RESUMO
Spatial interactions between cancer and immune cells, as well as the recognition of tumour antigens by cells of the immune system, play a key role in the immune response against solid tumours. The existing mathematical models generally focus only on one of these key aspects. We present here a spatial stochastic individual-based model that explicitly captures antigen expression and recognition. In our model, each cancer cell is characterised by an antigen profile which can change over time due to either epimutations or mutations. The immune response against the cancer cells is initiated by the dendritic cells that recognise the tumour antigens and present them to the cytotoxic T cells. Consequently, T cells become activated against the tumour cells expressing such antigens. Moreover, the differences in movement between inactive and active immune cells are explicitly taken into account by the model. Computational simulations of our model clarify the conditions for the emergence of tumour clearance, dormancy or escape, and allow us to assess the impact of antigenic heterogeneity of cancer cells on the efficacy of immune action. Ultimately, our results highlight the complex interplay between spatial interactions and adaptive mechanisms that underpins the immune response against solid tumours, and suggest how this may be exploited to further develop cancer immunotherapies.
Assuntos
Antígenos de Neoplasias/imunologia , Modelos Biológicos , Neoplasias/imunologia , Linhagem Celular Tumoral , Simulação por Computador , Humanos , Mutação/genética , Probabilidade , Receptores de Antígenos de Linfócitos T/metabolismo , Processos EstocásticosRESUMO
Increasing evidence supports the hypothesis that TGFb1 signalling may be mediated by high temperature requirement A1 (HtrA1) serine protease, acting on important regulatory mechanisms such as cell proliferation and mobility. Evidence is now accumulating to suggest that HtrA1 is involved in the development and progression of several pathologies. The aim of this study was to evaluate: i) if HtrA1 and TGFb1 expressions differ in eutopic and ectopic endometrium in women with endometriosis; ii) if HtrA1 correlates to TGFb1, pSmad and Ki67. This study was carried out including 10 women with ovarian endometriosis (cases) and 10 women with non endometriotic diseases (controls). Endometrial tissue underwent immunohistochemical H-score analysis for HtrA1, TGFb1, pSmad and Ki67 molecules. Data evaluation was performed by a nonparametric Kruskal-Wallis test and Spearman correlation was applied to evaluate the relationship among the molecules investigated in the epithelial and in the stromal compartment. The HtrA1 was significant decreased in ectopic and eutopic endometrium of women with endometriosis when compared with control endometrium in epithelial compartment. TGFb1was significantly increased in eutopic endometrium and decreased in ectopic endometrium in epithelial and stromal compartment. In addition, Ki67 was significant increased and an increase, but not significant, was detected for pSMAd2 in eutopic and ectopic endometrium compared to control one. In summary, the significant direct correlation between TGFb1 and pSmad2 as well as between HtrA1 and TGFb1 and the very significant increase of Ki67 in stromal compartment of eutopic endometrium suggest a possible involvement of HtrA1 in the pathogenesis of endometriosis.
Assuntos
Endometriose , Endométrio , Antígeno Ki-67/metabolismo , Serina Endopeptidases/metabolismo , Proteína Smad2/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Adulto , Endometriose/metabolismo , Endometriose/patologia , Endométrio/metabolismo , Endométrio/patologia , Feminino , Serina Peptidase 1 de Requerimento de Alta Temperatura A , HumanosRESUMO
INTRODUCTION: Chorioamnionitis is a gestational pathological condition characterized by acute inflammation of the amniochorionic membranes and placentas leading to high concentrations of IL-1ß, Il-6, Il-8 and TGF-ß in the amniotic fluid. In normal conditions, the permeability of foeto-maternal barrier is due to the assembly and maintenance of different cellular junctional domains. METHODS: In the present study, first we aimed to evaluate the protein expression (by immunohistochemistry and western blotting) and mRNA (by real time PCR) levels of the molecular components of tight junctions (Zonula occludens-1 and occludin), and of adherent junctions (VE-cadherin and ß-catenin) in placentas from chorioamnionitis compared to that in normal pregnancies. RESULTS: Western blotting results showed a significant down-regulation of occludin in placentas affected with chorioamnionitis. No differences were detected for the other proteins analysed. We evaluated whether occludin expression was regulated by IL-1ß, IL-6, IL-8 and TGF-ß by means of in vitro studies using HUVEC cultures and demonstrated a key role of IL-1ß and TGF-ß in the disappearance of occludin at cellular border. CONCLUSIONS: We conclude by suggesting a pivotal role of these two cytokines in facilitating intra-placental infection via para-cellular way due to the disassembly of tight junctions at trophoblastic and endothelial cells in placental tissues.
