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1.
Osteoarthritis Cartilage ; 24(7): 1223-34, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-26851450

RESUMO

OBJECTIVE: Semicarbazide-sensitive amine oxidase (SSAO) catalyzes the oxidation of primary amines into ammonia and reactive species (hydrogen peroxide, aldehydes). It is highly expressed in mammalian tissues, especially in vascular smooth muscle cells and adipocytes, where it plays a role in cell differentiation and glucose transport. The study aims at characterizing the expression and the activity of SSAO in rat and human articular cartilage of the knee, and to investigate its potential role in chondrocyte terminal differentiation. DESIGN: SSAO expression was examined by immunohistochemistry and western blot. Enzyme activity was measured using radiolabeled benzylamine as a substrate. Primary cell cultures of rat chondrocytes were treated for 21 days by a specific SSAO inhibitor, LJP 1586. Terminal chondrocyte differentiation markers were quantified by RT-qPCR. The basal and IL1ß-stimulated glucose transport was monitored by the entrance of (3)[H]2-deoxyglucose in chondrocytes. RESULTS: SSAO was expressed in chondrocytes of rat and human articular cartilage. SSAO expression was significantly enhanced during the hypertrophic differentiation of chondrocytes characterized by an increase in MMP13 and in alkaline phosphatase (ALP) expressions. SSAO inhibition delayed the late stage of chondrocyte differentiation without cell survival alteration and diminished the basal and IL1ß-stimulated glucose transport. Interestingly, SSAO activity was strongly increased in human osteoarthritic cartilage. CONCLUSIONS: SSAO was expressed as an active form in rat and human cartilage. The results suggest the involvement of SSAO in rat chondrocyte terminal differentiation via a modulation of the glucose transport. In man, the increased SSAO activity detected in osteoarthritic patients may trigger hypertrophy and cartilage degeneration.


Assuntos
Cartilagem Articular , Adipócitos , Amina Oxidase (contendo Cobre) , Animais , Diferenciação Celular , Condrócitos , Humanos , Ratos
2.
Toxicol Appl Pharmacol ; 264(3): 395-403, 2012 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-22959462

RESUMO

Caffeine is a definite factor of intrauterine growth retardation (IUGR). Previously, we have confirmed that prenatal caffeine ingestion inhibits the development of hypothalamic-pituitary-adrenal (HPA) axis, and alters the glucose and lipid metabolism in IUGR fetal rats. In this study, we aimed to verify a programmed alteration of neuroendocrine metabolism in prenatal caffeine ingested-offspring rats. The results showed that prenatal caffeine (120 mg/kg.day) ingestion caused low body weight and high IUGR rate of pups; the concentrations of blood adrenocorticotropic hormone (ACTH) and corticosterone in caffeine group were significantly increased in the early postnatal period followed by falling in late stage; the level of blood glucose was unchanged, while blood total cholesterol (TCH) and triglyceride (TG) were markedly enhanced in adult. After chronic stress, the concentrations and the gain rates of blood ACTH and corticosterone were obviously increased, meanwhile, the blood glucose increased while the TCH and TG decreased in caffeine group. Further, the hippocampal mineralocorticoid receptor (MR) expression in caffeine group was initially decreased and subsequently increased after birth. After chronic stress, the 11ß-hydroxysteroid dehydrogenase-1, glucocorticoid receptor (GR), MR as well as the MR/GR ratio were all significantly decreased. These results suggested that prenatal caffeine ingestion induced the dysfunction of HPA axis and associated neuroendocrine metabolic programmed alteration in IUGR offspring rats, which might be related with the functional injury of hippocampus. These observations provide a valuable experimental basis for explaining the susceptibility of IUGR offspring to metabolic syndrome and associated diseases.


Assuntos
Cafeína/efeitos adversos , Retardo do Crescimento Fetal/induzido quimicamente , Sistema Hipotálamo-Hipofisário/efeitos dos fármacos , Sistema Hipófise-Suprarrenal/efeitos dos fármacos , Animais , Peso ao Nascer , Glicemia , Feminino , Hipocampo/efeitos dos fármacos , Hipocampo/patologia , Metabolismo dos Lipídeos , Masculino , Gravidez , Efeitos Tardios da Exposição Pré-Natal , Ratos , Estresse Fisiológico , Fatores de Tempo , Aumento de Peso
4.
FEBS Lett ; 580(8): 1953-8, 2006 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-16529747

RESUMO

We investigated the role of the stop transfer sequence of human UGT1A6 in ER assembly and enzyme activity. We found that this sequence was able to address and translocate the upstream lumenal domain into microsomal membranes in vitro co- and posttranslationally. The signal activity of this sequence was further demonstrated in HeLa cells by its ability to target and maintain the CD4 protein deleted from both the N-terminal signal peptide and C-terminal transmembrane domain into the ER. We showed that total or partial deletion of the stop transfer sequence of UGT1A6 severely impaired enzyme activity highlighting its importance in both membrane assembly and function.


