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1.
Protein Expr Purif ; 167: 105542, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31756375

RESUMO

Retinol binding protein IV (RBP) functions as the principal carrier of retinol (Vitamin A) in the blood, where RBP circulates bound to another serum protein, transthyretin. Isolation of pure RBP from the transthyretin complex in human serum can be difficult, but expression of RBP in recombinant systems can circumvent these purification issues. Human recombinant RBP has previously been successfully expressed and purified from E. coli, but recovery of active protein typically requires extensive processing steps, such as denaturing and refolding, and complex purification steps, such as multi-modal chromatography. Furthermore, these methods produce recombinant proteins, often tagged, that display different functional and structural characteristics across systems. In this work, we optimized downstream processing by use of an intein-based expression system in E. coli to produce tag-free, human recombinant RBP (rRBP) with intact native amino termini at yields of up to ~15 mg/L off column. The novel method requires solubilization of inclusion bodies and subsequent oxidative refolding in the presence of retinol, but importantly allows for one-step chromatographic purification that yields high purity rRBP with no N-terminal Met or other tag. Previously reported purification methods typically require two or more chromatographic separation steps to recover tag-free rRBP. Given the interest in mechanistic understanding of RBP transport of retinol in health and disease, we characterized our purified product extensively to confirm rRBP is both structurally and functionally a suitable replacement for serum-derived RBP.


Assuntos
Proteínas Recombinantes/isolamento & purificação , Proteínas de Ligação ao Retinol/biossíntese , Proteínas de Ligação ao Retinol/isolamento & purificação , Escherichia coli/genética , Escherichia coli/metabolismo , Genes Bacterianos , Humanos , Inteínas/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas de Ligação ao Retinol/química , Vitamina A/sangue , Vitamina A/química
2.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 35(7): 653-658, 2019 Jul.
Artigo em Chinês | MEDLINE | ID: mdl-31537250

RESUMO

Objective To produce rabbit polyclonal antibodies against human retinol-binding protein (RBP). Methods RBP cDNA was amplified by reverse transcription polymerase chain reaction (RT-PCR) and then the amplified products were inserted into prokaryotic expression vector pET-28a(+) to construct recombinant plasmid pET-28a(+)-RBP. The established plasmid was then transformed into E. coli. Isopropylthio-ß-D-thiogalactoside (IPTG) was used to induce the expression of recombinant protein His-RBP in E. coli. The expression products were identified by SDS-PAGE from different clones of E. coli to screen positive bacteria, followed by amplifying culture. His-RBP protein was purified from the expression products of positive clones. The purified recombinant His-RBP was used to immunize New Zealand white rabbits. Antisera were acquired after four times of booster immunization. The prepared purified polyclonal antibodies were identified by SDS-PAGE, ELISA and Western blotting. Results We successfully constructed the recombinant plasmid pET-28a(+)-RBP, and acquired recombinant protein His-RBP of high purity. ELISA showed that the antibody titer reached 1:512 000. Conclusion The rabbit polyclonal antibodies against human RBP have been successfully prepared.


Assuntos
Anticorpos/metabolismo , Escherichia coli , Proteínas de Ligação ao Retinol/biossíntese , Animais , Western Blotting , Ensaio de Imunoadsorção Enzimática , Vetores Genéticos , Humanos , Plasmídeos , Coelhos
3.
Cancer Res ; 74(21): 6341-51, 2014 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-25237067

RESUMO

Vitamin A, retinol, circulates in blood bound to retinol-binding protein (RBP). At some tissues, RBP is recognized by STRA6, a plasma membrane protein that serves a dual role: it transports retinol from extracellular RBP into cells and it transduces a signaling cascade mediated by the Janus kinase JAK2 and the transcription factors STAT3 and STAT5. We show here that expression of RBP and STRA6 is markedly upregulated in human breast and colon tumors, that holo-RBP/STRA6 signaling promotes oncogenic properties, and that STRA6 expression is critical for tumor formation by colon carcinoma cells in vivo. The holo-RBP/STRA6 pathway also efficiently induces fibroblasts to undergo oncogenic transformation, rendering them highly tumorigenic. These data establish that holo-RBP and its receptor STRA6 are potent oncogenes and suggest that the pathway is a novel target for therapy of some human cancers.