Assuntos
Corioamnionite/fisiopatologia , Interleucina-1beta/fisiologia , Placenta/fisiologia , Junções Íntimas/fisiologia , Fator de Crescimento Transformador beta/fisiologia , Antígenos CD/genética , Antígenos CD/metabolismo , Caderinas/genética , Caderinas/metabolismo , Estudos de Casos e Controles , Permeabilidade da Membrana Celular , Corioamnionite/genética , Corioamnionite/patologia , Citocinas/metabolismo , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Imuno-Histoquímica , Troca Materno-Fetal , Ocludina/genética , Ocludina/metabolismo , Placenta/fisiopatologia , Gravidez , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Junções Íntimas/patologia , Proteína da Zônula de Oclusão-1/genética , Proteína da Zônula de Oclusão-1/metabolismo , beta Catenina/genética , beta Catenina/metabolismoRESUMO
We evaluated the presence of HtrA1 in maternal plasma of normal pregnancies and of pregnancies complicated by preeclampsia (PE) without and with Intrauterine Growth Restriction (IUGR). We demonstrate that HtrA1 maternal plasma levels show significant different concentrations in first, second and third trimester of gestation and that HtrA1 concentration increases in maternal plasma of gestations complicated by PE with IUGR compared with control maternal plasma matched for gestational age. Based on these data high maternal plasma levels of HtrA1 could be considered as a possible marker of an occurring IUGR in preeclamptic women.
Assuntos
Retardo do Crescimento Fetal/sangue , Pré-Eclâmpsia/sangue , Serina Endopeptidases/sangue , Adulto , Biomarcadores/sangue , Diagnóstico Precoce , Ensaio de Imunoadsorção Enzimática , Feminino , Retardo do Crescimento Fetal/diagnóstico , Idade Gestacional , Serina Peptidase 1 de Requerimento de Alta Temperatura A , Humanos , Gravidez , Adulto JovemRESUMO
Preeclampsia (PE) is a serious disorder of human pregnancy, it is often associated with fetal growth restriction (FGR) which is a failure of the fetus to reach its own growth potential. Activator protein-1 (AP-1) is a family of transcription factors inducible in response to a variety of extracellular stimuli and functions. AP-1 plays a complex role in the regulation of different fundamental cellular processes, including cell proliferation, survival, death and transformation. We investigate the expression pattern of AP-1 transcription factors in normal placentas during gestation and in placentas from PE without and with FGR using semiquantitative RT-PCR and immunohistochemistry techniques. The most interesting data concern the alterations of protein expression patterns of c-fos, Jun D and c-jun in normal gestation as well as in PE and PE-FGR pathologies. In addition, alterations but not significant changes are detected in mRNA expressions for these transcription factors. We strongly suggest that c-fos is implicated in regulating invasiveness mechanism of extravillous trophoblast in normal gestation as well as in PE placentas. In addition, we suggest that the opposite modulation of Jun D and c-jun in PE and PE-FGR supports the recent hypothesis that PE and PE-FGR could be considered two pathologies with different origin (maternal and placental) each of which has a different molecular pattern of expression.