Assuntos
Retículo Endoplasmático/metabolismo , Glucuronosiltransferase/química , Glucuronosiltransferase/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Processamento de Proteína Pós-Traducional , Sinais Direcionadores de Proteínas/fisiologia , Região 5'-Flanqueadora/genética , Sequência de Aminoácidos , Retículo Endoplasmático/enzimologia , Células HeLa , Humanos , Dados de Sequência Molecular , Pichia/metabolismo , Estrutura Terciária de Proteína , Transporte Proteico , Deleção de Sequência/genética
5.
Clin Hemorheol Microcirc ; 64(4): 613-633, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27791997

RESUMO

Most human tissues do not regenerate spontaneously, which is why "cell therapy" are promising alternative treatments. The Principe is simple: patients' or donors' cells are collected and introduced into the injured tissues or organs directly or in a porous 3D material, with or without modification of their properties. This concept of regenerative medicine is an emerging field which can be defined as "the way to improve health and quality of life by restoring, maintaining, or enhancing tissue and organ functions".There is an extraordinarily wide range of opportunities for clinical applications: artheropathies, diabetes, cartilage defects, bone repair, burns, livers or bladder regeneration, organs reconstruction (lung, heart, liver ...) neurodegenerative disorders, sepsis ...  Different stem cells (SC) with different potential can be used and characterised (totipotent, mesenchymal of different origins, especially those present in tissues...). Today it is undeniable that cells like bone marrow, adipose tissue or Wharton Jelly stem cells, are of potential interest for clinical applications because they are easily separated and prepared and no ethical problems are involved in their use.In this paper some potential clinical applications in the vascular field are considered: peripheral arteriopathy in diabetic patients, cardiac insufficiency, traitment of erectile dysfunction, or organ regeneration with liver as example. But the regeneration of tissue or organ is and will remain a challenge for the future development of cell therapy. Many problems remain to be solved that could lead to the development of innovative strategies to facilitate cell differentiation, increase the yield of cells and ensure a standardised product, overcome the risks of teratogenic effects and/or immune reactions, enable grafting via direct cell or biotissue transplantation and avoid legal issues involved in national regulations.


Assuntos
Medicina Regenerativa , Células-Tronco/metabolismo , Humanos , Qualidade de Vida , Células-Tronco/citologia , Engenharia Tecidual
6.
Biochim Biophys Acta ; 882(3): 469-72, 1986 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-3089296

RESUMO

The inductive effects of racemic 2-phenylpropionic acid and its isomers on rat liver bilirubin UDP-glucuronosyltransferase activity and lauric acid 12-hydroxylation (cytochrome P-452-dependent) were compared. The (S)-(+)-enantiomer and the racemic mixture gave the greatest induction of both enzyme activities, whereas (R)-(-)-2-phenylpropionic acid produced increases of only one-third of those of its antipode. The determination of the enantiomeric composition of the excreted 2-phenylpropionic acid after a single oral dose indicated that the (R)-(-)-enantiomer given as such or in the racemate was inverted to its antipode, which strongly suggests that (S)-(+)-2-phenylpropionic acid is responsible for the inductive effects observed. The demonstration of the same stereospecificity for the induction of bilirubin UDPglucuronosyltransferase and lauric acid 12-hydroxylation further indicates a close mechanistic link between these two processes.


