RESUMO
A diverse T cell receptor (TCR) repertoire is essential for protection against a variety of pathogens, and TCR repertoire size is believed to decline with age. However, the precise size of human TCR repertoires, in both total and subsets of T cells, as well as their changes with age, are not fully characterized. We conducted a longitudinal analysis of the human blood TCRα and TCRß repertoire of CD4+ and CD8+ T cell subsets using a unique molecular identifier-based (UMI-based) RNA-seq method. Thorough analysis of 1.9 × 108 T cells yielded the lower estimate of TCR repertoire richness in an adult at 3.8 × 108. Alterations of the TCR repertoire with age were observed in all 4 subsets of T cells. The greatest reduction was observed in naive CD8+ T cells, while the greatest clonal expansion was in memory CD8+ T cells, and the highest increased retention of TCR sequences was in memory CD8+ T cells. Our results demonstrated that age-related TCR repertoire attrition is subset specific and more profound for CD8+ than CD4+ T cells, suggesting that aging has a more profound effect on cytotoxic as opposed to helper T cell functions. This may explain the increased susceptibility of older adults to novel infections.
Assuntos
Linfócitos T CD8-Positivos , Subpopulações de Linfócitos T , Adulto , Idoso , Linfócitos T CD4-Positivos , Humanos , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T alfa-beta/genéticaRESUMO
OBJECTIVE: Impairment of the thrombomodulin-protein C anticoagulant pathway has been implicated in pathological thrombosis associated with malignancy. Patients who receive proteasome inhibitors as part of their chemotherapeutic regimen appear to be at decreased risk for thromboembolic events. We investigated the effects of proteasome inhibitors on endothelial thrombomodulin expression and function. METHODS AND RESULTS: Proteasome inhibitors as a class markedly induced the expression of thrombomodulin and enhanced the protein C activating capacity of endothelial cells. Thrombomodulin upregulation was independent of NF-kappaB signaling, a principal target of proteasome inhibitors, but was instead a direct consequence of increased expression of the Krüppel-like transcription factors, KLF2 and KLF4. These effects were confirmed in vivo, where systemic administration of a proteasome inhibitor enhanced thrombomodulin expression that was paralleled by changes in the expression of KLF2 and KLF4. CONCLUSIONS: These findings identify a novel mechanism of action of proteasome inhibitors that may help to explain their clinically observed thromboprotective effects.
Assuntos
Ácidos Borônicos/farmacologia , Células Endoteliais/efeitos dos fármacos , Fatores de Transcrição Kruppel-Like/fisiologia , Inibidores de Proteases/farmacologia , Inibidores de Proteassoma , Pirazinas/farmacologia , Trombomodulina/genética , Animais , Bortezomib , Células Cultivadas , Células Endoteliais/metabolismo , Humanos , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Masculino , Camundongos , Camundongos Endogâmicos C3H , NF-kappa B/antagonistas & inibidores , Proteína C/fisiologia , Trombomodulina/fisiologiaRESUMO
Vascular endothelial cells respond to biomechanical forces, such as cyclic stretch and shear stress, by altering gene expression. Since endothelial-derived prostanoids, such as prostacyclin and thromboxane A(2), are key mediators of endothelial function, we investigated the effects of cyclic stretch on the expression of genes in human umbilical vein endothelial cells controlling prostanoid synthesis: cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), prostacyclin synthase (PGIS) and thromboxane A(2) synthase (TXAS). COX-2 and TXAS mRNAs were upregulated by cyclic stretch for 24h. In contrast, PGIS mRNA was decreased and stretch had no effect on COX-1 mRNA expression. We further show that stretch-induced upregulation of COX-2 is mediated by activation of the NF-kappabeta signaling pathway.