Assuntos
Retardo do Crescimento Fetal/metabolismo , Placenta/metabolismo , Pré-Eclâmpsia/metabolismo , Fator de Transcrição AP-1/metabolismo , Adulto , Feminino , Retardo do Crescimento Fetal/genética , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Recém-Nascido , Pré-Eclâmpsia/genética , Gravidez , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Proto-Oncogênicas c-jun/metabolismo , RNA/química , RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estatísticas não Paramétricas , Fator de Transcrição AP-1/genética , Adulto JovemRESUMO
BACKGROUND: Preliminary data has shown temperament differences in workers of a few professions, particularly in artists. METHODS: 3805 subjects (75.5% female, mean 32.4+/-9.8 years) of 23 broad professional areas answered a web-survey with the Combined Emotional and Affective Temperament Scale (CEATS). RESULTS: Educational level was correlated with drive and control, was lower in depressives and apathetics and higher in euthymics and hyperthymics. Fear was lower in administration and communications and higher in computing and office workers. Drive was lower in those unemployed and at home and higher in fitness and administration. Control was lower in arts and higher in teaching and health caring. Anger was lower in subjects in the areas of teaching and health caring and higher in human studies and unemployed. For affective temperament scores: depressive was lower in fitness and higher in human studies; anxious and apathetic scores were lower in fitness and higher in unemployed subjects; cyclothymic was lower in health caring and higher in unemployed; euthymic score was lower in human studies and higher in fitness; irritable was lower in religion and higher in unemployed; labile was lower in health caring and higher in unemployed; disinhibited was lower in engineering and higher in communications and arts; hyperthymic was lower in human studies and high in fitness. LIMITATION: Convenience sample by the internet and most subjects assessed the instruments through a psychoeducational website for bipolar spectrum disorders, which may have biased the absolute scores of emotional temperaments. CONCLUSIONS: Professional areas and educational level are associated with distinct emotional and affective profiles.
Assuntos
Afeto , Emoções , Ocupações/estatística & dados numéricos , Temperamento , Adolescente , Adulto , Depressão/psicologia , Impulso (Psicologia) , Escolaridade , Medo , Feminino , Humanos , Humor Irritável , Masculino , Pessoa de Meia-Idade , Personalidade , Testes de Personalidade , Adulto JovemRESUMO
The syndecan family consists of four distinct membrane glycoproteins in mammals. Syndecans control cell proliferation, differentiation, adhesion and migration through participation in cell-cell interactions, anchorage of cells to the extracellular environment, and modulation of multiple growth factors. Therefore, syndecans may play a pivotal role in the regulation of cell behaviour depending on the cellular microenvironment. Here, we demonstrate that syndecan-1, syndecan-2 and syndecan-4 are expressed in fetal membrane tissue with different immunolocalizations. Syndecan-1 is expressed in the amniotic epithelium, localizing at basolateral cell surfaces. Syndecan-2 and syndecan-4, in contrast, are mostly localized in intracellular compartments, in the extravillous cytotrophoblastic cells and in some fibroblasts of the chorionic plate as well as in the amniotic epithelial cells. In the latter, syndecan-4 is mainly localized in the apical part of the cells. Our results strongly suggest a key role of syndecan-1, syndecan-2 and syndecan-4 in the determination of structural and functional characteristics of human amnion and chorionic plate. Since the solute exchanges between fetus and mother take place in fetal membranes, our data suggest that syndecans are important players in the placenta for the establishment of the fetal-maternal inter-communication.
