH in retinol-deficient animals relative to basal levels was similar to that observed in control as well as retinoic acid-fed, retinol-deficient rats. These results show that while basal T production in retinol-depleted rats is decreased, LH-stimulated T synthesis is unaffected. Furthermore, retinoic acid, in the absence of retinol, can support T production, suggesting that contrary to present dogma, retinoic acid plays a role in testis.

Changes in chick oviduct chromatin antigenicity following estrogen withdrawal.

A microcomplement fixation assay was used to study changes in the antigenicity of chick oviduct chromatin following estrogen withdrawal. Our findings indicate that following estrogen withdrawal, chromatin from previously stimulated oviducts lost those antigenic determinants which were characteristic of the stimulated state. Withdrawal did not, however, result in the reappearance of antigenic sites characteristic of the unstimulated oviduct. These results suggest that estrogen stimulation of the immature chick results in an irreversible alteration of the oviduct chromatin.

Opposing effects of retinoic acid and dexamethasone on cellular retinol-binding protein ribonucleic acid levels in the rat.

Cellular retinol-binding protein (CRBP) is a potential mediator of vitamin A action. To determine whether retinoic acid and dexamethasone administration, alone and in combination, influence CRBP gene expression, adult female vitamin A-sufficient Sprague-Dawley rats randomly received 1) all-trans retinoic acid (100 micrograms) by intragastric intubation, 2) dexamethasone (2 micrograms/g BW) by ip injection, or 3) both all-trans retinoic acid and dexamethasone in the same doses. Control animals received either cottonseed oil by intragastric intubation or saline by ip injection. Six hours after treatment, lung and liver tissue were collected for Northern blot analysis with the radiolabeled cDNA specific for rat CRBP. Retinoic acid administration increased the amount of lung CRBP mRNA only, whereas dexamethasone decreased both lung and liver CRBP mRNA abundance. In animals treated with both retinoic acid and dexamethasone, CRBP mRNA abundance was also reduced. We conclude that CRBP gene expression can be modulated by both retinoic acid and dexamethasone in the vitamin A-sufficient animal. In the whole animal, our findings indicate that dexamethasone not only represses CRBP gene expression, but also opposes the effect of retinoic acid.

Autoradiographic localization of serum retinol-binding protein in rat testis.

An interstitial target site in the rat testis for vitamin A has been detected using radioactively labeled rretinol-binding protein (RBP) in autoradiography at the light microscope level. RBP purified from rat serum was iodinated by the lactoperoxidase procedure and was also complexes with [3H]-retinol. The radioactively labeled RBP was administered to rat testis tissue by a direct intratesticular injection, an iv injection, and through an in vitro incubation of decapsulated tissue in medium containing the labeled protein. Localization on the autoradiograms of both [125I]RBP and [3H]retinol-RBP was interstitial; there was no interaction of RBP with cells in or on the seminiferous tubules. The patterns of localization were identical in normal rats and vitamin A-deficient rats. [3H]etinol uncomplexed with RBP (administered with rat serum albumin) was distributed evenly throughout testicular tissue, with no specific localization. Although the labeling of the cells on the autoradiograms was not visible diminished by the presence of excess unlabeled RBP, an in vitro binding assay of a crude interstitial cell preparation using [125I]RBP displayed saturable binding, indicative of receptors for RBP. An in vitro binding assay of Sertoli cells in culture with [125I]RBP demonstrated no specific binding, nor did [125I]RBP in autoradiography of Sertoli cell monolayers display interaction with these cells. Results from this investigation show that the plasma transport protein for vitamin A, RBP, interacts initially with cells in the interstitium of the testis. (Endocrinology 108: 658, 1981)

Mainstream and sidestream cigarette smoke exposure increases retinol in guinea pig lungs.

We have studied in guinea pigs the effects of cigarette smoke exposure on vitamin A (retinol) levels in plasma, lung, lung lavage, and liver. Smoke was generated from 1R3F cigarettes in a smoke exposure instrument designed by University of Kentucky Tobacco and Health Research Institute. Three-week-old male guinea pigs were exposed to mainstream, sidestream, or sham smoke, generated twice daily from three cigarettes for 6 weeks. In addition, some animals were kept as room controls for some time. After 6 weeks of smoke exposure, some animals were allowed to recover for 6 weeks without smoke. After 6 weeks of smoking, the plasma retinol levels were lower in both smoke exposed groups when compared to the values in the sham group. Furthermore, in comparison to the sham group, the mainstream and sidestream smoke exposed groups showed a 7.6- and 8.3-fold increase in the levels of lung retinol, respectively. After the 6-week recovery period, plasma retinol of both smoke-exposed groups reached the control levels. In contrast, withdrawal of smoking did not show such an effect on the lung retinol level in both mainstream or sidestream groups. Electronmicroscopy of the lungs showed deleterious alterations in the morphology of the lungs in both mainstream and sidestream groups. Although the mechanism(s) involved in the elevation of retinol content of the lung due to smoke exposure remains to be elucidated, it is of interest that elevation of retinol content and alteration of lung morphology occurred not only in the mainstream smoke exposed but also in the sidestream group.

Immunocytochemical localization of cellular retinol binding protein in the rat retina.

