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1.
Toxicology ; 290(2-3): 178-86, 2011 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-21946100

RESUMO

Long-term exposure to pharmacological agents can select for cells that overexpress efflux transporters. We previously showed that mouse J774 macrophages cultivated for a prolonged period of time with toxic concentrations of the fluoroquinolone ciprofloxacin overexpress the efflux transporter Mrp4 and display a reduced accumulation of this antibiotic, but no change in the accumulation of moxifloxacin, a closely related molecule (Antimicrob. Agents Chemother. [2006] 50, 1689-1695 and [2009] 53, 2410-2416). Because of this striking difference between the two fluoroquinolones, we have now examined the modifications in the expression of ABC efflux transporters induced by the prolonged exposure of J774 macrophages to high concentrations of moxifloxacin. The resulting cell line showed (i) no difference in the accumulation of moxifloxacin but an increased accumulation and decreased efflux of ciprofloxacin; (ii) an overexpression of the multidrug transporters Abcb1a (P-gp), Abcc2 (Mrp2) and Abcg2 (Bcrp1), and a decreased expression of Abcc4 (Mrp4). While P-gp and Bcrp1 were functional, they did not modify the cellular accumulation of fluoroquinolones. The data show that exposing cells to high concentrations of a drug that is not affected by active efflux can trigger a pleiotropic response leading to a modulation in the expression of several transporters. These changes, however, are not sufficient to protect cells against the toxicity that fluoroquinolones may exert at large concentrations. They could also cause unanticipated drug interactions in vivo, should the drug exposure grossly exceed what is anticipated from its current registered use.


Assuntos
Transportadores de Cassetes de Ligação de ATP/efeitos dos fármacos , Anti-Infecciosos/toxicidade , Compostos Aza/toxicidade , Ciprofloxacina/toxicidade , Macrófagos/efeitos dos fármacos , Quinolinas/toxicidade , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Anti-Infecciosos/administração & dosagem , Anti-Infecciosos/farmacocinética , Compostos Aza/administração & dosagem , Compostos Aza/farmacocinética , Linhagem Celular , Ciprofloxacina/administração & dosagem , Ciprofloxacina/farmacocinética , Relação Dose-Resposta a Droga , Fluoroquinolonas , Regulação da Expressão Gênica/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Moxifloxacina , Quinolinas/administração & dosagem , Quinolinas/farmacocinética
2.
Haematologica ; 96(9): 1293-301, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21606172

RESUMO

BACKGROUND: A major issue in the treatment of acute myeloid leukemia is resistance to chemotherapeutic drugs. An increasing number of ATP-Binding-Cassette transporters have been demonstrated to cause resistance to cancer drugs. The aim of this study was to highlight the putative role of other ATP-Binding-Cassette transporters in primary chemoresistant acute myeloid leukemia. DESIGN AND METHODS: In the first part of this study, using taqman custom arrays, we analyzed the relative expression levels of 49 ATP-Binding-Cassette genes in 51 patients divided into two extreme cohorts, one very sensitive and one very resistant to chemotherapy. In the second part of this study, we evaluated the prognostic impact, in a cohort of 281 patients, of ATP-Binding-Cassette genes selected in the first part of the study. RESULTS: In the first part of the study, six genes (ATP-Binding-CassetteA2, ATP-Binding-CassetteB1, ATP-Binding-CassetteB6, ATP-Binding-CassettC13, ATP-Binding-CassetteG1, and ATP-Binding-CassetteG2) were significantly over-expressed in the resistant group compared with the sensitive group. In the second cohort, overexpression of 5 of these 6 ATP-Binding-Cassette genes was correlated with outcome in univariate analysis, and only the well-known ATP-Binding-CassetteB1 and G2, and the new ATP-Binding-CassetteG1 in multivariate analysis. Prognosis decreased remarkably with the number of these over-expressed ABC genes. Complete remission was achieved in 71%, 59%, 54%, and 0%, (P=0.0011) and resistance disease in 21%, 37%, 43%, and 100% (P<0.0001) of patients over-expressing 0, 1, 2, or 3, ABC genes, respectively. The number of ATP-Binding-Cassette genes expressed, among ATP-Binding-CassetteB1, G1, and G2, was the strongest prognostic factor correlated, in multivariate analysis, with achievement of complete remission (P=0.01), resistant disease (P=0.01), and overall survival (P=0.02). CONCLUSIONS: Using expression profiling, we have emphasized the diversity of ATP-Binding-Cassette transporters that cooperate to promote chemoresistance rather than overexpression of single transporters and the putative role of new ATP-Binding-Cassette tranporters, such as ATP-Binding-CassetteG1. Modulation of these multiple transporters might be required to eradicate leukemic cells.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Resistencia a Medicamentos Antineoplásicos/genética , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Análise por Conglomerados , Estudos de Coortes , Feminino , Perfilação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica , Humanos , Leucemia Mieloide Aguda/diagnóstico , Masculino , Pessoa de Meia-Idade , Avaliação de Resultados em Cuidados de Saúde , Prognóstico , Adulto Jovem
3.
J Gen Virol ; 90(Pt 5): 1294-1301, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19264641