Assuntos
Bilirrubina/metabolismo , Glucuronosiltransferase/metabolismo , Fígado/enzimologia , Fenilpropionatos/farmacologia , Animais , Peso Corporal/efeitos dos fármacos , Citocromos/metabolismo , Hidroxilação , Isomerismo , Ácidos Láuricos/metabolismo , Masculino , Tamanho do Órgão/efeitos dos fármacos , Ratos
7.
Biochim Biophys Acta ; 883(2): 190-6, 1986 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-3091075

RESUMO

Bilirubin UDPglucuronosyltransferase of rat or human liver microsomes was inhibited, in vitro, by triphenylacetic acid and by structurally related arylcarboxylic acids. This inhibition appeared to be competitive towards bilirubin, and mixed-type towards UDPglucuronic acid. A decrease in the number of phenyl rings or the absence of the carboxyl group in the molecule gave structures which did not affect enzyme activity, showing that both the triphenyl moiety and the carboxyl group were necessary for the inhibition. On the other hand, successive additions of methylene groups in the aliphatic chain progressively increased inhibitory potency. Kappi,bilirubin for triphenylacetic acid was 96 microM compared with 5 microM for 7,7,7-triphenylheptanoic acid. The inhibition of bilirubin UDPglucuronosyltransferase was not due to displacement of bilirubin from albumin. On the basis of these results an attempt was made to delineate the molecular events leading to glucuronidation of bilirubin.


Assuntos
Glucuronosiltransferase/antagonistas & inibidores , Fenilacetatos/farmacologia , Animais , Digitonina/farmacologia , Humanos , Cinética , Masculino , Microssomos Hepáticos/enzimologia , Ratos , Ratos Endogâmicos
8.
Biochim Biophys Acta ; 1035(1): 12-9, 1990 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-2116906

RESUMO

The effect of thyroid hormones and chemically related compounds, on the activity of UDP-glucuronosyltransferases (EC 2.4.1.17) and cytochrome P-450-dependent monooxygenases in rat liver microsomes was investigated. The animals were thyroidectomized and treated with different doses of the drugs for 3 weeks. Opposite effects were observed depending on the isoenzyme of UDP-glucuronosyltransferase considered. While 3,3',5-triiodo-L-thyronine, 3,3',5-triiodothyroacetic acid, 3,3',5-triiodothyropropionic acid, isopropyldiiodothyronine and L- and D-thyroxine strongly increased 4-nitrophenol glucuronidation in a dose-dependent fashion, they decreased markedly bilirubin glucuronidation. However, the activity toward nopol, a monoterpenoid alcohol, was not significantly changed regardless of which compound or dose was used. Variation of UDP-glucuronosyltransferase observed with 4-nitrophenol and bilirubin was related to the thyromimetic effect of the drugs estimated from the increase in alpha-glycerophosphate dehydrogenase. Thyronine and 3,5-diiodo-L-tyrosine, which did not enhance this activity, also failed to affect glucuronidation. Variations in UDP-glucuronosyltransferase activity were more likely due to changes in protein expression rather than changes in enzyme latency, since lipid organization of the microsomal membrane, as estimated from the mean anisotropy of 1,6-diphenyl-1,3,5-hexatriene by fluorescence polarization was not significantly modified by the drug administration. Although some of the drugs could significantly decrease the triacylglycerol and cholesterol contents in plasma, all failed to affect lauric acid hydroxylation. The activities of catalase, palmitoyl-CoA dehydrogenase (CN- insensitive) and carnitine acetyltransferase in the fraction enriched in peroxisomes were also not significantly affected by treatment with the thyroid hormone LT3. In contrast, the activity of 7-ethoxycoumarine O-deethylase was increased by large doses of thyronine and by 3,3',5-triiodothyropropionic acid. The concentration of total cytochrome P-450 was decreased in a dose-dependent fashion by all the compounds used, except thyronine. Finally, significant correlations were observed between glucuronidation of bilirubin and 4-nitrophenol and the content in cytochrome P-450. This suggests a possible coordinate regulation of the two processes, which depends on the physicochemical characteristics of the thyroid hormones and related compounds.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Glucuronosiltransferase/metabolismo , Isoenzimas/metabolismo , Microssomos Hepáticos/enzimologia , Oxigenases/metabolismo , Hormônios Tireóideos/farmacologia , O-Dealquilase 7-Alcoxicumarina/metabolismo , Animais , Bilirrubina/metabolismo , Colesterol/sangue , Polarização de Fluorescência , Glicerolfosfato Desidrogenase/metabolismo , Lipídeos/sangue , Masculino , Microssomos Hepáticos/efeitos dos fármacos , Mitocôndrias Hepáticas/enzimologia , Nitrofenóis/farmacologia , Ratos , Ratos Endogâmicos
9.
Biochim Biophys Acta ; 842(2-3): 202-13, 1985 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-3931693