Assuntos
Transformação Celular Neoplásica/genética , Neoplasias do Colo/genética , Proteínas de Membrana/genética , Vitamina A/metabolismo , Neoplasias do Colo/patologia , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Células Hep G2 , Humanos , Janus Quinase 2/genética , Proteínas de Membrana/metabolismo , Proteínas de Ligação ao Retinol/biossíntese , Proteínas de Ligação ao Retinol/genética , Transdução de Sinais/genética
4.
Pediatr Surg Int ; 30(10): 1017-22, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24972641

RESUMO

BACKGROUND: It has been shown that pulmonary retinol level is decreased during lung morphogenesis in the nitrofen-induced PH in congenital diaphragmatic hernia (CDH). Placenta has a major role in the retinol homeostasis in fetal life. Since there is no fetal retinol synthesis, maternal retinol has to cross the placenta. Placenta is the main fetal retinol store where retinol is stored in retinyl-ester formation. Trophoblasts have to produce its own retinol-binding protein (RBP) for retinol transport from placenta to fetus. Recently, we demonstrated that trophoblastic RBP expression is decreased in the nitrofen model of CDH. The aim of this study was to investigate the retinol transfer from mother to the placenta in nitrofen model of CDH. METHODS: Pregnant rats were exposed to either olive oil or nitrofen on day 9 of gestation (D9). Fetal placenta harvested on D21 and divided into two groups: control (n = 11) and nitrofen with CDH (n = 11). Retinoid levels in placenta were measured using HPLC. Immunohistochemistry was performed to evaluate trophoblastic expression of main RSP genes. RESULTS: Total retinol levels in the placenta were significantly increased in CDH placenta compared to control placenta. The retinyl-ester levels were significantly increased in CDH placenta compared to control placenta. Markedly, decreased immunoreactivity of retinoid signaling pathway was observed in trophoblast cells in CDH compared to control placenta. CONCLUSIONS: Increased placental retinol levels show that retinol is transferred from mother to placenta and stored in the placenta in nitrofen model of CDH during lung morphogenesis. Nitrofen may disturb the mobilization of retinol from placenta to fetal circulation causing PH in CDH.


Assuntos
Hérnias Diafragmáticas Congênitas/metabolismo , Pulmão/embriologia , Morfogênese , Éteres Fenílicos/farmacologia , Placenta/metabolismo , Vitamina A/metabolismo , Animais , Cromatografia Líquida de Alta Pressão/métodos , Modelos Animais de Doenças , Feminino , Azeite de Oliva , Óleos de Plantas/administração & dosagem , Gravidez , Ratos , Ratos Sprague-Dawley , Proteínas de Ligação ao Retinol/biossíntese
5.
J Dairy Sci ; 95(12): 7097-104, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23040032

RESUMO

Retinol-binding protein (RBP) is the main transport system for retinol in circulation, is a relatively small protein with one binding site for retinol in the all-trans form, and is bound to transthyretin. The objectives of this study were to characterize the temporal pattern of bovine hepatic mRNA expression of RBP during the periparturient period and to determine if a relationship exists between the expression of RBP and that of tumor necrosis factor (TNF)-α in dairy cows. In experiment 1, we assessed hepatic mRNA expression of RBP during the periparturient period. Liver tissues were sampled from periparturient dairy cows (n=9) at -21, -4, +1, +7, and +21 relative to parturition and frozen in liquid N(2). Total RNA was extracted from each tissue sample and cDNA was generated. Gene expressions of RBP and ß-actin (as a housekeeping gene) were measured as relative quantity using reverse transcription-PCR. Data were analyzed using cycle threshold values, adjusted to ß-actin, and significance was determined at P<0.05. Serum samples (-21, -4, +1, +7, and +21 relative to parturition) were analyzed for retinol concentration using a standard HPLC-based method. Cows had variable expression of hepatic RBP and serum retinol over the transition period, with a decline near parturition and a rebound toward prepartum levels later in lactation. In experiment 2, liver and visceral (intestinal) adipose tissues were sampled from dairy cows (n=28) at slaughter. Expression of RBP and TNF-α was detected in all samples and variations among cows were highly significant for both genes. Across tissues, expression of RBP was positively correlated with that of TNF-α (r=0.60). Within adipose tissue, expression of RBP and TNF-α was weakly correlated (r=0.23), whereas in hepatic tissue, expression was strongly correlated (r=0.62). In experiment 3, late-lactation dairy Holstein cows were blocked by parity and feed intake, and randomly assigned to control, recombinant bovine (rb)TNF challenge, or pair-fed control treatment (n=5/treatment). Cows were injected with either rbTNF (subcutaneous injection of 2 µg/kg of body weight in saline) or sterile saline (control and pair-fed control animals) once daily for 7d. Liver biopsy was performed on d 7 and samples were processed for expression of RBP and TNF-α. Although TNF challenge caused an upregulation of hepatic TNF-α expression, as expected, it did not alter hepatic RBP expression. Overall, the temporal pattern of hepatic RBP gene expression during the periparturient period followed, to a great extent, that of plasma retinol. Although a strong positive correlation was previously detected between bovine hepatic RBP and TNF-α transcripts, rbTNF challenge did not cause alter RBP expression. These observations collectively imply that regulation of RBP at the transcription level is influenced by physiological state but may be independent from that of transthyretin, which is altered by proinflammatory stimuli (such as TNF-α) via induction of transcription factor nuclear factor-interleukin 6.