Assuntos
Âmnio/metabolismo , Córion/metabolismo , Placenta/metabolismo , Sindecana-1/metabolismo , Sindecana-2/metabolismo , Sindecana-4/metabolismo , Âmnio/citologia , Córion/citologia , Feminino , Fibroblastos/metabolismo , Humanos , Técnicas Imunoenzimáticas , Placenta/citologia , GravidezRESUMO
BACKGROUND: Temperament relates to emotions and the prevailing mood or affective temperament. Uric acid (UA) is the end-product of purine metabolism and has been associated with psychological features such as high energy/drive, positive affect, achievement, good performance, higher social status and leadership. METHODS: 129 subjects (44 males, 85 females) completed with the Combined Emotional and Affective Temperaments Scale, serum UA levels and a general health questionnaire. RESULTS: In the whole sample, serum UA levels were significantly correlated with disinhibition (r=0.36, p<0.001) and drive (r=0.25, p<0.01), but not with control, anger or any of the affective temperament scores. Among males, we found correlations at trend level (p>0.05 and <0.07) for control (r=0.27), irritable (r=0.29) and hyperthymic (r=0.27) affective temperaments. Among females, a significant correlation was found only with disinhibition (r=0.34, p=0.001). The top tertile of males (serum UA>6.0 mg/ml, n=16) had significantly higher drive (29.9+/-5.9x26.0+/-3.6, p=0.01) and higher control at trend level (21.2+/-3.1x19.3+/-2.9, p=0.054) than other males. Among women, the top tertile (serum UA>4.0 mg/ml, n=29) showed higher disinhibition scores (20.7+/-4.9x17.9+/-3.6, p<0.01) and more frequent choices of hyperthymic (8/26x6/59, p=0.023) and irritable temperaments (7/26x5/59, p=0.031) than the rest of the sample. Controlling for daily intake of meat and grains, which could lead to higher UA levels, did not change these results. LIMITATIONS: Small sample size for males. CONCLUSIONS: Externalized traits of temperament are associated with higher serum UA levels both in men and women.
Assuntos
Afeto/fisiologia , Emoções/fisiologia , Temperamento/fisiologia , Ácido Úrico/sangue , Adulto , Ira/fisiologia , Nível de Alerta/fisiologia , Brasil , Impulso (Psicologia) , Feminino , Humanos , Inibição Psicológica , Controle Interno-Externo , Masculino , Pessoa de Meia-Idade , Inventário de Personalidade/estatística & dados numéricos , Psicometria , Valores de Referência , Fatores SexuaisRESUMO
Basic fibroblast growth factor (bFGF) is a heparin-binding cationic protein involved in a variety of pathological conditions including angiogenesis and solid tumour growth. The basic fibroblast growth factor receptor (FGFR) family comprises at least 4 high affinity tyrosine kinase receptors that require syndecans for their function. Mounting evidence indicates that syndecans, that bind both bFGF and their FGFRs, will act as stimulators, whereas syndecans that only bind bFGF will act as inhibitors of signaling by sequestering the growth factor. Recent findings have highlighted the importance of syndecans in urological cancers. The aim of this study is to investigate the expression of bFGF, its receptors (R1 and R2) and syndecans (1-4) in invasive urothelial carcinoma and normal-looking urothelium by Western blotting, RT-PCR, and immunohistochemistry analyses. Interestingly, bFGF, FGFR1 and FGFR2 protein levels statistically increased in bladder cancer tissues. mRNA of FGFR1 and syndecans (1-4), showed a statistically significant increase while an mRNA increase in the other molecules analysed was not significant. bFGF, its receptors and syndecan immunostaining were mainly present in the urothelium both in normal-looking tissues and urothelial neoplastic cells. In conclusion, our data report that the bFGF, FGFR and syndecan expressions are altered in bladder tumours.