Assuntos
Ciclo-Oxigenase 2/genética , Endotélio Vascular/enzimologia , Regulação Enzimológica da Expressão Gênica , Mecanotransdução Celular/genética , NF-kappa B/metabolismo , Estresse Mecânico , Células Cultivadas , Humanos , Resistência ao CisalhamentoRESUMO
We previously isolated a bisphenol A (BPA)-binding protein from rat brain and showed that it was identical to the protein disulfide isomerase (PDI), which also serves as a 3,3',5-triiodo-l-thyronine (T3)-binding protein. In this study, we investigated the effects of BPA on the production of growth hormone (GH) in GH3 cells and examined the possible involvement of PDI in this process. When administered singly, BPA and T3 each induced GH release in GH3 cells. When cells were treated with the combination of BPA and T3, the release of GH was much greater than that by T3 alone, but GH mRNA and promoter activity were not increased. These results suggested that the synergistic effect of T3 plus BPA on GH release was due to posttranslational regulation. Over-expression of PDI suppressed GH mRNA expression and GH release, suggesting that PDI modulates the T3-induced gene expression. We conclude that BPA can disrupt the thyroid hormone function in GH3 cells, and GH release induced by T3 is influenced by expression of PDI.
Assuntos
Poluentes Ocupacionais do Ar/antagonistas & inibidores , Hormônio do Crescimento/metabolismo , Fenóis/antagonistas & inibidores , Hipófise/enzimologia , Isomerases de Dissulfetos de Proteínas/metabolismo , Tri-Iodotironina/antagonistas & inibidores , Poluentes Ocupacionais do Ar/farmacologia , Animais , Compostos Benzidrílicos , Linhagem Celular Tumoral , Expressão Gênica , Hormônio do Crescimento/genética , Fenóis/farmacologia , Hipófise/efeitos dos fármacos , Isomerases de Dissulfetos de Proteínas/genética , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Ratos , Tri-Iodotironina/metabolismo , Tri-Iodotironina/farmacologiaRESUMO
Bisphenol A [2,2-bis-(4-hydroxyphenyl) propane; BPA] is a versatile industrial material for plastic products, but is increasingly being recognized as a pervasive industrial pollutant as well. Accumulating evidence indicates that the environmental contaminant BPA is one of the endocrine-disrupting chemicals that potentially can adversely affect humans as well as wildlife. To define the molecular aspects of BPA action, we first investigated the molecules with which it physically interacts. High BPA-binding activity was detected in the P2 membrane fraction prepared from rat brains. As determined by SDS-PAGE analysis, the molecular mass of a BPA-binding protein purified from the rat brain P2 fraction was 53 kDa. The N-terminal amino acid sequence of the purified BPA-binding protein was identical with that of the rat protein disulfide isomerase (PDI), which is a multifunctional protein that is critically involved in the folding, assembly, and shedding of many cellular proteins via its isomerase activity in addition to being considered to function as an intracellular hormone reservoir. The Kd value of BPA binding to recombinant rat PDI was 22.6 +/- 6.6 microm. Importantly, the binding activity of L-T3 and 17beta-estradiol hormones to PDI was competitively inhibited by BPA in addition to abolishing its isomerase activities. In this paper we report that the ubiquitous and multifunctional protein PDI is a target of BPA and propose that binding to PDI and subsequent inhibition of PDI activity might be mechanistically responsible for various actions of BPA.
Assuntos
Estradiol/metabolismo , Fenóis/farmacologia , Isomerases de Dissulfetos de Proteínas/antagonistas & inibidores , Tri-Iodotironina/metabolismo , Animais , Compostos Benzidrílicos , Proteínas de Transporte/isolamento & purificação , Proteínas de Transporte/metabolismo , Masculino , Fenóis/metabolismo , Ligação Proteica , Isomerases de Dissulfetos de Proteínas/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/metabolismoRESUMO
The TCR repertoire serves as a reservoir of TCRs for recognizing all potential pathogens. Two major types of T cells, CD4(+) and CD8(+), that use the same genetic elements and process to generate a functional TCR differ in their recognition of peptide bound to MHC class II and I, respectively. However, it is currently unclear to what extent the TCR repertoire of CD4(+) and CD8(+) T cells is different. Here, we report a comparative analysis of the TCRß repertoires of CD4(+) and CD8(+) T cells by use of a 5' rapid amplification of cDNA ends-PCR-sequencing method. We found that TCRß richness of CD4(+) T cells ranges from 1.2 to 9.8 × 10(4) and is approximately 5 times greater, on average, than that of CD8(+) T cells in each study subject. Furthermore, there was little overlap in TCRß sequences between CD4(+) (0.3%) and CD8(+) (1.3%) T cells. Further analysis showed that CD4(+) and CD8(+) T cells exhibited distinct preferences for certain amino acids in the CDR3, and this was confirmed further by a support vector machine classifier, suggesting that there are distinct and discernible differences between TCRß CDR3 in CD4(+) and CD8(+) T cells. Finally, we identified 5-12% of the unique TCRßs that share an identical CDR3 with different variable genes. Together, our findings reveal the distinct features of the TCRß repertoire between CD4(+) and CD8(+) T cells and could potentially be used to evaluate the competency of T cell immunity.