Assuntos
Tecido Adiposo/metabolismo , Fígado/metabolismo , Proteínas de Ligação ao Retinol/biossíntese , Fator de Necrose Tumoral alfa/biossíntese , Actinas/biossíntese , Animais , Bovinos , Feminino , Expressão Gênica/fisiologia , Período Periparto/metabolismo , Período Periparto/fisiologia , Gravidez , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Vitamina A/sangue
6.
Pediatr Surg Int ; 28(2): 143-8, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22015466

RESUMO

BACKGROUND: Retinoids play a key role in lung development. Retinoid signaling pathway has been shown to be disrupted in the nitrofen model of congenital diaphragmatic hernia (CDH) but the exact mechanism is not clearly understood. Retinol-binding protein (RBP) and transthyretin (TTR) are transport proteins for delivery of retinol to the tissues via circulation. Previous studies have shown that pulmonary retinol levels are decreased during lung morphogenesis in the nitrofen CDH model. In human newborns with CDH, both retinol and RBP levels are decreased. It has been reported that maternal RBP does not cross the placenta and the fetus produces its own RBP by trophoblast. RBP and TTR synthesized in the fetus are essential for retinol transport to the developing organs including lung morphogenesis. We hypothesized that nitrofen interferes with the trophoblastic expression of RBP and TTR during lung morphogenesis and designed this study to examine the trophoblastic expression of RBP and TTR, and the total level of RBP and TTR in the lung in the nitrofen model of CDH. METHODS: Pregnant rats were exposed to either olive oil or nitrofen on day 9 of gestation (D9). Fetal lungs and placenta harvested on D21 and divided into two groups: control (n = 8) and nitrofen with CDH (n = 8). Total lung RBP and TTR levels using protein extraction were compared with enzyme linked immunoassay (ELISA). Immunohistochemistry was performed to evaluate trophoblastic RBP and TTR expression. RESULTS: Total protein levels of lung RBP and TTR were significantly lower in CDH (0.26 ± 0.003 and 6.4 ± 0.5 µg/mL) compared with controls (0.4 ± 0.001 and 9.9 ± 1.6 µg/mL, p < 0.05). In the control group, immunohistochemical staining showed strong immunoreactivity of RBP and TTR in the trophoblast compared to CDH group. CONCLUSIONS: Decreased trophoblast expression of retinol transport proteins suggest that nitrofen may interfere with the fetal retinol transport resulting in reduced pulmonary RBP and TTR levels and causing pulmonary hypoplasia in CDH.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Pulmão/embriologia , Pré-Albumina/biossíntese , Prenhez , RNA/genética , Proteínas de Ligação ao Retinol/biossíntese , Trofoblastos/metabolismo , Animais , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Feminino , Hérnia Diafragmática/induzido quimicamente , Hérnia Diafragmática/embriologia , Hérnia Diafragmática/genética , Hérnias Diafragmáticas Congênitas , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Morfogênese/efeitos dos fármacos , Morfogênese/genética , Éteres Fenílicos/toxicidade , Gravidez , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real , Trofoblastos/efeitos dos fármacos
7.
J Neurosci ; 31(21): 7900-9, 2011 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-21613504

RESUMO

Rapid regeneration of the visual pigment following its photoactivation is critical for the function of cone photoreceptors throughout the day. Though the reactions of the visual cycle in the retinal pigment epithelium (RPE) that recycle chromophore for rod pigment regeneration are well characterized, the corresponding mechanisms that enable rapid regeneration of cone pigment are poorly understood. A key remaining question is the relative contribution of the recently discovered cone-specific retina visual cycle and the classic RPE-dependent visual cycle to mammalian cone pigment regeneration. In addition, it is not clear what role, if any, the abundant interphotoreceptor retinoid-binding protein (IRBP) presumed to facilitate the traffic of chromophore, plays in accelerating mammalian cone pigment regeneration. To address these issues, we used transretinal recordings to evaluate M/L-cone pigment regeneration in isolated retinas and eyecups from control and IRBP-deficient mice. Remarkably, the mouse retina promoted M/L-cone dark adaptation eightfold faster than the RPE. However, complete cone recovery required both visual cycles. We conclude that the retina visual cycle is critical for the initial rapid regeneration of mouse M/L-cone pigment during dark adaptation, whereas the slower RPE visual cycle is required to complete the process. While the deletion of IRBP reduced the amplitude and slowed the kinetics of mouse M/L-cone photoresponses, cone adaptation in bright, steady light and the kinetics of cone dark adaptation were not affected in isolated retina or in intact eyecup. Thus, IRBP does not accelerate cone pigment regeneration and is not critical for the function of mouse M/L-cones in bright light.