Assuntos
Carcinoma/química , Fator 2 de Crescimento de Fibroblastos/análise , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/análise , Sindecanas/análise , Neoplasias da Bexiga Urinária/química , Idoso , Western Blotting , Carcinoma/genética , Carcinoma/patologia , Carcinoma/cirurgia , Cistectomia , Feminino , Fator 2 de Crescimento de Fibroblastos/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , RNA Mensageiro/análise , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/análise , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sindecana-1/análise , Sindecana-2/análise , Sindecana-3/análise , Sindecana-4/análise , Sindecanas/genética , Regulação para Cima , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/cirurgia , Urotélio/químicaRESUMO
Preeclampsia (PE) and intrauterine growth restriction (IUGR) are pregnancy-specific disorders that have in common abnormal placental implantation, a marked proliferation of villous cytotrophoblastic cells and focal necrosis of the syncytiotrophoblast. Several studies show an ischemic placenta with a high-resistance vasculature, which cannot deliver an adequate blood supply to the feto-placental unit. The cause of PE is a matter of debate, but recently studies in mice suggest that the primary feto-placental lesions are sufficient to initiate the disease. HtrA1, a member of the family of HtrA proteins, is a secreted multidomain protein with serine protease activity. It is expressed in first and third trimester of gestation. In specimens from the first trimester of gestation, immunostaining for HtrA1 is generally found in both layers of villous trophoblast, syncytiotrophoblast and cytotrophoblast. Cytoplasm of extravillous trophoblast and extracellular matrix of cell islands and cell columns are labeled for HtrA1. Specimens from third trimester of gestation show a more intense positivity for HtrA1 in the syncytiotrophoblast than in cytotrophoblast. The extravillous trophoblast and the decidual cells, is positive for HtrA1. The purpose of this study is to investigate the expression pattern of HtrA1 in placentas from PE without IUGR (maternal PE) and with IUGR (fetal PE) by quantitative western blotting and immunohistochemistry. By quantitative western blotting analysis we observed a significant upregulation of approximately 30 kDa HtrA1 form in PE. Differently, we detected a significant total HtrA1 down-regulation in PE-IUGR. Moreover, immunostaining for HtrA1 was positive in the villous trophoblast, in the syncytial knots and irregularly in the fetal vessel walls in PE placentas while immunostaining for HtrA1was present particularly in the syncytial knots in PE-IUGR placentas. In conclusion, we suggest that the approximately 30 kDa HtrA1 form can be correlated to maternal PE while that the significant down-regulation of total HtrA1 can be correlated to placental PE. These HtrA1 alterations could be considered as possible markers to discriminate placental PE from maternal PE.
Assuntos
Retardo do Crescimento Fetal/metabolismo , Pré-Eclâmpsia/metabolismo , Serina Endopeptidases/metabolismo , Trofoblastos/metabolismo , Biomarcadores/metabolismo , Western Blotting , Regulação para Baixo , Feminino , Retardo do Crescimento Fetal/patologia , Técnica Direta de Fluorescência para Anticorpo , Técnica Indireta de Fluorescência para Anticorpo , Serina Peptidase 1 de Requerimento de Alta Temperatura A , Humanos , Técnicas Imunoenzimáticas , Pré-Eclâmpsia/patologia , Gravidez , Isoformas de Proteínas , Trofoblastos/patologiaRESUMO
There is evidence that alpha-smooth muscle actin (alpha-SMA) is a protein that plays a pivotal role in the production of contractile forces and it is induced by transforming growth factor-beta1 (TGF-beta1). We have analysed the expression of alpha-SMA, TGF-beta1, its receptor RI and the activator phospho-Smad2 in (a) fetal growth restriction pre-eclamptic placentae characterised by early onset and absence of end diastolic velocities in the umbilical arteries (FGR-AED) and (b) control placentae accurately matched for gestational age. The study was performed by immunohistochemical, quantitative Western blotting, ELISA, RT-PCR and in vitro analyses. We found that TGF-beta1 stimulates alpha-SMA production in chorionic villi cultured in vitro. In addition, we observed that in vivo TGF-beta1 concentration is significantly higher in FGR-AED placental samples than in control placentae and that this growth factor could have a paracrine action on villous stroma myofibroblasts expressing TGF-beta1 receptors and phospho-Smad2. Indeed, we report that alpha-SMA undergoes a redistribution in FGR-AED placental villous tree, i.e. we show that alpha-SMA is enhanced in medium and small stem villi and significantly decreased in the peripheral villi. Our data allow us to consider TGF-beta1 and alpha-SMA as key molecules related to FGR-AED placental villous tree phenotypic changes responsible for increased impedance to blood flow observable in this pathology.