RESUMO

The inductive potency of carboxylic acids, structurally related to clofibrate, on bilirubin UDPglucuronosyltransferase was investigated in the rat. For this purpose, structure-induction relationships were established using ten different arylcarboxylic or chlorophenoxycarboxylic acids. 4'-Chlorophenoxyacetic, -propionic and -isobutyric (clofibric) acids progressively increased hepatic glucuronidation of bilirubin (17%, 43%, 60% greater than controls, respectively) after a 5-day treatment in rat (100 mg/kg per day). 2-Phenylpropionic acid also enhanced bilirubin UDPglucuronosyltransferase activity (50%) in contrast to phenylacetic acid. The other compounds did not, or only slightly, affect this parameter. These results indicate that specific structural features are required for the induction property. Moreover, a good correlation (r = 0.962) was found between the extent of induction and the physiochemical descriptors which characterize the electronic state of the molecules, when analysed by multidimensional regression. Fluorescence polarization revealed that the compounds tested, especially clofibric acid, did not affect, in vivo or in vitro, the anisotropy of two different probes embedded in the microsomal membranes. Finally, since the interaction of the carboxylic acids with the membranes did not modify the latency state of bilirubin UDPglucuronosyltransferase, it was concluded that the increase in enzyme activity was due more to a real induction than to activation of bilirubin UDPglucuronosyltransferase. A close linkage was established between bilirubin UDPglucuronosyltransferase induction and that of cytochrome P-452, as shown by enhanced omega-oxidation of lauric acid. This led to the hypothesis that both processes could be under coordinate regulation and mediated by a molecular interaction depending on the physicochemical properties of the carboxylic acids.


Assuntos
Bilirrubina/metabolismo , Clofibrato/análogos & derivados , Clofibrato/farmacologia , Glucuronosiltransferase/metabolismo , Ácidos Láuricos/metabolismo , Microssomos Hepáticos/metabolismo , Animais , Sistema Enzimático do Citocromo P-450/metabolismo , Indução Enzimática , Glucuronatos/metabolismo , Hidroxilação , Cinética , Masculino , Ratos , Ratos Endogâmicos , Relação Estrutura-Atividade , Termodinâmica
10.
Biochim Biophys Acta ; 1139(1-2): 20-4, 1992 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-1610916

RESUMO

The molecular form of UDP-glucuronosyltransferase involved in the catalysis of 3'-azido-3'-deoxythymidine (AZT)-5'-O-glucuronide was characterized in human liver microsomes. The specific activity (1.3 nmol/min per mg protein) in transplantable liver was more than 2-times higher than in post-mortem fragments. Liver microsomes from patients suffering Crigler-Najjar syndrome, who are genetically deficient in bilirubin UDP-glucuronosyltransferase, could also glucuronidate AZT to a similar extent, thus indicating that this protein was not involved in that process. A genetically engineered V79 cell line stably expressing a cDNA which encodes a human isozyme active towards 1-naphthol was unable to glucuronidate AZT. Clinically used drugs, most of them being glucuronidated, were tested as potential inhibitors of the glucuronidation of AZT in human liver microsomes. The drugs chemically related to 2-phenylpropionic acid, naproxen and flurbiprofen, and the steroid compounds testosterone, estrone and ethynylestradiol strongly inhibited AZT glucuronidation. Codeine and morphine also decreased the reaction rate although to a lower extent. Except estrone which elicited a partial competitive inhibition, ethynylestradiol, flurbiprofen naproxen and testosterone could competitively inhibit AZT glucuronidation with an apparent Ki of 38, 50, 172 and 250 microM, respectively. The results suggest that these drugs were substrates of the same isozyme(s) involved in AZT glucuronidation. Probenecid was a weak inhibitor of the reaction (Ki 900 microM), only when non-disrupted microsomes were used. This drug may compete with the anion carrier system involved in the microsomal uptake of UDP-glucuronic acid.


Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Estrona/farmacologia , Etinilestradiol/farmacologia , Glucuronosiltransferase/antagonistas & inibidores , Microssomos Hepáticos/enzimologia , Testosterona/farmacologia , Zidovudina/metabolismo , Flurbiprofeno/farmacologia , Humanos , Microssomos Hepáticos/efeitos dos fármacos , Naproxeno/farmacologia , Fenilpropionatos/farmacologia , Probenecid/farmacologia
11.
Biochim Biophys Acta ; 1243(1): 9-14, 1995 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-7827113

