Cellular retinol binding protein 1 transfection reduces proliferation and AKT-related gene expression in H460 non-small lung cancer cells.

In recent years, new treatments with novel action mechanisms have been explored for advanced non-small cell lung cancer (NSCLC). Retinoids promote cancer cell differentiation and death and their trafficking and action is mediated from specific cytoplasmic and nuclear receptors, respectively. The purpose of this study was to investigate the effect of Cellular retinol binding protein-1 (CRBP-1) transfection in H460 human NSCLC cell line, normally not expressing CRBP-1. H460 cells were transfected by using a vector pTargeT Mammalian expression system carrying the whole sequence of CRBP-1 gene. For proliferation and apoptosis studies, cells were treated with different concentrations of all-trans Retinoic Acid (atRA) and retinol. AKT-related gene expression was analyzed by using western blot and Signosis array and results analysed by one-way analysis of variance (ANOVA) or by t-student test. CRBP-1+ showed reduced proliferation and viability in basal condition and after atRA treatment when compared to empty-transfected H460 cells. Reduced proliferation in CRBP-1+ H460 cells associated to the down-regulation of pAKT/pERK/pEGFR-related genes. In particular, gene array documented the down-regulation of AKT and Stat-3-related genes, including M-Tor, Akt1, Akt2, Akt3, Foxo1, p27, Jun. Restoration of CRBP-1 expression in H460 cells reduced proliferation and viability in both basal condition and after atRA treatment, likely by down-regulating AKT-related gene level. Further studies are needed to better clarify how those CRBP-1-related intracellular pathways contribute to counteract NSCLC progression in order to suggest a potential tool to improve efficacy of retinoid anti lung cancer adjuvant therapy.

Identification of RBP4 from bighead carp (Hypophthalmichthys nobilis) / silver carp (Hypophthalmichthys molitrix) and effects of CpG ODN on RBP4 expression under A. hydrophila challenge.

Retinol-binding protein 4 (RBP4) is known as a highly conserved adipokine for immune activation. Aeromonas hydrophila (A. hydrophila) is the most common zoonotic pathogen in aquaculture, which causes serious economic losses to aquaculture, especially to bighead carp (Hypophthalmichthys nobilis, H. nobilis) and silver carp (Hypophthalmichthys molitrix, H. molitrix). Recent studies along with our previous findings have shown that synthetic oligodeoxynucleotides containing CpG motifs (CpG ODN) can play a good role in aquatic animals against infection. In order to clarify the relationship between CpG ODN and RBP4 under A. hydrophila infection, firstly, full-length RBP4 cDNAs from H. nobilis and H. molitrix were cloned. And characteristics of RBP4, including sequence and structure, tissue distribution and genetic evolution were analyzed. In addition, mRNA expression levels of RBP4, cytokine, toll-like receptors (TLRs), morbidity and survival rates of H. nobilis and H. molitrix were observed post CpG ODN immunization or following challenge. The results indicated that hn/hm_RBP4 (RBP4 genes obtained from H. nobilis and H. molitrix) had the highest homology with Megalobrama amblycephala. Distribution data showed that the expression level of hn_RBP4 mRNA was higher than that of hm_RBP4. After CpG ODN immunization followed by A.hydrophila challenge, significantly higher survival was observed in both carps, together with up-regulated RBP4 expression. Meanwhile, hn/hm_IL-1ß level was relatively flat (and decreased), hn/hm_IFN-γ, hn/hm_TLR4 and hn/hm_TLR9 levels increased significantly, but hn/hm_STRA6 showed no significant change, compared with control. Moreover, CpG ODN immunization could induce stronger immune protective responses (higher IFN-γ/gentle IL-1ß level and lower morbidity/higher survival rate) against A. hydrophila in H. nobilis, along with higher RBP4 level, when compared with that in H. molitrix. These results demonstrated that RBP4 was well involved in the immune protection of CpG ODN. Based on the results, we speculated that in the case of A. hydrophila infection, TLR9 signaling pathway was activated by CpG ODN. Subsequently, CpG ODN up-regulated RBP4, and RBP4 activated TLR4 signaling pathway. Then TLR4 and TLR9 synergistically improved the anti-infection responses. Our findings have good significance for improving resistance to pathogen infection in freshwater fish.