Assuntos
Opsinas dos Cones/biossíntese , Proteínas do Olho/biossíntese , Estimulação Luminosa/métodos , Células Fotorreceptoras Retinianas Cones/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Pigmentos da Retina/biossíntese , Proteínas de Ligação ao Retinol/biossíntese , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Retina/metabolismo , Fatores de Tempo , Percepção Visual/fisiologia
8.
Methods Mol Biol ; 652: 55-73, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20552421

RESUMO

The retinal pigment epithelium (RPE) occupies a strategic position within the eye, given its location between the neurosensory retina and the vascular bed (choroid) that nourishes the photoreceptor cells (rods and cones). Among the many attributes of this versatile monolayer of cells is its unique ability to convert vitamin A (retinol) into the prosthetic group (11-cis-retinal) for the rod and cone opsins, the photopigments essential for vision. It does so by absorbing retinol via a receptor-mediated process that involves the interaction of a carrier protein secreted by the liver, retinol-binding protein (RBP), and a receptor/channel that is the gene product of STRA6 (stimulated by retinoic acid 6). Following its uptake through the basolateral plasma membrane of the RPE, retinol encounters a brigade of binding proteins, membrane-bound receptors, and enzymes that mediate its multi-step conversion to 11-cis-retinal and the transport of this visual chromophore to the light-sensitive photoreceptor cell outer segment, the portion of the cell that houses the phototransduction cascade. This process is iterative, repeating itself via the retinoid visual cycle. Most of the human genes that code for this cohort of proteins carry disease-causing mutations in humans. The consequences of these mutations range in severity from relatively mild dysfunction such as congenital stationary night blindness to total blindness. The RPE, although post-mitotic in situ, is capable of proliferation when removed from its native milieu. This offers one the opportunity to study the retinoid visual cycle in modular form, providing insights into this intriguing process in health and disease. This chapter describes a cell culture method whereby the entire visual cycle can be created in vitro.


Assuntos
Diferenciação Celular , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/metabolismo , Retinoides/metabolismo , Técnicas de Cultura de Tecidos/métodos , Animais , Transporte Biológico , Bovinos , Meios de Cultura , Escherichia coli/genética , Feminino , Humanos , Pré-Albumina/metabolismo , Gravidez , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Retinoides/isolamento & purificação , Proteínas de Ligação ao Retinol/biossíntese , Proteínas de Ligação ao Retinol/isolamento & purificação , Proteínas de Ligação ao Retinol/metabolismo
9.
Methods Mol Biol ; 652: 341-61, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20552439

RESUMO

STRA6 is a multitransmembrane domain protein that was recently identified as the cell-surface receptor for plasma retinol-binding protein (RBP), the vitamin A carrier protein in the blood. STRA6 binds to RBP with high affinity and mediates cellular uptake of vitamin A from RBP. It is not homologous to any known receptors, transporters, and channels, and it represents a new class of membrane transport protein. Consistent with the diverse physiological functions of vitamin A, STRA6 is widely expressed in diverse adult organs and throughout embryonic development. Mutations in human STRA6 that abolish its vitamin A uptake activity cause severe pathological phenotypes in many human organs including the eye, brain, lung, and heart. This chapter describes functional assays for STRA6 in live cells and on cellular membranes. These assays can be employed to study the mechanism of this new membrane transport mechanism and its roles in the physiology and pathology of many organs.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Proteínas de Membrana/metabolismo , Receptores de Superfície Celular/metabolismo , Vitamina A/metabolismo , Animais , Apoproteínas/biossíntese , Apoproteínas/química , Apoproteínas/isolamento & purificação , Apoproteínas/metabolismo , Transporte Biológico , Células COS , Membrana Celular/metabolismo , Sobrevivência Celular , Sistema Livre de Células/metabolismo , Chlorocebus aethiops , Ésteres , Humanos , Proteínas de Membrana/química , Imagem Molecular , Renaturação Proteica , Receptores de Superfície Celular/química , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Ligação ao Retinol/biossíntese , Proteínas de Ligação ao Retinol/química , Proteínas de Ligação ao Retinol/isolamento & purificação , Proteínas de Ligação ao Retinol/metabolismo , Solubilidade , Trítio/química
10.
J Neurochem ; 110(2): 486-95, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19457110