Cellular retinol binding protein (CRBP) was localized in the rat retina by means of light and electron microscopic immunocytochemistry. The peroxidase-antiperoxidase (PAP) method employed at the light microscopic level showed that CRBP is sharply localized to the retinal pigment epithelium (RPE). None was detectable in the epithelium of the pars plana or pars plicata of the ciliary body. Likewise, the photoreceptors were negative. Within the neural retina, the PAP method revealed a bilaminar staining pattern in the inner plexiform layer and staining within elements near the vitreal surface. Indirect immunoferritin electron microscopy demonstrated that CRBP is distributed uniformly within the RPE cytosol from basal infoldings to apical processes. The nucleoplasm also was stained, albeit, more lightly than the cytoplasm. Labeled elements in the inner plexiform layer and vitreal surface were identified as Müller cell processes and end feet, respectively. Interpretation of the results includes a dual role for CRBP in the RPE, namely its involvement in gene expression and transcytoplasmic transport of retinol.

Presence of cellular rentinol and retinoic acid binding proteins in experimental rumors.

Cellular retinol and retinoic acid binding proteins were detected in mouse skin papillomas, human adenocarcinoma HAD-1, Dunning Leukemia, Walker 256 carcinosarcoma and mammary adenocarcinoma MAC-1. A chondrosarcoma and Sarcoma 180 apparently contain only the cellular retinoic acid binding protein. Neither protein could be detected in Ehrlich carcinoma and L1210 leukemia. The presence of these proteins might be necessary for sensitivity to retinoid therapy.

Cellular vitamin A-binding proteins in the testis.

Vitamin A plays an important role in the testis, being essential for the maintenance of spermatogenesis. Studies on CRBP and CRABP suggest that both retinol and retinoic acid are involved in maintaining testicular function. The cellular location of the two proteins suggests that retinoic acid may be particularly involved in the later stages of germ cell differentiation, but retinol may be the form of vitamin A that the Sertoli cell receives initially. The requirement for vitamin A may be to regulate gene expression in the testis by direct interaction with the chromosomal material. Specific distinct binding sites for retinol and retinoic acid can be demonstrated in testicular nuclei and chromatin. These sites are only revealed when the two ligands are present in complex with their specific binding proteins, suggesting that these proteins may be required for the action of retinol and retinoic acid in some cells of the testis.

Localization of cellular retinol-binding protein and cellular retinoic acid-binding protein in the rat testis and epididymis.

The distribution of cellular retinol-binding protein (CRBP) and cellular retinoic acid-binding protein (CRABP) in rat testis and epididymis was examined by the peroxidase-antiperoxidase immunolocalization technique. In the testis, cellular retinol-binding protein was localized exclusively in the Sertoli cells. Staining varied with the stages of the seminiferous epithelium cycle and was maximal prior to the maturation divisions. Cellular retinoic acid-binding protein was localized exclusively in the germinal cells in the adluminal compartment. The results suggest that retinoic acid may be the retinoid form used by the germinal cells, and that Sertoli cells may use the cellular retinol-binding protein to transfer retinol from the basal to the adluminal compartment. In the epididymis, cellular retinol-binding protein was localized in the cytoplasm and stereocilia of the principal cells in the proximal caput epididymidis, while cellular retinoic acid-binding protein was localized in the spermatozoa and the stereocilia of the principal cells throughout the epididymis and in the epithelial cells of the distal vas deferens. Sperm staining intensity decreased from the initial segment to the cauda. The presence of high levels of cellular retinol-binding protein in the epithelial cells and high levels of cellular retinoic acid-binding protein in the spermatozoa of the caput epididymidis, known to be involved in the synthesis and secretion of factors necessary for sperm maturation, suggests that vitamin A may have a role in this process.

Vitamin A and lung development.

Several lines of evidence suggest that vitamin A is involved in lung development. They are: 1) Dietary deficiency of the fat-soluble vitamin A results in squamous metaplasia of the tracheal and bronchial epithelium. 2) Infants with vitamin A deficiency have a high incidence of respiratory problems. 3) Levels of two intracellular proteins binding specifically retinol (vitamin A alcohol) and retinoic acid (vitamin A acid) change dramatically during perinatal lung development. 4) Prematurely born infants hospitalized for respiratory problems have low serum concentrations of retinol and retinol-binding protein. 5) Postnatal supply of vitamin A by parenteral alimentation may not be adequate, as large quantities of vitamin A are absorbed by the tubing. Careful assessment of vitamin A status in postnatal nutritional management of premature infants is desirable.

Vitamin A status and airway infection in mechanically ventilated very-low-birth-weight neonates.

Vitamin A (retinol) plays an important role in immunity. Respiratory and enteral infections in children are associated with low serum vitamin A concentrations that improve during recovery. To test the hypothesis that airway infection in very-low-birth-weight (VLBW) neonates likewise may be associated with a change in vitamin A status, we examined 20 VLBW neonates (selection criteria: birth weight 700-1300 g, gestational age 26-30 weeks, need for supplemental oxygen and mechanical ventilation for > 72 hr after birth) who were enrolled in the control group of a randomized clinical trial of vitamin A supplementation reported earlier. We studied changes in weekly measurements of plasma concentrations of vitamin A and retinol-binding protein (RBP) during 4 weeks following enrollment in the trial (postnatal day 4) and compared changes between periods with and without airway infections. Seventeen infants had 22 episodes of documented airway infection. Staphylococcus epidermidis was the predominant organism. Plasma vitamin A concentrations decreased during 19 out of 22. With airway infection (mean change: -4.1 to -18.6 micrograms/dL), while they increased during 37 out of 58 periods without airway infection (mean change: -0.2 to +5.8 micrograms/dl; P < 0.001). The mean (+/- SD) plasma vitamin A concentrations before, during, 1 week after, and 2 weeks after an episode of airway infection were 20.9 +/- 8.3, 9.7 +/- 4.1, 12.8 +/- 8.9, and 16.2 +/- 7.2 micrograms/dL, respectively. The mean value during airway infection was significantly lower than those before and two weeks after airway infection (P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)