Inhibitory effect of chitooligosaccharides on retinol metabolism and bioavailability in mice.

This study investigated the intervention effects of chitooligosaccharides (COS) on retinol metabolism and included comparisons of the retinol level, retinol binding protein 4 (RBP4) content, key genes, and protein expression between mice on a COS-enriched diet and a normal diet. The results showed that COS markedly decreased the retinol and RBP4 concentrations in the serum and liver. Furthermore, COS suppressed the mRNA and protein expression of RBP4, cellular retinol binding protein 1 (CRBP1), lecithin: retinol acyltransferase (LRAT) and cytochrome P45026A1 (CYP26A1). In addition, COS inhibited the mRNA expression of stimulated by retinoic acid 6 (STRA6). However, the protein expression of STRA6 was not significantly decreased. Thus, COS reduced the retinol concentration in the serum and disrupted the metabolism of retinol. The intervention mechanism of COS on retinol metabolism may be attributed to the modulation of RBP4, CRBP1, LRAT, STRA6, and CYP26A1 expression at the mRNA and protein levels. PRACTICAL APPLICATIONS: Chitooligosaccharides (COS), known to be the degradation products of chitosan, have been found to induce pinkeye in industrial workers who participate in the manufacturing of COS. Meanwhile, 5% population with COS dietary supplement also have similar phenomenon. The aim of this study is to explore the possible mechanism underlay of this potential risk. The results of this study showed that high exposure to COS during manufacture influences retinol metabolism and leads to a decrease in retinol content, ultimately causing pinkeye. These findings provide new evidence for understanding COS-induced retinol metabolism alteration and drawing attention toward the prevention of potential risk in high-exposure populations.

Abnormal Cannabidiol Modulates Vitamin A Metabolism by Acting as a Competitive Inhibitor of CRBP1.

Cellular retinol-binding proteins (CRBPs) facilitate the uptake and intracellular transport of vitamin A. They integrate retinoid metabolism, playing an important role in regulating the synthesis of bioactive vitamin A metabolites. Thus, CRBPs constitute potential pharmacological targets to modulate cellular retinoid status that in turn may have applications in the treatment of certain immunological, metabolic, and ocular disorders. Here we identify abnormal cannabidiol (abn-CBD) as a nonretinoid inhibitor of cellular retinol-binding protein 1 (CRBP1). X-ray crystal structures of CRBP1 in complex with abn-CBD and its derivatives revealed a distinctive mode of protein-ligand interaction and provided a molecular basis for the high affinity and selectivity of this compound. We demonstrated that abn-CBD modulates the flux of retinoids via the retinoid cycle in vivo. Furthermore, the biological activity of abn-CBD was evidenced by its ability to protect against light-induced retinal damage in Balb/cJ mice. Altogether, our findings indicate that targeting selected CRBPs with a small-molecule inhibitor can potentially lead to the development of new therapeutic agents to counteract diseases with etiologies involving imbalance in retinoid metabolism or signaling.

The coordinated action of lecithin:retinol acyltransferase and cellular retinol-binding proteins for regulation of vitamin A esterification.

Vitamin A is a fat-soluble vitamin required for many physiological functions. The intracellular transport of vitamin A is assisted by proteins called cellular retinol-binding proteins (CRBP I/II). The absorption, storage and usage of vitamin A are regulated by a protein called lecithin:retinol acyltransferase (LRAT), a retinol-related enzyme that transfers an acyl group derived from an sn-1 position of phosphatidylcholine to retinol. LRAT is a member of the protein family which includes HRAS-like tumor suppressors (HRASLS). However, the HRASLS proteins never use retinol as an acyl acceptor. The mechanisms underlying the different substrate specificities between LRAT and HRASLS proteins are unknown. We propose in this report that LRAT physically interacts with CRBP and the LRAT-CRBP complex represents the binding pockets for both an acyl group and retinol, thus assuring the substrate specificity of LRAT.