RESUMO

Neuroimage analysis in alcoholic corpus callosum (CC) suggests that microstructural abnormalities are higher in the genu followed by the body and the splenium. Molecular mechanisms underlying these dysmorphologys are still unclear. Protein expression was performed using the CC body samples [(nine controls, seven uncomplicated, and six complicated (with liver cirrhosis) alcoholics] through proteomics approach. Thirty-nine protein spots in uncomplicated and 60 in complicated alcoholics were differentially altered compared with the control (p < 0.05). Comparison between alcoholic groups revealed that 40% more protein showed altered expression in complicated compared with uncomplicated. This result suggests that alcohol-related liver dysfunction has synergetic effects on brain protein expression. Subregional expression profiles indicate that the highest numbers of region-specific proteins were in the genus followed by the CC body and the splenium. Interestingly, abnormal thiamine cascade was strongly suggested in the genu, and to a lesser extent in the CC body, but no such cascade was observed in the splenium. Therefore, alcohol-induced microstructural damage detected by image analysis in the CC, possibly involves multiple biochemical mechanisms.


Assuntos
Alcoolismo/metabolismo , Corpo Caloso/fisiologia , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica/fisiologia , Biossíntese de Proteínas , Alcoolismo/genética , Alcoolismo/patologia , Corpo Caloso/patologia , Proteína Glial Fibrilar Ácida/biossíntese , Proteína Glial Fibrilar Ácida/genética , Humanos , Estresse Oxidativo/fisiologia , Biossíntese de Proteínas/genética , Proteômica/métodos , Proteínas de Ligação ao Retinol/biossíntese , Proteínas de Ligação ao Retinol/genética , Transdução de Sinais/fisiologia
11.
BMC Dev Biol ; 7: 138, 2007 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-18093305

RESUMO

BACKGROUND: Human retinoic acid teratogenesis results in malformations of dorsally derived hindbrain structures such as the cerebellum, noradrenergic hindbrain neurons and the precerebellar system. These structures originate from the rhombic lip and adjacent dorsal precursor pools that border the fourth ventricle roofplate. While retinoic acid synthesis is known to occur in the meninges that blanket the hindbrain, the particular sensitivity of only dorsal structures to disruptions in retinoid signalling is puzzling. We therefore looked for evidence within the neural tube for more spatiotemporally specific signalling pathways using an in situ hybridisation screen of known retinoic acid pathway transcripts. RESULTS: We find that there are highly restricted domains of retinoic acid synthesis and breakdown within specific hindbrain nuclei as well as the ventricular layer and roofplate. Intriguingly, transcripts of cellular retinoic acid binding protein 1 are always found at the interface between dividing and post-mitotic cells. By contrast to earlier stages of development, domains of synthesis and breakdown in post-mitotic neurons are co-localised. At the rhombic lip, expression of the mRNA for retinoic acid synthesising and catabolising enzymes is spatially highly organised with respect to the Cath1-positive precursors of migratory precerebellar neurons. CONCLUSION: The late developing hindbrain shows patterns of retinoic acid synthesis and use that are distinct from the well characterised phase of rostrocaudal patterning. Selected post-mitotic populations, such as the locus coeruleus, appear to both make and break down retinoic acid suggesting that a requirement for an autocrine, or at least a highly localised paracrine signalling network, might explain its acute sensitivity to retinoic acid disruption. At the rhombic lip, retinoic acid is likely to act as a dorsalising factor in parallel with other roofplate signalling pathways. While its precise role is unclear, retinoic acid is potentially well placed to regulate temporally determined cell fate decisions within the rhombic lip precursor pool.


Assuntos
Padronização Corporal , Rombencéfalo/embriologia , Transdução de Sinais , Tretinoína/metabolismo , Animais , Hidrocarboneto de Aril Hidroxilases/biossíntese , Embrião de Galinha , Citocromo P-450 CYP1B1 , Regulação da Expressão Gênica no Desenvolvimento , Imuno-Histoquímica , Hibridização In Situ , Retinal Desidrogenase/biossíntese , Proteínas de Ligação ao Retinol/biossíntese , Rombencéfalo/metabolismo
12.
Mol Immunol ; 44(13): 3291-6, 2007 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-17467057

RESUMO

Equine recurrent uveitis (ERU) is a valuable model for autoimmune diseases, since it develops frequently and occurs spontaneously. We investigated the overall expression level of three major retinal autoantigens in normal retinas and various ERU stages. Analysis of retinal proteomes of both, healthy and diseased retinas revealed an almost unaffected expression of IRBP, S-antigen and cRALBP in ERU cases. Validation of these findings with western blots and immunohistochemistry confirmed constant to increased expression of these autoantigens, although loss of their physiological expression sites within retina is evident. In contrast to stable expression of autoantigens, rhodopsin, the major component of phototransduction in photoreceptors, disappeared from destructed retinas. These results explain persistent uveitic attacks even in severely damaged eyes and draw the attention to further investigations of biological pathways and regulations in autoimmune target tissues.