RESUMO

The inhibitory effect of a series of novel structurally related compounds on the human UDP-glucuronosyltransferase UGT1*6 stably expressed in a V79 cell line was investigated. The inhibitors contain a lipophilic N-acyl phenylaminoalcohol residue and a uridine moiety connected by a spacer varying for each compound. The effects of these compounds on the glucuronidation reaction measured with 4-methylumbelliferone as substrate were determined. The best inhibitor of the series, D-DPMSU, had an IC50 of 39 microM in the assay conditions. Low Ki values were found toward both UDP-glucuronic acid and 4-methylumbelliferone (17 and 21 microM, respectively). The inhibition was competitive toward both substrates. A similar strong and competitive inhibitory effect was observed with two other inhibitors, DHPASU and DHPASiU. Another compound, D-DPASiU, showed a pure competitive inhibition towards UDP-glucuronic acid, but a non-competitive inhibition towards the acceptor substrate. These data and the optimization of the structures of the inhibitors by molecular modeling suggest that D-DPMSU and DHPASiU compounds may be transition state analog inhibitors of the recombinant UGT1*6 enzyme.


Assuntos
Glucuronosiltransferase/antagonistas & inibidores , Isoenzimas/antagonistas & inibidores , Fígado/enzimologia , Propanolaminas/farmacologia , Uridina/análogos & derivados , Sítios de Ligação , Simulação por Computador , Desoxiuridina/análogos & derivados , Desoxiuridina/farmacologia , Relação Dose-Resposta a Droga , Humanos , Himecromona/metabolismo , Cinética , Modelos Moleculares , Naftóis/metabolismo , Proteínas Recombinantes/antagonistas & inibidores , Sulfonas/farmacologia , Uridina/farmacologia
12.
Biochim Biophys Acta ; 1205(2): 336-45, 1994 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-8155717

RESUMO

The UDP-glucuronosyltransferases (UGT) involved in glucuronidation of endogenous and exogenous toxic compounds transfer the glucuronic acid residue from UDP-glucuronic acid (UDP-GlcUA), to various acceptor groups. A series of compounds that contain N-acyl phenylaminoalcohol derivatives linked to uridine or isopropylideneuridine were tested as UGT inhibitors. The potency of these inhibitors was determined by studying their effect on the photoaffinity labeling of rat liver microsomal UGTs by two photoaffinity probes, [beta-32P]5-azido-UDP-glucuronic acid (5N3UDP-GlcUA) and [beta-32P]5-azido-UDP-glucose (5N3UDP-Glc) and on the enzymatic formation of the two glucuronide conjugates (3-O- and carboxyl-specific) of lithocholic acid. All but one of the compounds tested proved to have an inhibitory effect on UGTs, both in the photoaffinity labeling system and in the enzymatic glucuronidation assay. In the photoaffinity labeling system, the inhibitors containing the isopropylidene moiety were less effective than their unprotected derivatives; however, the protected forms were, with one exception, more potent inhibitors of enzymatic activity. The photoaffinity labeling of UGTs with [beta-32P]5N3UDP-Glc was more susceptible to inhibition by all derivatives than that with [beta-32P]5N3UDP-GlcUA. The effect of one inhibitor, PP50B, on the two enzymatic activities involved in LA glucuronidation was extensively tested. A double-reciprocal plot suggested a competitive inhibition for UDP-GlcUA with an apparent Ki of 35 microM for LA 3-O-glucuronide formation and 94 microM for the carboxyl-linked glucuronide of the same substrate.


Assuntos
Glucuronosiltransferase/antagonistas & inibidores , Fígado/enzimologia , Uridina/análogos & derivados , Marcadores de Afinidade , Animais , Desoxiuridina/análogos & derivados , Desoxiuridina/farmacologia , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/enzimologia , Ácido Litocólico/metabolismo , Polissacarídeos Bacterianos/farmacologia , Propanolaminas/farmacologia , Ratos
13.
Biochim Biophys Acta ; 1283(2): 223-31, 1996 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-8809103