CRBP-1 expression in ovarian cancer: a potential therapeutic target.

BACKGROUND/AIM: Cellular retinol binding protein-1 regulates retinol bioavailability and contributes to cell differentiation maintenance, but its role in ovarian carcinogenesis remains uncertain. We investigated CRBP-1 expression in ovarian tumors and CRBP-1 signaling-regulated pathways. MATERIALS AND METHODS: We performed immunohistochemistry, methylation-specific PCR, gene copy number analysis in ovarian tumors and proliferation/apoptosis evaluation, gene array, blot and real-time PCR in CRBP-1-transfected A2780 ovarian cancer cells. RESULTS: CRBP-1 expression was reduced or absent in G2 and G3 ovarian carcinomas. CRBP-1 silencing in 60% of G2 and 66.7% of G3 carcinomas was due to CRBP-1 promoter methylation. A2780 CRBP-1-transfected cells showed increased retinol-induced apoptosis, retinoid-induced reduced clonogenicity and down-regulation of proliferation and transcription genes, including AKT1, AKT3, EGFR, FOS, JUN, STAT1 and STAT5A. CONCLUSION: CRBP-1 loss in G2/G3 ovarian carcinomas and increased apoptotic susceptibility to retinoids in CRBP-1-transfected-A2780 cells suggest CRBP-1 screening as a target to ensure efficacy of an adjuvant retinoid therapy.

Characterization of the expression profile of CRBP1 and CRBP3 gene in chicken.

In this study, we quantified the expression of CRBP1 and CRBP3 in Roman layer (R) and Erlang mountainous chickens (SD02 and SD03), to discern the tissue, breed and age-related expression patterns in order to discover potential involvement in egg production and other related reproduction traits. Real-time quantitative PCR assays were developed for accurate measurement of CRBP1 and CRBP3 mRNA levels in different tissues from chickens at four ages (12, 20, 32 and 45 weeks). We found that the CRBP1 and CRBP3 were expressed in all six tissues examined in all three breeds of chicken at 32 weeks. CRBP1 mRNA levels in SD02 kidneys were slightly higher than those in SD03 and R at 12 weeks, whereas, at the other three time points, the expression levels of CRBP1 in SD03 were higher than those in SD02 and R. In addition, there was higher hepatic expression of CRBP3 mRNA in layers (R) compared to broilers (SD02 and SD03) at 20 and 32 weeks. An age-related expression pattern of CRBP1 gene was evident in liver (P < 0.01), but not in pituitary (P > 0.05). Overall, the expression level of CRBP1 gene in kidney, ovary and oviduct at the different ages had a "decline-rise-decline" trend in all three breeds. In contrast, in pituitary, hypothalamus, liver and kidney CRBP3 mRNA expression levels were significantly different at various ages (P < 0.05) and exhibited a "rise-decline-rise" pattern in all three breeds. Our results show that the expression of CRBP1 and CRBP3 in chicken tissues exhibit specific developmental changes and age-related patterns.

Immunohistochemical expression of RBP2 and LSD1 in papillary thyroid carcinoma.