Assuntos
Autoantígenos/biossíntese , Autoantígenos/genética , Retina/imunologia , Uveíte/imunologia , Uveíte/metabolismo , Animais , Arrestina/biossíntese , Arrestina/genética , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Proteínas do Olho/biossíntese , Proteínas do Olho/genética , Regulação da Expressão Gênica/imunologia , Cavalos , Recidiva , Retina/metabolismo , Retina/patologia , Proteínas de Ligação ao Retinol/biossíntese , Proteínas de Ligação ao Retinol/genética , Rodopsina/biossíntese , Rodopsina/genética , Uveíte/patologia
13.
Diabetologia ; 50(4): 814-23, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17294166

RESUMO

AIMS/HYPOTHESIS: Levels of retinol binding protein (RBP4) are increased in the serum of insulin-resistant human subjects even before overt diabetes develops. RBP4 levels correlate with insulin resistance, BMI, WHR, dyslipidaemia and hypertension. Improvement of insulin sensitivity with exercise training is associated with reduction in serum RBP4 levels. Therefore serum RBP4 may be useful for early diagnosis of insulin resistance and for monitoring improvements in insulin sensitivity. We sought to determine the performance of assays for this application. SUBJECTS AND METHODS: We compared quantitative western blotting and three commercially available multiwell immunoassays in parallel measurements of RBP4 concentrations in serum from insulin-sensitive subjects and from insulin-resistant subjects with impaired glucose tolerance or type 2 diabetes. RESULTS: The assays yielded different absolute values and magnitudes of elevation of serum RBP4. Western blotting and a sandwich ELISA reported RBP4 concentrations that highly inversely correlated with insulin sensitivity measured by euglycaemic-hyperinsulinaemic clamp. However, western blotting yielded concentrations with a greater dynamic range and less overlap between control and insulin-resistant subjects. Two competitive enzyme-linked immunoassays undervalued serum RBP4 concentrations in insulin-resistant subjects, possibly due to assay saturation. Poor linearity of dilution also limited assay utility. All assays tested exhibited greater immunoreactivity with urinary (C-terminal proteolysed) RBP4 than with full-length RBP4, the predominant form in serum. CONCLUSIONS/INTERPRETATIONS: These findings support the use of quantitative western blotting standardised to full-length RBP4 protein as a 'gold standard' method for measuring serum RBP4 in insulin-resistant states. Other assays should use full-length RBP4 and be extensively cross-validated using other methods.


Assuntos
Imunoensaio/instrumentação , Resistência à Insulina , Insulina/metabolismo , Proteínas de Ligação ao Retinol/análise , Proteínas de Ligação ao Retinol/biossíntese , Glicemia/análise , Western Blotting , Diabetes Mellitus Tipo 2/sangue , Ensaio de Imunoadsorção Enzimática/métodos , Teste de Tolerância a Glucose , Humanos , Imunoensaio/métodos , Insulina/sangue , Síndrome Metabólica/metabolismo , Obesidade/sangue , Obesidade/metabolismo , Reprodutibilidade dos Testes , Proteínas Plasmáticas de Ligação ao Retinol , Vitamina A/metabolismo
14.
J Exp Med ; 203(12): 2727-35, 2006 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-17116738

RESUMO

The expression of self-antigen in the thymus is believed to be responsible for the deletion of autoreactive T lymphocytes, a critical process in the maintenance of unresponsiveness to self. The Autoimmune regulator (Aire) gene, which is defective in the disorder autoimmune polyglandular syndrome type 1, has been shown to promote the thymic expression of self-antigens. A clear link, however, between specific thymic self-antigens and a single autoimmune phenotype in this model has been lacking. We show that autoimmune eye disease in aire-deficient mice develops as a result of loss of thymic expression of a single eye antigen, interphotoreceptor retinoid-binding protein (IRBP). In addition, lack of IRBP expression solely in the thymus, even in the presence of aire expression, is sufficient to trigger spontaneous eye-specific autoimmunity. These results suggest that failure of thymic expression of selective single self-antigens can be sufficient to cause organ-specific autoimmune disease, even in otherwise self-tolerant individuals.