Assuntos
Actinas/metabolismo , Retardo do Crescimento Fetal/fisiopatologia , Placenta/fisiopatologia , Pré-Eclâmpsia/fisiopatologia , Complicações na Gravidez , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Adulto , Feminino , Feto , Regulação da Expressão Gênica , Humanos , Placenta/irrigação sanguínea , Gravidez , Proteínas Serina-Treonina Quinases/genética , RNA Mensageiro/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptores de Fatores de Crescimento Transformadores beta/genética , Transdução de Sinais , Fator de Crescimento Transformador beta1/genéticaRESUMO
Two dinucleoside polyphosphate NAD analogs, P1-(adenosine-5')-P3-(nicotinamide riboside-5')triphosphate (Np3A, 1) and P1-(adenosine-5')-P4-(nicotinamide riboside-5')tetraphosphate (Np4A, 2), were synthesized and tested as inhibitors of both microbial and human recombinant NMN adenylyltransferase. Compounds 1 and 2 proved to be selective inhibitors of microbial enzymes.
Assuntos
Inibidores Enzimáticos/síntese química , NAD/análogos & derivados , NAD/síntese química , Nicotinamida-Nucleotídeo Adenililtransferase/antagonistas & inibidores , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , NAD/química , NAD/farmacologia , Proteínas Recombinantes/antagonistas & inibidoresAssuntos
Nicotinamida-Nucleotídeo Adenililtransferase/isolamento & purificação , Nicotinamida-Nucleotídeo Adenililtransferase/metabolismo , Sulfolobus/enzimologia , Cromatografia/métodos , Cromatografia de Afinidade/métodos , Cromatografia por Troca Iônica/métodos , Ditiotreitol/farmacologia , Durapatita , Eletroforese em Gel de Poliacrilamida/métodos , Estabilidade Enzimática , Temperatura Alta , Cinética , Peso Molecular , Nicotinamida-Nucleotídeo Adenililtransferase/química , Concentração Osmolar , TermodinâmicaAssuntos
Mathanococcus/enzimologia , Nicotinamida-Nucleotídeo Adenililtransferase/química , Nicotinamida-Nucleotídeo Adenililtransferase/genética , Sequência de Aminoácidos , Cromatografia/métodos , Durapatita , Estabilidade Enzimática , Temperatura Alta , Cinética , Mathanococcus/genética , Mathanococcus/crescimento & desenvolvimento , Dados de Sequência Molecular , Peso Molecular , Nicotinamida-Nucleotídeo Adenililtransferase/isolamento & purificação , Nicotinamida-Nucleotídeo Adenililtransferase/metabolismo , Fases de Leitura Aberta , Subunidades Proteicas , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Sulfolobus/enzimologia , Sulfolobus/genética , TermodinâmicaRESUMO
BACKGROUND: Nicotinamide adenine dinucleotide (NAD(+)) is an essential cofactor involved in fundamental processes in cell metabolism. The enzyme nicotinamide mononucleotide adenylyltransferase (NMN AT) plays a key role in NAD(+) biosynthesis, catalysing the condensation of nicotinamide mononucleotide and ATP, and yielding NAD(+) and pyrophosphate. Given its vital role in cell life, the enzyme represents a possible target for the development of new antibacterial agents. RESULTS: The structure of NMN AT from Methanococcus jannaschii in complex with ATP has been solved by X-ray crystallography at 2.0 A resolution, using a combination of single isomorphous replacement and density modification techniques. The structure reveals a hexamer with 32 point group symmetry composed of alpha/beta topology subunits. The catalytic site is located in a deep cleft on the surface of each subunit, where one ATP molecule and one Mg(2+) are observed. A strictly conserved HXGH motif (in single-letter amino acid code) is involved in ATP binding and recognition. CONCLUSIONS: The structure of NMN AT closely resembles that of phosphopantetheine adenylyltransferase. Remarkably, in spite of the fact that the two enzymes share the same fold and hexameric assembly, a striking difference in their quaternary structure is observed. Moreover, on the basis of structural similarity including the HXGH motif, we identify NMN AT as a novel member of the newly proposed superfamily of nucleotidyltransferase alpha/beta phosphodiesterases. Our structural data suggest that the catalytic mechanism does not rely on the direct involvement of any protein residues and is likely to be carried out through optimal positioning of substrates and transition-state stabilisation, as is proposed for other members of the nucleotidyltransferase alpha/beta phosphodiesterase superfamily.