PURPOSE: To investigate the prognostic significance of LSD1 and RBP2 expression in patients with papillary thyroid carcinoma. MATERIALS AND METHODS: LSD1 and RBP2 expressions were detected by immunohistochemistry in surgically resected samples from thyroid adenoma, papillary thyroid carcinoma and paracancerous tissues. RESULTS: To be members of histone demethylases, LSD1 and RBP2 were both localized mainly to the thyroid cell nucleus. Despite the fact that both RBP2 and LSD1 expressions were higher in papillary thyroid carcinoma than in paracancerous tissues (U=-3.855, p=0.000; U=-5.575, p=0.000) and thyroid adenoma (U=-1.972, p=0.049; U=-3.190, p=0.001), they did not show us statistical correlation (r=-0.149, p=0.270). Like LSD1 (U=-2.286, p=0.022), RBP2 expression was less frequently in paracancerous tissues than in thyroid adenoma (U=-1.985, p=0.047). Neither LSD1 nor RBP2 expression was significantly associated with age, gender, stage status, tumor size, and lymph node metastases (p>0.05). CONCLUSIONS: Both LSD1 and RBP2 are well related with the occurrence and malignant transformation of papillary thyroid carcinoma. Though the positive expression of both LSD1 and RBP2 can be used to estimate the potentiality of thyroid carcinoma and help for the adjuvant treatment, LSD1 is a more sensitive molecular marker than RBP2 on thyroid cancer diagnosis.

Hypothalamic ventricular ependymal thyroid hormone deiodinases are an important element of circannual timing in the Siberian hamster (Phodopus sungorus).

Exposure to short days (SD) induces profound changes in the physiology and behaviour of Siberian hamsters, including gonadal regression and up to 30% loss in body weight. In a continuous SD environment after approximately 20 weeks, Siberian hamsters spontaneously revert to a long day (LD) phenotype, a phenomenon referred to as the photorefractory response. Previously we have identified a number of genes that are regulated by short photoperiod in the neuropil and ventricular ependymal (VE) cells of the hypothalamus, although their importance and contribution to photoperiod induced physiology is unclear. In this refractory model we hypothesised that the return to LD physiology involves reversal of SD expression levels of key hypothalamic genes to their LD values and thereby implicate genes required for LD physiology. Male Siberian hamsters were kept in either LD or SD for up to 39 weeks during which time SD hamster body weight decreased before increasing, after more than 20 weeks, back to LD values. Brain tissue was collected between 14 and 39 weeks for in situ hybridization to determine hypothalamic gene expression. In VE cells lining the third ventricle, expression of nestin, vimentin, Crbp1 and Gpr50 were down-regulated at 18 weeks in SD photoperiod, but expression was not restored to the LD level in photorefractory hamsters. Dio2, Mct8 and Tsh-r expression were altered by SD photoperiod and were fully restored, or even exceeded values found in LD hamsters in the refractory state. In hypothalamic nuclei, expression of Srif and Mc3r mRNAs was altered at 18 weeks in SD, but were similar to LD expression values in photorefractory hamsters. We conclude that in refractory hamsters not all VE cell functions are required to establish LD physiology. However, thyroid hormone signalling from ependymal cells and reversal of neuronal gene expression appear to be essential for the SD refractory response.

Associations of polymorphisms in four candidate genes with carcass and/or meat-quality traits in two meat-type chicken lines.

The associations between polymorphisms of five genes, calpain 1 (CAPN1), follicle stimulating hormone beta (FSHB), follicle stimulating hormone receptor (FSHR), peroxisome proliferator-activated receptor gamma (PPARG), and retinol binding protein 7 (RBP7), and live weight, carcass composition, and meat-quality traits were estimated from two meat-type chickens lines (n=311). Except for the variants of the FSHR gene, 11 SNPs of the other four genes and two diplotypes of PPARG were associated with one or more traits excluding shear factor (SF). SNP C31566680T of the CAPN1 gene was significantly associated with live weight (LW) carcass traits. The SNP A4580859C of FSHB gene was significantly associated with breast muscle weight (BrW) and LW. One of the PPARG SNPs, C5070948T, was associated with intramuscular fat content in breast (IMFbr). Diplotype P1 of the PPARG gene was significantly associated with LW and all carcass traits. P3 were significantly associated with abdominal fat weight (AbFW). SNPs in RBP7 were only associated with BrW. These results indicate that the four genes were associated with these traits and have promise as genetic markers for future marker-assisted selection. Supplementary materials for this paper are available online.