Assuntos
Autoantígenos/biossíntese , Doenças Autoimunes/imunologia , Doenças Autoimunes/prevenção & controle , Proteínas do Olho/biossíntese , Regulação da Expressão Gênica/imunologia , Proteínas de Ligação ao Retinol/biossíntese , Timo/imunologia , Timo/metabolismo , Fatores de Transcrição/biossíntese , Animais , Autoantígenos/genética , Autoantígenos/fisiologia , Proteínas do Olho/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Nus , Proteínas de Ligação ao Retinol/genética , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Uveíte/genética , Uveíte/imunologia , Uveíte/metabolismo , Proteína AIRE
15.
J Atheroscler Thromb ; 13(4): 209-15, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16908954

RESUMO

OBJECTIVE: We examined various factors possibly related to metabolic syndrome, particularly focusing on nutritional assessment proteins such as retinol binding protein (RBP) and transthyretin (TTR), and remnant lipoproteins. MATERIALS AND METHODS: Fasting serum lipid was analyzed in 58 Japanese adult volunteers (33 men and 25 women, 42.5 +/- 10.1 years old). RESULTS: The lipid profiles of the subjects were classified by lipoprotein polyacrylamide gel electrophoretic patterns into Types S (n = 10), A (n = 37), and N (n = 11), according to the method described in Internal Medicine 42: 244, 2003. RBP and TTR were significantly higher in Type N than in Types S and A. In multivariate analysis, RBP was accounted for by remnant-like particle-triglyceride (RLP-TG), interleukin 6, body mass index and low-density lipoprotein (LDL)-cholesterol (adjusted R2 = 0.621). TTR was accounted for by lipoprotein(a), adiponectin and RLP-TG (adjusted R2 = 0.415). Malondialdehyde-LDL was significantly accounted for by LDL-cholesterol and RLP-cholesterol (adjusted R2 = 0.601). Lipoprotein(a) and LDL-cholesterol were independent variables for oxidized LDL antigen (adjusted R2 = 0.620). High-sensitivity C-reactive protein was accounted for by interleukin 6, immunoreactive insulin and oxidized LDL antigen (adjusted R2 = 0.361). Uric acid and body mass index were independent variables for adiponectin (adjusted R2 = 0.429). CONCLUSION: RBP and TTR may be useful as convenient and simple clinical markers of overnutrition and possibly of metabolic syndrome.


Assuntos
Pré-Albumina/biossíntese , Proteínas de Ligação ao Retinol/biossíntese , Adulto , Proteína C-Reativa/biossíntese , Eletroforese em Gel de Poliacrilamida , Feminino , Humanos , Japão , Lipoproteínas/sangue , Lipoproteínas/química , Lipoproteínas LDL/química , Masculino , Malondialdeído/química , Síndrome Metabólica/sangue , Pessoa de Meia-Idade , Análise Multivariada
17.
Protein Eng Des Sel ; 19(8): 385-90, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16720693

RESUMO

Although Escherichia coli is in wide use for preparative protein expression, problems with the folding of the recombinant gene product and protein aggregation are frequently encountered. Apart from cytoplasmic expression, this is also true for secretion into the bacterial periplasm, the method of choice for the production of proteins that carry structural disulfide bonds. Here we report the construction of the helper plasmid pTUM4, which effects overexpression of four established periplasmic chaperones and folding catalysts: the thiol-disulfide oxidoreductases DsbA and DsbC that catalyze the formation and isomerization of disulfide bridges and the peptidyl-prolyl cis/trans-isomerases with chaperone activity, FkpA and SurA. pTUM4 carries a p15a origin of replication and a chloramphenicol resistance gene and, thus, it is compatible with many conventional expression vectors that use the ColEI origin and an ampicillin resistance. Its positive effects on the yield of soluble recombinant protein and the homogeneity of disulfide pattern are illustrated here using the human plasma retinol-binding protein as well as the extracellular carbohydrate recognition domain of the dendritic cell membrane receptor DC-SIGN. Hence, pTUM4 represents a novel helper vector which complements existing cytosolic chaperone coexpression plasmids and should be useful for the functional secretion of various recombinant proteins with hampered folding efficiency.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Periplasma/metabolismo , Dobramento de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Moléculas de Adesão Celular/biossíntese , Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/isolamento & purificação , Escherichia coli/enzimologia , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Vetores Genéticos , Humanos , Imunofilinas/genética , Imunofilinas/metabolismo , Lectinas Tipo C/biossíntese , Lectinas Tipo C/química , Lectinas Tipo C/isolamento & purificação , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Peptidilprolil Isomerase/genética , Peptidilprolil Isomerase/metabolismo , Plasmídeos/genética , Isomerases de Dissulfetos de Proteínas/genética , Isomerases de Dissulfetos de Proteínas/metabolismo , Receptores de Superfície Celular/biossíntese , Receptores de Superfície Celular/química , Receptores de Superfície Celular/isolamento & purificação , Proteínas Recombinantes/isolamento & purificação , Proteínas de Ligação ao Retinol/biossíntese , Proteínas de Ligação ao Retinol/química , Proteínas de Ligação ao Retinol/isolamento & purificação , Proteínas Plasmáticas de Ligação ao Retinol
18.
Clin Cancer Res ; 10(18 Pt 1): 6265-75, 2004 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-15448016

RESUMO

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.