Assuntos
Aminoácidos/análise , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Regiões Determinantes de Complementaridade/análise , Receptores de Antígenos de Linfócitos T alfa-beta/análise , Adulto , Idoso , Genes Codificadores da Cadeia beta de Receptores de Linfócitos T , Humanos , Pessoa de Meia-Idade , Receptores de Antígenos de Linfócitos T alfa-beta/químicaRESUMO
We first identified the transcriptional regulatory element of the CYP2D4 gene. CYP2D4 is of interest in brain pharmacology and physiology because this enzyme can be involved in the metabolism of endogenous and exogenous compounds, which act on the central nervous system. Transfection studies using a series of the CYP2D4 promoter luciferase constructs identified the transcriptional element of CYP2D4 in the sequence between nucleotides -116 and -90 (named the neural expression regulatory element, NERE). The nucleotide sequence of NERE was specific for the CYP2D4 gene. Within this region, two nuclear factor-binding sequences, Oct-1 and YY-1, were present. Oct-1 acts as the activator of the CYP2D4. The core sequence of the YY-1 binding motif partially overlapped that of the Oct-1 binding motif. YY-1 may act as the repressor of CYP2D4, which interferes with Oct-1 activation by its binding to NERE. We concluded that a novel transcriptional regulatory element NERE specifically regulates the expression of the CYP2D4. This regulation system may be involved in the unique distribution of this isoform, such as the expression in the brain.
Assuntos
Hidrocarboneto de Aril Hidroxilases/genética , Hidrocarboneto de Aril Hidroxilases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Sequências Reguladoras de Ácido Nucleico , Fatores de Transcrição/metabolismo , Transcrição Gênica , Sequência de Aminoácidos , Animais , Sítios de Ligação , Diferenciação Celular/genética , Proteínas de Ligação a DNA/genética , Fatores de Ligação de DNA Eritroide Específicos , Regulação da Expressão Gênica , Fator C1 de Célula Hospedeira , Dados de Sequência Molecular , Neurônios/citologia , Neurônios/fisiologia , Fator 1 de Transcrição de Octâmero , Células PC12 , Ratos , Fatores de Transcrição/genética , Ativação TranscricionalRESUMO
BACKGROUND: The influence of endocrine disrupting chemicals (EDCs) upon physiological functions and their mechanisms is of concern. We have previously demonstrated that protein disulfide isomerase (PDI) was a target molecule of bisphenol A (BPA), which is considered to be EDC. PDI plays a key role in protein folding as an isomerase and also possesses a 3,3',5-triiodo-L-thyronine (T3)-binding activity. Since PDI activities were inhibited by BPA in our previous study, BPA might have adverse effects on physiological functions via the inhibition of PDI activities. We conducted this study to identify the compounds which disturb PDI activities as well as BPA, and to discuss their structural characteristics. METHODS: We examined the effects of 22 suspected EDC on both the T3-binding activity and isomerase activity of rat recombinant PDI. RESULTS: Among the 22 compounds, only phenolic compounds, namely BPA, p-octylphenol, p-nonylphenol, 2,4-dichlorophenol, pentachlorophenol, tetrabromobisphenol A, and tetrachlorobisphenol A, inhibited T3 binding to PDI. Furthermore non-halogenated compounds among these phenolic compounds, such as BPA, p-octylphenol, and p-nonylphenol, showed inhibitory effects on the isomerase activity of PDI. CONCLUSIONS: Our results suggest that phenolic groups might have important inhibitory effects on the T3-binding activity of PDI, and that compounds with phenolic groups might have the same effects on PDI. Furthermore, non-halogenated phenolic compounds had inhibitory effects on the isomerase activities of PDI in addition to T3-binding activity, indicating that these compounds might also have adverse effects on protein folding, which PDI participates in by catalyzing rearrangements of disulfide bonds as an isomerase.