RDH10 is the primary enzyme responsible for the first step of embryonic Vitamin A metabolism and retinoic acid synthesis.

Retinoic acid (atRA) signaling is essential for regulating embryonic development, and atRA levels must be tightly controlled in order to prevent congenital abnormalities and fetal death which can result from both excessive and insufficient atRA signaling. Cellular enzymes synthesize atRA from Vitamin A, which is obtained from dietary sources. Embryos express multiple enzymes that are biochemically capable of catalyzing the initial step of Vitamin A oxidation, but the precise contribution of these enzymes to embryonic atRA synthesis remains unknown. Using Rdh10(trex)-mutant embryos, dietary supplementation of retinaldehyde, and retinol dehydrogenase (RDH) activity assays, we demonstrate that RDH10 is the primary RDH responsible for the first step of embryonic Vitamin A oxidation. Moreover, we show that this initial step of atRA synthesis occurs predominantly in a membrane-bound cellular compartment, which prevents inhibition by the cytosolic cellular retinol-binding protein (RBP1). These studies reveal that widely expressed cytosolic enzymes with RDH activity play a very limited role in embryonic atRA synthesis under normal dietary conditions. This provides a breakthrough in understanding the precise cellular mechanisms that regulate Vitamin A metabolism and the synthesis of the essential embryonic regulatory molecule atRA.

Effect of Cordyceps sinensis on renal function of patients with chronic allograft nephropathy.

OBJECTIVE: To investigate the effect of Cordyceps sinensis (Bailing capsule, fermented agent of C. sinensis) on renal function of patients with chronic allograft nephropathy (CAN). METHODS: A total of 231 CAN patients who underwent transplantation between 2005 and 2008 and experienced chronic graft dysfunction were randomly divided into 2 groups. Patients in group A (n = 122) were treated with immunosuppressive agents and C. sinensis (2.0 g/day, 3 times a day), while patients in group B (n = 109) were treated with traditional immunosuppressive drugs. Serum creatinine (SCr), blood urea nitrogen (BUN), creatinine clearance rate (C(Cr)) and urinary protein in 24 h (24-hour Upro) of all patients were measured before and after treatment. Urinary concentrations of transforming growth factor (TGF)-ß(1), retinol-binding protein (RBP) and ß(2)-microglobulin (ß(2)-MG) were detected at the same time. RESULTS: After 6-month treatment with C. sinensis, SCr and C(Cr) in group A were significantly improved (p < 0.05), while there was no significant improvement observed for group B. There was no significant change in BUN in groups A and B (p > 0.05). 24-hour Upro, RBP and ß(2)-MG were lower in group A after treatment with C. sinensis (p < 0.05 or p < 0.01), and urinary TGF-ß(1) in group A was significantly lower than the values before C. sinensis treatment (p < 0.05), but showed no change in patients of group B. In group A, renal function had improved in 72 cases, stabilized in 38 cases, and worsened in 12 cases. In group B, renal function had improved in 14 cases, stabilized in 50 cases, and worsened in 45 cases (p < 0.05). CONCLUSION: C. sinensis therapy is advantageous in improving renal function of CAN patients by retarding CAN progression.

Uptake of all-trans retinoic acid-containing aerosol by inhalation to lungs in a guinea pig model system--a pilot study.