RESUMO

Previous studies have documented the presence of protein-mediated transport of UDP-glucuronic acid (UDP-GlcUA) in rat liver endoplasmic reticulum (ER). Measurement of uptake at varying concentrations of high specific activity [beta-32P]UDP-GlcUA has revealed the presence of a two component UDP-GlcUA transporting system. Transport at low substrate concentrations occurred predominantly via a high affinity component (K(m) = 1.6 microM), whereas a low affinity component (K(m) = 38 microM) predominated at high substrate concentrations. The K(m) for the high affinity system is in agreement with that previously published, while the low affinity component is a new finding. The uptake of UDP-GlcUA was temperature-sensitive, time dependent, and saturable for both components. The high affinity transport was affected by trans-stimulation and cis-inhibition by UDP-N-acetylglucosamine (UDP-GlcNAc); however, the same concentrations of UDP-GlcNAc had less effect on the low affinity system. In order to further study the two transport components, various inhibitors of anion transport carriers were tested. The high affinity component was strongly inhibited by 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS) and furosemide, while the low affinity system was less sensitive to these reagents. Dose-dependent inhibition by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) was found for both transport systems. Probenecid was found to be a weak inhibitor of both components of the UDP-GlcUA uptake. Finally, the major metabolite of 3'-azido-3'-deoxythymidine, 3'-azido-3'-deoxythymidine monophosphate (AZTMP), was able to inhibit the uptake of UDP-GlcUA by both components. The results indicate the presence of two carrier-mediated UDP-glucuronic acid transporting components in rat liver ER.


Assuntos
Proteínas de Transporte/metabolismo , Retículo Endoplasmático/metabolismo , Fígado/ultraestrutura , Uridina Difosfato Ácido Glucurônico/metabolismo , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-dissulfônico/farmacologia , Animais , Transporte Biológico/efeitos dos fármacos , Furosemida/farmacologia , Cinética , Masculino , Concentração Osmolar , Probenecid/farmacologia , Ratos , Ratos Sprague-Dawley
14.
Stem Cells Int ; 2015: 734731, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26300923

RESUMO

Since the 1960s and the therapeutic use of hematopoietic stem cells of bone marrow origin, there has been an increasing interest in the study of undifferentiated progenitors that have the ability to proliferate and differentiate into various tissues. Stem cells (SC) with different potency can be isolated and characterised. Despite the promise of embryonic stem cells, in many cases, adult or even fetal stem cells provide a more interesting approach for clinical applications. It is undeniable that mesenchymal stem cells (MSC) from bone marrow, adipose tissue, or Wharton's Jelly are of potential interest for clinical applications in regenerative medicine because they are easily available without ethical problems for their uses. During the last 10 years, these multipotent cells have generated considerable interest and have particularly been shown to escape to allogeneic immune response and be capable of immunomodulatory activity. These properties may be of a great interest for regenerative medicine. Different clinical applications are under study (cardiac insufficiency, atherosclerosis, stroke, bone and cartilage deterioration, diabetes, urology, liver, ophthalmology, and organ's reconstruction). This review focuses mainly on tissue and organ regeneration using SC and in particular MSC.

15.
Protein Sci ; 10(1): 200-11, 2001 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-11266607

RESUMO

Cellular retinoic acid-binding proteins (CRABPs) are carrier proteins thought to play a crucial role in the transport and metabolism of all-trans-retinoic acid (atRA) and its derivatives within the cell. This report describes a novel photoaffinity-based binding assay involving competition between potential ligands of CRABP and [(3)H]atRA or [(3)H]-9-cis-RA for binding to the atRA-binding sites of CRABP I and II. Photoaffinity labeling of purified CRABPs with [(3)H]atRA was light- and concentration-dependent, saturable, and protected by several retinoids in a concentration-dependent manner, indicating that binding occurred in the CRABP atRA-binding site. Structure-function relationship studies demonstrated that oxidative changes to the atRA beta-ionone ring did not affect ligand potency. However, derivatives lacking a terminal carboxyl group and some cis isomers did not bind to CRABPs. These studies also identified two novel ligands for CRABPs: 5,6-epoxy-RA and retinoyl-beta-D-glucuronide (RAG). The labeling of both CRABPs with 9-cis-RA occurred with much lower affinity. Experimental evidence excluded nonspecific binding of RAG to CRABPs and UDP-glucuronosyltransferases, the enzymes responsible for RAG synthesis. These results established that RAG is an effective ligand of CRABPs. Therefore, photoaffinity labeling with [(3)H]atRA can be used to identify new ligands for CRABP and retinoid nuclear receptors and also provide information concerning the identity of amino acid(s) localized in the atRA-binding site of these proteins.