Assuntos
Disruptores Endócrinos/toxicidade , Poluentes Ambientais/toxicidade , Fenóis/farmacologia , Isomerases de Dissulfetos de Proteínas/antagonistas & inibidores , Isomerases de Dissulfetos de Proteínas/metabolismo , Animais , Compostos Benzidrílicos , Clorofenóis/farmacologia , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/toxicidade , Masculino , Pentaclorofenol/farmacologia , Bifenil Polibromatos/farmacologia , Dobramento de Proteína , Ratos , Ratos Sprague-Dawley , Receptores dos Hormônios Tireóideos/metabolismo , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/metabolismo , Tri-Iodotironina Reversa/metabolismoRESUMO
mRNA of cytochrome P450 21-hydroxylase (P450c21) is expressed in the brain, but little is known about the enzymatic properties of P450c21 in the brain. In the present study, we showed, by using various recombinant cytochrome P450 (CYP)2D enzymes and anti-CYP2D4- or P450c21-specific antibodies, that rat brain microsomal steroid 21-hydroxylation is catalyzed not by P450c21, but by CYP2D isoforms. Rat CYP2D4 and human CYP2D6, which are the predominant CYP2D isoforms in the brain, possess 21-hydroxylation activity for both progesterone and 17alpha-hydroxyprogesterone. In rat brain microsomes, these activities were not inhibited by anti-P450c21 antibodies, but they were effectively inhibited by the CYP2D-specific chemical inhibitor quinidine and by anti-CYP2D4 antibodies. mRNA and protein of CYP2D4 were expressed throughout the brain, especially in cerebellum, striatum, pons, and medulla oblongata, whereas the mRNA and protein levels of P450c21 were extremely low or undetectable. These results support the idea that CYP2D4, not P450c21, works as steroid 21-hydroxylase in the brain. Allopregnanolone, a representative gamma-aminobutyric acid receptor modulator, was also hydroxylated at the C-21 position by recombinant CYP2D4 and CYP2D6. Rat brain microsomal allopregnanolone 21-hydroxylation was inhibited by fluoxetine with an IC(50) value of 2 microm, suggesting the possibility that the brain CYP2D isoforms regulate levels of neurosteroids such as allopregnanolone, and that this regulation is modified by central nervous system-active drugs such as fluoxetine.
Assuntos
Hidrocarboneto de Aril Hidroxilases/metabolismo , Encéfalo/enzimologia , Citocromo P-450 CYP2D6/metabolismo , Oxigenases de Função Mista/metabolismo , 17-alfa-Hidroxiprogesterona/metabolismo , Animais , Hidrocarboneto de Aril Hidroxilases/análise , Hidrocarboneto de Aril Hidroxilases/genética , Western Blotting , Química Encefálica , Citocromo P-450 CYP2D6/análise , Citocromo P-450 CYP2D6/genética , Fluoxetina/farmacologia , Humanos , Hidroxilação , Masculino , Microssomos/enzimologia , Oxigenases de Função Mista/análise , Oxigenases de Função Mista/genética , Pregnanolona/metabolismo , Progesterona/metabolismo , RNA Mensageiro/análise , Ratos , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Esteroide 21-Hidroxilase/análise , Esteroide 21-Hidroxilase/genética , Esteroide 21-Hidroxilase/metabolismo , Especificidade por Substrato , Distribuição TecidualRESUMO
The ligand-binding characteristics of rat and human CYP2D isoforms, i.e., rat CYP2D1-4 and human CYP2D6, were investigated by measuring IC(50) values of 11 known CYP2D6 ligands using 7-methoxy-4-(aminomethyl)coumarin (MAMC) as substrate. Like CYP2D6, all rat CYP2D isozymes catalyzed the O-demethylation of MAMC with K(m) and V(max) values ranging between 78 and 145 microM and 0.048 and 1.122 min(-1), respectively. To rationalize observed differences in the experimentally determined IC(50) values, homology models of the CYP2D isoforms were constructed. A homology model of CYP2D6 was generated on the basis of crystallized rabbit CYP2C5 and was validated on its ability to reproduce binding orientations corresponding to metabolic profiles of the substrates and to remain stable during unrestrained molecular dynamics simulations at 300 K. Twenty-two active site residues, sharing up to 59% sequence identity, were identified in the CYP2D binding pockets and included CYP2D6 residues Phe120, Glu216, and Asp301. Electrostatic potential calculations displayed large differences in the negative charge of the CYP2D active sites, which was consistent with observed differences in absolute IC(50) values. MD studies on the binding mode of sparteine, quinidine, and quinine in CYP2D2 and CYP2D6 furthermore concurred well with experimentally determined IC(50) values and metabolic profiles. The current study thus provides new insights into differences in the active site topology of the investigated CYP2D isoforms.