Systemic therapies with retinoic acid (RA) can result in toxic side effects without yielding biologically effective levels in target tissues such as lung. The authors adapted a PARI LC Star nebulizer to create a tubular system for short-term inhalation treatment of guinea pigs using a water-miscible formulation of all-trans RA (ATRA) or vehicle. Based on the initial average weight, animals received an estimated average ATRA doses of either 0.32 mg·kg(-1) (low dose, 1.4 mM), or 0.62 mg·kg(-1) (medium dose, 2.8 mM), or 1.26 mg·kg(-1) (high dose, 5.6 mM) 20 minutes per day for 6 consecutive days. This system led to a rise of ATRA levels in lung, but not liver or plasma. Cellular lung levels of retinol, retinyl palmitate, and retinyl stearate also appeared to be unaffected (245.6 ± 10.7, 47.4 ± 3.4, and 132.8 ± 7.7 ng·g(-1) wet weight, respectively). The application of this aerosolized ATRA also induced a dose-dependent protein expression of the cellular retinol-binding protein 1 (CRBP-1) in lung, without apparent harmful side effects.

A global metabolite profiling approach to identify protein-metabolite interactions.

Understanding the biochemical functions of proteins is an important factor in elucidating their cellular and physiological functions. Due to the predominance of biopolymer interactions in biology, many methods have been designed to interrogate and identify biologically relevant interactions that proteins make to DNA, RNA, and other proteins. Complementary approaches that can elucidate binding interactions between proteins and small molecule metabolites will impact the understanding of protein-metabolite interactions and fill a need that is outside the scope of current methods. Here, we demonstrate the ability to identify natural protein-metabolite interactions from complex metabolite mixtures by combining a protein-mediated small molecule enrichment step with a global metabolite profiling platform.

Characterization of cellular retinoid-binding proteins in human myometrium during pregnancy.

Many complementary or competing signalling pathways bear an influence on the myometrium at any one time, and because the retinoic acid signalling pathway influences differentiation of a wide array of human tissues, this may be one of the determinants of myometrial differentiation during pregnancy. We have explored the novel hypothesis that the retinoids may act as important regulators in controlling the differentiated state of the human myometrium during pregnancy by characterizing the expression profiles for cellular retinoid-binding proteins CRBPI, CRABPI and CRABPII in non-pregnant, pregnant (non-labouring) and labouring human myometrium taken from the functionally distinct upper and lower uterine segments. In addition, we have investigated the effect of all-trans retinoic acid (ATRA) on the expression of several retinoic acid response genes including cyclooxygenase-2 (COX-2) and connexin-43 (Cx-43). Different spatial and temporal patterns of expression were observed for CRBPI, CRABPI and CRABPII within the upper and lower uterine segments through the three trimesters of pregnancy and in labour. Furthermore, the expression of COX-2, Cx-43, CRABPI, the transcription factor c-Jun and the retinoic acid receptor RARbeta altered in response to different concentrations of ATRA, suggesting that the differential expression of cellular retinoid-binding proteins may lead to different levels of retinoic acid being delivered to its nuclear targets, leading to the differential expression of specific target genes within the myometrium during pregnancy.

Integrating protein structures and precomputed genealogies in the Magnum database: examples with cellular retinoid binding proteins.

BACKGROUND: When accurate models for the divergent evolution of protein sequences are integrated with complementary biological information, such as folded protein structures, analyses of the combined data often lead to new hypotheses about molecular physiology. This represents an excellent example of how bioinformatics can be used to guide experimental research. However, progress in this direction has been slowed by the lack of a publicly available resource suitable for general use. RESULTS: The precomputed Magnum database offers a solution to this problem for ca. 1,800 full-length protein families with at least one crystal structure. The Magnum deliverables include 1) multiple sequence alignments, 2) mapping of alignment sites to crystal structure sites, 3) phylogenetic trees, 4) inferred ancestral sequences at internal tree nodes, and 5) amino acid replacements along tree branches. Comprehensive evaluations revealed that the automated procedures used to construct Magnum produced accurate models of how proteins divergently evolve, or genealogies, and correctly integrated these with the structural data. To demonstrate Magnum's capabilities, we asked for amino acid replacements requiring three nucleotide substitutions, located at internal protein structure sites, and occurring on short phylogenetic tree branches. In the cellular retinoid binding protein family a site that potentially modulates ligand binding affinity was discovered. Recruitment of cellular retinol binding protein to function as a lens crystallin in the diurnal gecko afforded another opportunity to showcase the predictive value of a browsable database containing branch replacement patterns integrated with protein structures. CONCLUSION: We integrated two areas of protein science, evolution and structure, on a large scale and created a precomputed database, known as Magnum, which is the first freely available resource of its kind. Magnum provides evolutionary and structural bioinformatics resources that are useful for identifying experimentally testable hypotheses about the molecular basis of protein behaviors and functions, as illustrated with the examples from the cellular retinoid binding proteins.