Assuntos
Anticarcinógenos/sangue , Sistema Enzimático do Citocromo P-450/sangue , Fenretinida/análogos & derivados , Fenretinida/sangue , Fenretinida/farmacocinética , Neoplasias Ovarianas/sangue , Proteínas de Ligação ao Retinol/biossíntese , Western Blotting , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Sistema Enzimático do Citocromo P-450/metabolismo , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Feminino , Fenretinida/metabolismo , Vetores Genéticos , Humanos , Immunoblotting , Espectrometria de Massas , Neoplasias Ovarianas/metabolismo , Oxigênio/química , RNA Mensageiro/metabolismo , Ácido Retinoico 4 Hidroxilase , Proteínas Celulares de Ligação ao Retinol , Proteínas Plasmáticas de Ligação ao Retinol , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e Especificidade , Fatores de Tempo , Transfecção , Tretinoína/farmacologia , Regulação para Cima
19.
Cell Tissue Res ; 316(1): 131-9, 2004 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-14963765

RESUMO

Exposure to estrogenic substances during a time window, the so-called "critical period," in perinatal life causes an irregular development of the genital tract that leads to ovary-independent proliferation and cornification in the vaginal epithelium in mice. We have previously demonstrated that retinol inhibits the irreversible effects of estrogen on the vagina. Here, mice kept in a vitamin-A-deficient condition during perinatal life were shown to be more sensitive to the harmful effects of estrogen. In addition, expression of mRNA for retinol binding protein type 2 (CRBP2), a "small intestine-specific" cytosolic protein that captures intracellular retinal and retinol, was detected in the vaginal epithelium. Induction of increased expression of CRBP2 mRNA by estrogen was also evident in the uterus and epididymis. Both estradiol-17beta and diethylstilbestrol markedly increased the tissue content of CRBP2 mRNA in the vagina and uterus during the neonatal "critical period" but not after 15 days of age. These results taken together imply that estrogen disrupts the local vitamin A balance by an induction of CRBP2 gene expression in the epithelium in the developing mouse genital tract, and that retinoid imbalance may contribute to the genesis of irreversible effects of estrogen on the vagina.


Assuntos
Estradiol/administração & dosagem , Proteínas de Ligação ao Retinol/biossíntese , Útero/metabolismo , Vagina/metabolismo , Vitamina A/administração & dosagem , Animais , Animais Recém-Nascidos , Dietilestilbestrol/administração & dosagem , Epitélio/metabolismo , Estrogênios não Esteroides/administração & dosagem , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Hibridização In Situ , Camundongos , RNA Mensageiro , Proteínas Celulares de Ligação ao Retinol , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Útero/crescimento & desenvolvimento , Vagina/crescimento & desenvolvimento , Deficiência de Vitamina A/metabolismo
20.
Yi Chuan ; 26(2): 257-62, 2004 Mar.
Artigo em Chinês | MEDLINE | ID: mdl-15639998

RESUMO

Retinol-binding proteins (RBPs) are a kind of circulating carrier proteins for serum and cellular retinol and retinol acid, which are lipid-soluble vitamins, and are members of hydrophobic binding protein family. Serum RBPs were synthesized primarily in liver, then was released into blood streams, and then to various tissues. Under the interaction with substances such as retinol, pre-albumin and the receptors of cellular surface, they play important roles in storage, metabolism of VitA and transport of VitA to the target cells. Cellular RBPs play the similar function as serum RBPs in intracell. This review introduces action mechanism, tissue localization and developmental expression of retinol-binding proteins. This review also introduces the structure, chromosome mapping and their relationships with reproductive performance of retinol-binding protein genes.


Assuntos
Cruzamento , Fígado/metabolismo , Proteínas de Ligação ao Retinol/genética , Vitamina A/metabolismo , Animais , Mapeamento Cromossômico , Feminino , Regulação da Expressão Gênica , Humanos , Rim/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Proteínas de Ligação ao Retinol/biossíntese , Proteínas de Ligação ao Retinol/química , Proteínas de Ligação ao Retinol/fisiologia , Proteínas Celulares de Ligação ao Retinol , Útero/metabolismo
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