Assuntos
Sistema Enzimático do Citocromo P-450/química , Inibidores Enzimáticos/química , Oxirredutases do Álcool , Sequência de Aminoácidos , Animais , Hidrocarboneto de Aril Hidroxilases/química , Sítios de Ligação , Cumarínicos/química , Citocromo P-450 CYP2D6/química , Família 2 do Citocromo P450 , Humanos , Isoenzimas/química , Cinética , Ligantes , Oxigenases de Função Mista/química , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Quinidina/química , Quinina/química , Coelhos , Ratos , Homologia de Sequência de Aminoácidos , Esparteína/química , Especificidade da EspécieRESUMO
We investigated the biological effects of the active oxygen produced by P450s. First, we identified which isoforms of P450 efficiently produced active oxygen using electron spin resonance. Eight forms of P450 purified from rat liver were used. Of these, CYP1A2, 2B1, 2C11 and 3A2 produced hydroxyl radicals efficiently. Phenobarbital (PB) which is a typical inducer of CYP2B1 and 3A2 induced production of hydroxyl radicals by rat liver and ketoconazole, an inhibitor of P450, inhibited production of hydroxyl radicals in vitro. PB is a tumor promoter as well as the P450-inducer. We investigated oxidation of the genomic DNA by the hydroxyl radicals produced by PB-inducible P450 in vitro and in vivo. 8-hydroxy-2'-deoxyguanosine (8-OHdG), a biomarker of DNA oxidation in vivo was assayed by HPLC. PB strongly induced the production of 8-OHdG in the rat liver. While ketoconazole inhibited the production of 8-OHdG in vivo. These results suggest that active oxygen produced by P450 oxidized genomic DNA and induction of P450 increased oxidative stress that may contribute to tumor initiation and promotion.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , DNA/metabolismo , Fígado/enzimologia , Oxigênio/metabolismo , Fenobarbital/farmacologia , Animais , Hidrocarboneto de Aril Hidroxilases/metabolismo , Cromatografia Líquida de Alta Pressão , Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP2B1/metabolismo , Citocromo P-450 CYP3A , Família 2 do Citocromo P450 , Espectroscopia de Ressonância de Spin Eletrônica , Antagonistas de Aminoácidos Excitatórios/farmacologia , Radical Hidroxila , Imunoquímica , Imuno-Histoquímica , Cetoconazol/farmacologia , Peroxidação de Lipídeos , Fígado/metabolismo , Proteínas de Membrana/metabolismo , Microssomos Hepáticos/metabolismo , Isoformas de Proteínas , Proteínas Proto-Oncogênicas c-jun/biossíntese , Proteínas Proto-Oncogênicas c-myc/biossíntese , Ratos , Ratos Sprague-Dawley , Esteroide 16-alfa-Hidroxilase/metabolismo , Superóxidos/metabolismoRESUMO
Metabolic activities toward endogenous substrates in the brain, progesterone and p-tyramine, by cytochrome P450 2D6.2 (CYP2D6.2), CYP2D6.10A, CYP2D6.10C, and P34S, G42R, R296C, and S486T mutants expressed in recombinant Saccharomyces cerevisiae were compared with those by CYP2D6.1 (wild-type) in order to clarify the effects of genetic polymorphism of CYP2D6 on the metabolism of neuroactive steroids and amines in the brain. For the 6beta-hydroxylation of progesterone, the V(max) values for CYP2D6.2, CYP2D6.10A, and the P34S and G42R mutants, were less than half of those for CYP2D6.1, and CYP2D6.10C had a higher K(m) and a lower V(max) than the wild-type. The V(max)/K(m) values for CYP2D6.10A, CYP2D6.10C, and the P34S and G42R mutants were 12-31% of that for CYP2D6. The 16alpha-hydroxylation and 21-hydroxylation of progesterone by CYP2D6.10A, CYP2D6.10C, and the P34S and G42R mutants were not detected, and the R296C mutant had a higher K(m) for