Downregulation of Otx2 in the dedifferentiated RPE cells of regenerating newt retina.

Cynops pyrrhogaster (the Japanese common newt) regenerates neural retina from retinal pigmented epithelium (RPE) cells. Otx2 is a transcription factor that is involved in RPE cell differentiation. To understand the role of Otx2 during transdifferentiation of RPE cells, we cloned a Cynops Otx2 cDNA, and explored its expression by RT-PCR, immunohistochemistry and in situ hybridization. The expression of Otx2 was compared with the localization of a proliferating cell marker (PCNA), RPE cell markers (RPE65, CRBP) and an RPE and Muller glial cell marker (CRALBP). At the early stage of regeneration, 2 to 3 cell layered regenerating retina consisting of pigmented cells uniformly expressed Otx2 and other markers. Following this stage, 4-cell layered regenerating retina consisted of two distinct layers, pigmented monolayer (the outer layer) attached to Bruch's membrane and presumptive neural retina (the inner layers). In the outer layer, Otx2 and CRBP expression was maintained and majority of cells lost PCNA expression. Some of cells maintained RPE65. In the inner layers, expression of Otx2, CRBP and RPE65 was downregulated, but a majority of those cells maintained PCNA expression. These results indicate that spatiotemporal regulation of Otx2 expression is consistent with those of RPE markers. Otx2 may play a pivotal role in maintenance and specification of RPE cells during neural retina regeneration. In contrast to RPE cell markers, CRALBP was expressed in both the pigmented and the de-pigmented layers. This observation implicates the appearance of Muller glial cells in an early phase of regenerating retina.

Cellular retinol-binding protein-1 expression in endometrial stromal cells: physiopathological and diagnostic implications.

AIMS: Cellular retinol-binding protein-1 (CRBP-1) contributes to the maintenance of the differentiated state of the endometrium through retinol bioavailability regulation. The aim was to analyse CRBP-1 expression in endometrial stromal cells at eutopic and ectopic sites in different physiopathological conditions. METHODS AND RESULTS: Antibodies to CRBP-1, CD10 and alpha-smooth muscle actin were applied to proliferative (n = 10), secretory (n = 9) and atrophic (n = 7) endometrium, decidua (n = 4), adenomyosis (n = 5), endometriosis (n = 10), endometrial polyps (n = 9), simple endometrial hyperplasia (n = 6), well-differentiated endometrioid carcinoma (n = 6) and submucosal leiomyomas (n = 5). In some cases, Western blotting and reverse transcription-polymerase chain reaction were also applied. CRBP-1 was expressed by eutopic and ectopic endometrial stromal cells more markedly during the late secretory phase and in decidua of pregnancy. CRBP-1 expression was low in the stroma of atrophic endometrium and absent in myometrium, leiomyomas and cervical stroma. CD10 immunoreactivity was weak in atrophic endometrium and in decidua. CONCLUSIONS: CRBP-1 expression characterizes endometrial stromal cells at eutopic and ectopic sites and appears to be more specific than CD10. The level of CRBP-1 varies in intensity according to hormonal variations, reaching its maximum in predecidua and decidua. Thus, immunodetection of CRBP-1 may help to elucidate the physiopathological changes which occur in endometrial stroma and can also be applied as an adjuvant stromal marker.