Assuntos
Receptores do Ácido Retinoico/química , Tretinoína/química , Alitretinoína , Animais , Glucuronatos/química , Glucuronosiltransferase/química , Humanos , Ácido Litocólico/química , Microssomos Hepáticos/química , Marcadores de Fotoafinidade , Ratos , Proteínas Recombinantes/química , Relação Estrutura-Atividade , Trítio
16.
J Invest Dermatol ; 94(6): 749-52, 1990 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-2113070

RESUMO

The metabolic capacity of reconstituted epidermis from the outer root sheath cells of human hair follicles was determined. It was found that this epidermis possesses enzymes involved in both phase I (oxidation) and phase II (conjugation) reactions for drug biotransformation. The use of model substrates allowed the characterization of several isoenzymes. The homogenate fraction contained membrane-bound mixed-function oxydases (cytochrome P-450 dependent) involved in the O-dealkylation of 7-ethoxy-, 7-pentoxy-, and 7-benzoxyresorufin, NADPH cytochrome c (P-450) reductase, testosterone 5 alpha-reductase, and UDP-glucuronosyltransferases, which conjugate 1-naphthol and bilirubin. One isoform of each glutathione S-transferase, steroid-, and arylsulfatases, acting on estrone- and 4-methylumbelliferone sulfates, was detected. Additionally, the activity of two distinct forms of epoxide hydrolases, which hydrate cis- and trans-stilbene oxides, could be measured. The presence of these drug metabolizing enzymes in the reconstituted epidermis indicates that it has a potential to serve as a model to study epidermal drug metabolism in vitro.


Assuntos
Epiderme , Modelos Biológicos , Preparações Farmacêuticas/metabolismo , Arilsulfatases/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Epiderme/enzimologia , Epiderme/metabolismo , Compostos de Epóxi/metabolismo , Glucuronosiltransferase/metabolismo , Cabelo/metabolismo , Técnicas Histológicas , Humanos , Oxirredutases/metabolismo , Oxigenases/metabolismo , Esteril-Sulfatase
17.
Pharmacogenetics ; 7(4): 255-69, 1997 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-9295054

RESUMO

This review represents an update of the nomenclature system for the UDP glucuronosyltransferase gene superfamily, which is based on divergent evolution. Since the previous review in 1991, sequences of many related UDP glycosyltransferases from lower organisms have appeared in the database, which expand our database considerably. At latest count, in animals, yeast, plants and bacteria there are 110 distinct cDNAs/genes whose protein products all contain a characteristic 'signature sequence' and, thus, are regarded as members of the same superfamily. Comparison of a relatedness tree of proteins leads to the definition of 33 families. It should be emphasized that at least six cloned UDP-GlcNAc N-acetylglucosaminyltransferases are not sufficiently homologous to be included as members of this superfamily and may represent an example of convergent evolution. For naming each gene, it is recommended that the root symbol UGT for human (Ugt for mouse and Drosophila), denoting 'UDP glycosyltransferase,' be followed by an Arabic number representing the family, a letter designating the subfamily, and an Arabic numeral denoting the individual gene within the family or subfamily, e.g. 'human UGT2B4' and 'mouse Ugt2b5'. We recommend the name 'UDP glycosyltransferase' because many of the proteins do not preferentially use UDP glucuronic acid, or their nucleotide sugar preference is unknown. Whereas the gene is italicized, the corresponding cDNA, transcript, protein and enzyme activity should be written with upper-case letters and without italics, e.g. 'human or mouse UGT1A1.' The UGT1 gene (spanning > 500 kb) contains at least 12 promoters/first exons, which can be spliced and joined with common exons 2 through 5, leading to different N-terminal halves but identical C-terminal halves of the gene products; in this scheme each first exon is regarded as a distinct gene (e.g. UGT1A1, UGT1A2, ... UGT1A12). When an orthologous gene between species cannot be identified with certainty, as occurs in the UGT2B subfamily, sequential naming of the genes is being carried out chronologically as they become characterized. We suggest that the Human Gene Nomenclature Guidelines (http://www.gene.acl.ac.uk/nomenclature/guidelines.html++ +) be used for all species other than the mouse and Drosophila. Thirty published human UGT1A1 mutant alleles responsible for clinical hyperbilirubinemias are listed herein, and given numbers following an asterisk (e.g. UGT1A1*30) consistent with the Human Gene Nomenclature Guidelines. It is anticipated that this UGT gene nomenclature system will require updating on a regular basis.