the 16alpha-hydroxylation and a lower V(max) for the 21-hydroxylation than those for CYP2D6.1. For dopamine formation from p-tyramine, the K(m) values for CYP2D6.2 and the R296C mutant were higher than those for CYP2D6.1, CYP2D6.10A, and CYP2D6.10C had a higher K(m) and a lower V(max) than the wild-type. The V(max)/K(m) values for CYP2D6.2, CYP2D6.10A, CYP2D6.10C and the P34S, G42R and R296C mutants were less than 45% of those for the wild-type. These results suggest the possibility that the polymorphism of CYP2D6, including CYP2D6*2, CYP2D6*10 and CYP2D6*12, might affect an individual behavior and the central nervous system through endogenous compounds, such as neuroactive steroids and tyramine, in the brain.
Assuntos
Inibidores da Captação Adrenérgica/metabolismo , Citocromo P-450 CYP2D6/genética , Citocromo P-450 CYP2D6/metabolismo , Polimorfismo Genético , Progesterona/metabolismo , Tiramina/metabolismo , Encéfalo/metabolismo , Dopamina/biossíntese , Feminino , Humanos , Microssomos/química , Microssomos/metabolismo , Oxigenases de Função Mista/metabolismoRESUMO
CYP2D1, 2D2, 2D3, and 2D4 are major CYP2D isoforms expressed in the rat. In humans, only CYP2D6 is expressed. In rat brain, the mRNA for CYP2D4 is most abundant in cerebellum, striatum, pons and medulla oblongata. In human brain, CYP2D6 mRNA expression was detected in all regions with highest levels observed in cerebellum. CYP2D isoforms are involved in the metabolism of not only xenobiotics such as antidepressants, beta-adrenergic blockers, antiarrhysthmics, and antihypertensives, but also endogenous compounds such as trace amine and neurosteroids. Among 11 isoforms of human recombinant P450s, only CYP2D6 exhibited an ability to efficiently convert tyramine which exists in the brain, to dopamine. CYP2D4 and CYP2D6 which are the predominant CYP2D isoforms in the rat and human brain, respectively, possess 21-hydroxylation activity for both progesterone and allopregnanolone. CYP2D4, not P450c21, works as a steroid 21-hydroxylase in the brain. These results suggested that CYP2D in the brain may be involved in the metabolism of neuronal amines and steroids and in the regulation of the central nervous system.
RESUMO
The participation of cytochrome P-450 (CYP) isoforms in the metabolism of selegiline was investigated. Experiments using recombinant CYP isoforms expressed in human lymphoblastoid cells showed CYP2B6 to be the major CYP isoform involved with the metabolism of selegiline. CYP1A2 and CYP3A4 also contributed to the metabolism of selegiline but their catalytic activities were much less than that of CYP2B6. CYP2B6 had a higher affinity for both N-depropagylation (K(m)=21.4 microM) and N-demethylation (K(m)=25.2 microM) of selegiline than CYP3A4 and CYP1A2. In immunoinhibition studies using mixed human hepatic microsomes, selegiline N-depropagylation activity was most strongly inhibited by anti-CYP2B and anti-CYP3A antibodies, while selegiline N-demethylation activity was most inhibited by anti-CYP2B antibody. In CYP2B6-rich human hepatic microsomes, anti-CYP2B antibody had the strongest inhibitory effects on both activities. Selegiline inhibited CYP2B6-mediated (S)-mephenytoin N-demethylation activity and CYP2C19-mediated (S)-mephenytoin 4'-hydroxylation activity. These findings suggest that attention should be paid to the drug-drug interaction associated with CYP2B6 and CYP2C19. In conclusion, CYP2B6 participates in the metabolism of selegiline but the degree of its contribution varies with the level of its expression in human liver.