Assuntos
Mathanococcus/enzimologia , Nicotinamida-Nucleotídeo Adenililtransferase/química , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , NAD/biossíntese , Nicotinamida-Nucleotídeo Adenililtransferase/metabolismo , Conformação Proteica , Estrutura Secundária de Proteína , Subunidades Proteicas , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
The first identification and characterization of a catalytic activity associated with NadR protein is reported. A computer-aided search for sequence similarity revealed the presence in NadR of a 29-residue region highly conserved among known nicotinamide mononucleotide adenylyltransferases. The Escherichia coli nadR gene was cloned into a T7-based vector and overexpressed. In addition to functionally specific DNA binding properties, the homogeneous recombinant protein catalyzes NAD synthesis from nicotinamide mononucleotide and ATP.
Assuntos
Proteínas de Bactérias , Nicotinamida-Nucleotídeo Adenililtransferase/metabolismo , Proteínas Repressoras/metabolismo , Sequência de Aminoácidos , Clonagem Molecular , DNA/metabolismo , Escherichia coli , Expressão Gênica , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/isolamento & purificaçãoRESUMO
Increasing evidence on the importance of fluctuations in NAD+ levels in the living cell is accumulating. Therefore a deeper knowledge on the regulation of coenzyme synthesis and recycling is required. In this context the study of NMN adenylyltransferase (EC 2.7.7.1),. a key enzyme in the NAD+ biosynthetic pathway, assumes a remarkable relevance. We have previously purified to homogeneity and characterized the protein from the thermophilic archaeon Sulfolobus solfataricus. The determination of partial sequence of the S. solfataricus enzyme, together with the recent availability of the genome sequence of the archaeon Methanococcus jannaschii, allowed us, based on sequence similarity, to identify the M. jannaschii NMN adenylyltransferase gene. As far as we know from literature, this is the first report on the NMN adenylyltransferase gene.
Assuntos
Nicotinamida-Nucleotídeo Adenililtransferase/genética , Sulfolobus/enzimologia , Sulfolobus/genética , Sequência de Aminoácidos , Eletroforese em Gel de Poliacrilamida , Dados de Sequência Molecular , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
Synechocystis sp. slr0787 open reading frame encodes a 339 residue polypeptide with a predicted molecular mass of 38.5 kDa. Its deduced amino acid sequence shows extensive homology with known separate sequences of proteins from the thermophilic archaeon Methanococcus jannaschii. The N-terminal domain is highly homologous to the archaeal NMN adenylyltransferase, which catalyzes NAD synthesis from NMN and ATP. The C-terminal domain shares homology with the archaeal ADP-ribose pyrophosphatase, a member of the 'Nudix' hydrolase family. The slr0787 gene has been cloned into a T7-based vector for expression in Escherichia coli cells. The recombinant protein has been purified to homogeneity and demonstrated to possess both NMN adenylyltransferase and ADP-ribose pyrophosphatase activities. Both activities have been characterized and compared to their archaeal counterparts.