Identification of the fenretinide metabolite 4-oxo-fenretinide present in human plasma and formed in human ovarian carcinoma cells through induction of cytochrome P450 26A1.

PURPOSE: The synthetic retinoid fenretinide (4-HPR) exhibits preventive and therapeutic activity against ovarian tumors. An unidentified polar metabolite was previously found in 4-HPR-treated subjects and in A2780 human ovarian carcinoma cells continuously treated with 4-HPR (A2780/HPR). The metabolite and the enzyme involved in its formation in tumor cells are herein identified. EXPERIMENTAL DESIGN: The metabolite was identified by mass spectrometry in A2780/HPR cell extracts and in plasma from 11 women participating in a phase III trial and treated with 200 mg/d 4-HPR for 5 years. The expression of proteins involved in retinoid metabolism and transport, cytochrome P450 26A1 (CYP26A1), cellular retinol-binding protein I (CRBP-I), and cellular retinoic acid-binding protein I and II (CRABP-I, CRABP-II) were evaluated in tumor cells by reverse transcription-PCR and Western blot analyses. Overexpression of CYP26A1 and retinoic acid receptors (RARs) in A2780 cells were obtained by cDNAs transfection. RESULTS: The polar metabolite was 4-oxo-N-(4-hydroxyphenyl)retinamide (4-oxo-4-HPR) i.e., an oxidized form of 4-HPR with modification in position 4 of the cyclohexene ring. 4-oxo-4-HPR plasma levels were slightly lower (0.52 +/- 0.17 micromol/L) than those of the parent drug (0.84 +/- 0.53 micromol/L) and of the already identified metabolite N-(4-methoxyphenyl)retinamide (1.13 +/- 0.85 micromol/L). In A2780/HPR cells continuously treated with 4-HPR and producing 4-oxo-4-HPR, CYP26A1 and CRBP-I were markedly up-regulated compared with A2780 untreated cells. In A2780 cells, not producing 4-oxo-4-HPR, overexpression of CYP26A1 caused formation of 4-oxo-4-HPR, which was associated with no change in 4-HPR sensitivity. Moreover, the addition of 4-oxo-4-HPR to A2780 cells inhibited cell proliferation. Elevated levels of CYP26A1 protein and metabolism of 4-HPR to 4-oxo-4-HPR were found in A2780 cells transfected with RARbeta and to a lesser extent in those transfected with RARgamma. CONCLUSIONS: A new metabolite of 4-HPR, 4-oxo-4-HPR, present in human plasma and in tumor cells, has been identified. The formation of this biologically active metabolite in tumor cells was due to CYP26A1 induction and was influenced by RAR expression. Moreover evidence was provided that 4-HPR up-modulates the expression of CRBP-I transcript, which is lost during ovarian carcinogenesis.

Complexes between recombinant intracellular carriers of vitamin A and their specific ligands investigated by electrospray-mass spectrometry.

The intracellular carriers of vitamin A, cellular retinol-binding protein type I, cellular retinol-binding protein type II and cellular retinoic acid-binding protein type I are members of the intracellular lipid-binding proteins family, in which the ligand-binding cavity is located in the interior of a barrel-like structure. The dissociation constants of the specific complexes in water solutions around neutrality are very low (in the 0.1 to 10 nM range). Because of their high stability, they represent ideal systems to verify the adequacy of electrospray ionization-mass spectrometry in the analysis of non-covalent protein-ligand complexes. The electrospray interface parameters were varied to detect the presence of species not present in solution but generated as artefacts during transfer of complexes from the condensed state to the gas-phase. The results clearly indicate that mass-spectrometry data reflect the situation present in solution only if the electrospray conditions are carefully selected. In particular, the values of cone voltage and temperature compatible with persistence of the complexes in the gas phase were determined for each vitamin A carrier. Lack of correlation between complex stability in solution and in the gas phase is attributable to the specific and differential effects of the two environments on protein conformation and ligand-protein interactions.