RESUMO
Nine different antibodies against P450 isoforms were prepared using purified cytochrome P450s (P450) expressed in E. coli. Purified isozymes were injected into rabbits to raise specific antibody. The resulting antibodies were characterized for their specificity and sensitivity through each particular P450 enzyme-mediated probe reaction.Anti-CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2D6, CYP2E1, and CYP3A4 antibodies proved to be strong immunoinhibitors with inhibitory effects specific to their corresponding antigen. Antiserum derived from the CYP2C19-immunized rabbits was reacted with CYP2C9 as well as CYP2C19 and immunoabsorbed with membrane-bound CYP2C9 expressed in E. coli. Antibody specific for CYP2C19 was obtained. Anti-CYP2C19 together with the anti-CYP2C8 and anti-CYP2C9 can be very useful for determining the contribution of a particular P450 in the metabolism of a drug. The developed inhibitory antibodies will serve as in vitro-specific tools for evaluating the quantitative contribution of individual P450 enzymes to drug metabolism.
RESUMO
We investigated the expressional regulation of cytochrome P450 (CYP) 2D isoforms in rat brain by toluene. Toluene (10 mmol/kg) was injected intraperitoneally into male rats daily for 3 days. CYP2D4 level in the brain was determined by Western blotting. The level of CYP2D4 in the brain and the catalytic activity specific for this isoform were significantly increased by toluene administration. The expressions of CYP2D mRNAs in the brain were quantified by real time PCR. The mRNA level of CYP2D4 increased similar to the expression of the protein and enzymatic activity. These results showed that CYP2D4 was transcriptionally induced by toluene administration for 3 days. This is the first report that subacute exposure to toluene activated the expression of CYP2D4 mRNA in the brain. The response of CYP2D4 expression to toluene is thought to relates to the regulation of the physiological and pharmacological functions of CYP2D isoforms in the brain.
Assuntos
Hidrocarboneto de Aril Hidroxilases/genética , Encéfalo/enzimologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Oxigenases de Função Mista/genética , Tolueno/farmacologia , Animais , Hidrocarboneto de Aril Hidroxilases/análise , Etanolaminas/metabolismo , Masculino , Oxigenases de Função Mista/análise , RNA Mensageiro/análise , Ratos , Ratos Sprague-DawleyRESUMO
Because cholera toxin (CT) is responsible for most of the symptoms induced by Vibrio cholerae infection, detection of CT is critical for diagnosis of the disease. In this study, we constructed an immunochromatographic test strip for detection of CT (CT-IC) with polyclonal antibodies developed against purified recombinant whole CT protein. The detection limit of the CT-IC was 10 ng/mL of purified recombinant CT, and it could detect the CT in culture supernatant of all 15 toxigenic V. cholerae isolates examined, whereas no false-positive signal was detected in all 5 nontoxigenic V. cholerae isolates examined. The specificity of the CT-IC was examined with recombinant heat-labile toxin (LT), which shares high homology with CT, and it was revealed that the minimum detection limit for LT was 100 times higher than that for CT. In addition, lt gene-positive enterotoxigenic Escherichia coli (ETEC) was examined by CT-IC. The false-positive signals were observed in 3 out of 12 ETEC isolates, but these signals were considerably faint. The CT-IC did not develop false-positive signals with all 7 V. parahaemolyticus isolates. These results showed the high specificity of CT-IC and the feasible use of it for the detection and surveillance of toxigenic V. cholerae.