Assuntos
Proteínas de Bactérias/genética , Cianobactérias/enzimologia , Nicotinamida-Nucleotídeo Adenililtransferase/genética , Pirofosfatases/genética , Sequência de Aminoácidos , Proteínas de Bactérias/química , Clonagem Molecular , Estabilidade Enzimática , Cinética , Magnésio/farmacologia , Manganês/farmacologia , Mathanococcus/enzimologia , Dados de Sequência Molecular , Nicotinamida-Nucleotídeo Adenililtransferase/química , Pirofosfatases/química , Proteínas Recombinantes/genética , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , TemperaturaRESUMO
The enzyme nicotinamide mononucleotide (NMN) adenylyltransferase (EC 2.7.7.1) catalyzes the synthesis of NAD+ and nicotinic acid adenine dinucleotide. It has been purified to homogeneity from cellular extracts of the thermophilic archaeon Sulfolobus solfataricus. Through a database search, a highly significant match was found between its N-terminal sequence and a hypothetical protein coded by the thermophilic archaeon Methanococcus jannaschii MJ0541 open reading frame (GenBank accession no. U67503). The MJ0541 gene was isolated, cloned into a T7-based vector, and expressed in Escherichia coli cells, yielding a high level of thermophilic NMN adenylyltransferase activity. The expressed protein was purified to homogeneity by a single-step chromatographic procedure. Both the subunit molecular mass and the N-terminal sequence of the pure recombinant protein were as expected from the deduced amino acid sequence of the MJ0541 open reading frame-encoded protein. Molecular and kinetic properties of the enzymes from both archaea are reported and compared with those already known for the mesophilic eukaryotic NMN adenylyltransferase.
Assuntos
Proteínas Arqueais/isolamento & purificação , Mathanococcus/enzimologia , Nicotinamida-Nucleotídeo Adenililtransferase/isolamento & purificação , Sulfolobus/enzimologia , Sequência de Aminoácidos , Aminoácidos/análise , Proteínas Arqueais/genética , Clonagem Molecular , Estabilidade Enzimática , Escherichia coli/genética , Temperatura Alta , Mathanococcus/genética , Dados de Sequência Molecular , Nicotinamida-Nucleotídeo Adenililtransferase/genética , Proteínas Recombinantes/isolamento & purificação , Análise de Sequência , Homologia de Sequência de AminoácidosRESUMO
Nineteen Brazilian HIV-infected hemophiliacs and their stable heterosexual sexual partners were studied with the aim of assessing the rate of HIV transmission in this at risk group. The mean length of relationship between couples was 7.4 years. The hemophiliac men were Class II (n = 6), III (n = 11) and IVa (n = 2) of the CDC classification. They had decreased CD4+ and elevated CD8+ cell numbers; five had p24 antigenemia. We found 3 HIV-infected women (15.8 percent) by routine and confirmatory tests, a prevalence similar to that seen in other countries. They were asymptomatic and had no detectable p24 antigenemia. The 3 seropositive women's partners were Class II and III-CDC, and had normal CD4+ and CD8+ values and no p24 antigenemia. All seronegative women also had normal CD4+ and CD8+ numbers, except for elevated CD8+ cells in three of them, but immune abnormalities had already been seen in some seronegative partners at high risk for HIV infection. Our results reinforce previous suggestions that heterosexual transmission to stable female partners occurs preferentially early after initiation of sexual exposure, and possibly when the transmitter had high levels of viremia and regular sexual activity.
PIP: In Brazil, hemophiliacs who received coagulation factor concentrates during 1980-85 have rates of HIV exceeding 50% and rates of heterosexual transmission to their partners in the range of 10-20%. This study investigated the clinical course of HIV infection in 19 male patients from a hematology center in Sao Paulo, Brazil, with hemophilia A or B and their stable, asymptomatic female sexual partners. The mean duration of the relationship was 7.4 years. Compared with 15 normal adult subjects used as controls, CD8+ cell counts of hemophiliacs were significantly higher while CD4+ cell values were significantly reduced. Three sexual partners (15.8%) were HIV-positive, implying a transmission rate of 2.1 per 100 person-years. All female partners were in Centers for Disease Control Class II. Their male partners were in Classes II and III and had normal CD4+ and CD8+ levels. Neither males nor females had p24 antigenemia. Fragments of HIV particles were present in several HIV-negative female partners. These findings suggests early HIV transmission, when the transmitter has high levels of viremia, to stable female partners of hemophiliacs.