Assuntos
Evolução Molecular , Genes , Glucuronosiltransferase/genética , Família Multigênica , Terminologia como Assunto , Sequência de Aminoácidos , Animais , Glucuronosiltransferase/química , Humanos , Dados de Sequência Molecular , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
18.
FEBS Lett ; 454(3): 187-91, 1999 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-10431804

RESUMO

UDP-glucuronosyltransferase 1A6 (UGT1A6) is a membrane glycoprotein of the endoplasmic reticulum playing a key role in drug metabolism. It is synthesized as a precursor with an N-terminal cleavable signal peptide. We demonstrate that deletion of the signal peptide sequence does not prevent membrane targeting and integration of this human isoform when expressed in an in vitro transcription-translation system, as shown by N-glycosylation, resistance to alkaline treatment and protease protection. Furthermore, UGT1A6 lacking the signal peptide (UGT1A6delta sp) was targeted to the endoplasmic reticulum in mammalian cells as shown by immunofluorescence microscopy and was catalytically active with kinetic constants for 4-methylumbelliferone glucuronidation similar to that of the wild-type. These results provide evidence that the signal peptide is not essential for the membrane assembly and activity of UGT1A6 suggesting that additional topogenic element(s) mediate(s) this process.


Assuntos
Glucuronosiltransferase/metabolismo , Fígado/enzimologia , Animais , Transporte Biológico , Linhagem Celular , Cricetinae , Retículo Endoplasmático/metabolismo , Glucuronosiltransferase/genética , Humanos , Fígado/ultraestrutura , Deleção de Sequência
19.
FEBS Lett ; 339(1-2): 195-9, 1994 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-8313973

RESUMO

The membrane-bound human liver UDP-glucuronosyltransferase UGT1*6 was expressed in Escherichia coli. Exchange of the natural signal peptide by the bacterial signal peptides of pclB or OmpT proteins considerably increased the level of expression and, as the natural signal peptide, targeted the protein to the membranes. The extent of maturation of SpelB-UGT1*6 precursor was about 30%. No processing of sOmpT-UGT1*6 occurred but the processing rate of this precursor could be significantly increased by mutagenesis of the first two amino acid residues of the mature sequence. These expression vectors allowed us to produce high levels of recombinant mature UGT1*6 required for further structural studies.


Assuntos
Proteínas de Bactérias/genética , Escherichia coli/genética , Expressão Gênica/efeitos dos fármacos , Glucuronosiltransferase/genética , Precursores de Proteínas/genética , Sinais Direcionadores de Proteínas/genética , Sequência de Aminoácidos , Proteínas de Bactérias/fisiologia , Sequência de Bases , Membrana Celular/enzimologia , Clonagem Molecular , Vetores Genéticos , Glucuronosiltransferase/biossíntese , Humanos , Dados de Sequência Molecular , Peso Molecular , Plasmídeos , Sinais Direcionadores de Proteínas/fisiologia , Proteínas Recombinantes de Fusão , Proteínas Recombinantes/biossíntese
20.
Free Radic Biol Med ; 37(7): 1051-63, 2004 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-15336321

RESUMO

We have investigated the effects of mild oxidative conditions on drug-metabolizing enzyme activity in rat cultured astrocytes. These experimental conditions promoting an oxidative environment were obtained by short exposure to a low concentration of menadione (5 microM) for a short duration (15 min). This resulted in the rapid and transient production of reactive oxygen species (+130%), associated with a decrease in GSH cellular content (-24%), and an increase in total protein oxidation (+26%), but promoted neither PGE(2) nor NO production. This treatment induced a rapid and persistent decrease in astrocyte glucuronidation activities, which was totally prevented by N-acetyl-l-cysteine. These oxidative conditions also affected the specific UGT1A6 activity measured in transfected V79-1A6 cells. Finally, the subsequent recovery of astrocyte glucuronidation activity may result from upregulation of UGT1A6 expression (+62%) as shown by RT-PCR and gene reporter assay. These results show that the catalytic properties and expression of cerebral UGT1A6 are highly sensitive to the redox environment. The protective effect of N-acetyl-l-cysteine suggests both a direct action of reactive oxygen species on the protein and a more delayed action on the transcriptional regulation of UGT1A6. These results suggest that cerebral metabolism can be altered by physiological or pathological redox modifications.


Assuntos
Astrócitos/metabolismo , Glucuronatos/metabolismo , Animais , Apoptose/efeitos dos fármacos , Astrócitos/citologia , Astrócitos/efeitos dos fármacos , Sequência de Bases , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cricetinae , Feminino , Glucuronosiltransferase/química , Glucuronosiltransferase/metabolismo , Glutationa/metabolismo , Peróxido de Hidrogênio/farmacologia , Inflamação/metabolismo , Masculino , Oxirredução/efeitos dos fármacos , Regiões Promotoras Genéticas , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Vitamina K 3/farmacologia
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