Assuntos
Anticorpos Antibacterianos/química , Toxina da Cólera/análise , Fitas Reagentes/química , Vibrio cholerae , Anticorpos Antibacterianos/imunologia , Toxina da Cólera/química , Toxina da Cólera/imunologia , Cromatografia de Afinidade , Escherichia coli Enterotoxigênica , HumanosRESUMO
An intense effort has been launched to develop improved anthrax vaccines that confer rapid, long lasting protection preferably with an extended stability profile amenable for stockpiling. Protective antigen (PA)-based vaccines are most favored as immune responses directed against PA are singularly protective, although the actual protective mechanism remains to be unraveled. Herein we show that contrary to the prevailing view, an efficacious PA-based vaccine confers protection against inhalation anthrax by preventing the establishment of a toxin-releasing systemic infection. Equally importantly, antibodies measured by the in vitro lethal toxin neutralization activity assay (TNA) that is considered as a reliable correlate of protection, especially for PA protein-based vaccines adjuvanted with aluminum salts appear to be not absolutely essential for this protective immune response.
Assuntos
Vacinas contra Antraz/imunologia , Antraz/imunologia , Antraz/prevenção & controle , Antígenos de Bactérias/imunologia , Toxinas Bacterianas/imunologia , Infecções Respiratórias/imunologia , Infecções Respiratórias/prevenção & controle , Animais , Anticorpos Antibacterianos/sangue , Anticorpos Neutralizantes/sangue , Feminino , Humanos , CoelhosRESUMO
ADP- ribosylation factor (ARF) proteins play a pivotal role in the regulation of membrane traffic and the organization of the cytoskeleton that are crucial to fundamental cellular processes, such as intracellular sorting/trafficking of newly synthesized proteins and endocytosis/exocytosis. In epithelial junctions, the ARF proteins are intimately associated with the dynamics of transmembrane proteins, such as E- cadherin and beta- 1 integrin, and the adaptor proteins such as paxillin. In addition, ARF proteins play a key regulatory role in the remodeling of actin cytoskeleton necessary for the formation of membrane ruffles and protrusions in association with phospholipase D and members of the Rho GTPase family. These activities of ARF proteins influence not only the formation, stability and functional integrity of epithelial junctions but also the cell motility as well. In this review, I have attempted to provide a compendium of evidence that has contributed to our evolving understanding of these ARF proteins as well as their regulators (guanine nucleotide exchange factors and GTPase- activating proteins) in the regulation of epithelial junctions. In addition, I have also highlighted potential mechanisms as to how these intricate regulatory pathways are regulated both spatially and temporally.
Assuntos
Fatores de Ribosilação do ADP/fisiologia , Células Epiteliais/fisiologia , Junções Íntimas/fisiologia , Animais , HumanosRESUMO
The 55-kDa TNFR1 (type I tumor necrosis factor receptor) can be released to the extracellular space by two mechanisms, the proteolytic cleavage and shedding of soluble receptor ectodomains and the release of full-length receptors within exosome-like vesicles. We have shown that the brefeldin A-inhibited guanine nucleotide exchange protein BIG2 associates with TNFR1 and selectively modulates the release of TNFR1 exosome-like vesicles via an ARF1- and ARF3-dependent mechanism. Here, we assessed the role of BIG2 A kinase-anchoring protein (AKAP) domains in the regulation of TNFR1 exosome-like vesicle release from human vascular endothelial cells. We show that 8-bromo-cyclic AMP induced the release of full-length, 55-kDa TNFR1 within exosome-like vesicles via a protein kinase A (PKA)-dependent mechanism. Using RNA interference to decrease specifically the levels of individual PKA regulatory subunits, we demonstrate that RIIbeta modulates both the constitutive and cAMP-induced release of TNFR1 exosome-like vesicles. Consistent with its AKAP function, BIG2 was required for the cAMP-induced PKA-dependent release of TNFR1 exosome-like vesicles via a mechanism that involved the binding of RIIbeta to BIG2 AKAP domains B and C. We conclude that both the constitutive and cAMP-induced release of TNFR1 exosome-like vesicles occur via PKA-dependent pathways that are regulated by the anchoring of RIIbeta to BIG2 via AKAP domains B and C. Thus, BIG2 regulates TNFR1 exosome-like vesicle release by two distinct mechanisms, as a guanine nucleotide exchange protein that activates class I ADP-ribosylation factors and as an AKAP for RIIbeta that localizes PKA signaling within cellular TNFR1 trafficking pathways.