RESUMO

At present, there is no effective therapy for any of the neurodegenerative amyloidoses, despite renewed efforts to identify compounds active against the various implicated pathogenetic molecules. We have screened a library of 2960 natural and synthetic compounds in two cell lines chronically infected with mouse prions, and have identified eight new inhibitors of prion replication in vitro. They belong to two distinct chemical families that have not previously been recognised as effective in the field of transmissible spongiform encephalopathies: seven are 3-aminosteroids and one is a derivative of erythromycin A with an oxime functionality. Our results suggest that these aminosteroids inhibit prion replication by triggering a common target, possibly implicated in the regulatory pathways of cellular prion protein metabolism. Furthermore, using a quantitative approach for the study of protein stability, it was shown that the erythromycin A derivative altered prion protein stability by direct interaction. Such direct targeting of this amyloid precursor might provide new clues for the understanding of prion diseases and, more importantly, help to define new molecules that are active against prion diseases.


Assuntos
Precursor de Proteína beta-Amiloide/antagonistas & inibidores , Príons/antagonistas & inibidores , Animais , Bioensaio/métodos , Linhagem Celular , Camundongos , Príons/efeitos dos fármacos , Príons/fisiologia , Bibliotecas de Moléculas Pequenas
4.
J Org Chem ; 72(17): 6421-6, 2007 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-17655358

RESUMO

In this paper we report the rearrangement of spirocyclohexadienones into dihydrotropones in basic conditions as a new method for the preparation of seven-membered ring ketones, which are key building blocks for the synthesis of tropoloalkaloids. DFT calculations and deuterium labeling studies support the mechanism we propose for this rearrangement, involving the ring opening of a spirocyclopropane intermediate followed by successive base-catalyzed 1,3-hydrogen shifts. The X-ray structure of the resulting dihydrotropone shows near-perfect planarity and the conjugation gain is likely to be the driving force of the reaction.


Assuntos
Hexanos/química , Tropolona/análogos & derivados , Ciclização , Hidrogênio/química , Espectroscopia de Ressonância Magnética , Tropolona/química , Difração de Raios X
5.
Eur Biophys J ; 37(1): 95-104, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17522855

RESUMO

Most proteins comprise several domains and/or participate in functional complexes. Owing to ongoing structural genomic projects, it is likely that it will soon be possible to predict, with reasonable accuracy, the conserved regions of most structural domains. Under these circumstances, it will be important to have methods, based on simple-to-acquire experimental data, that allow to build and refine structures of multi-domain proteins or of protein complexes from homology models of the individual domains/proteins. It has been recently shown that small angle X-ray scattering (SAXS) and NMR residual dipolar coupling (RDC) data can be combined to determine the architecture of such objects when the X-ray structures of the domains are known and can be considered as rigid objects. We developed a simple genetic algorithm to achieve the same goal, but by using homology models of the domains considered as deformable objects. We applied it to two model systems, an S1KH bi-domain of the NusA protein and the gammaS-crystallin protein. Despite its simplicity our algorithm is able to generate good solutions when driven by SAXS and RDC data.


Assuntos
Algoritmos , Espectroscopia de Ressonância Magnética/métodos , Modelos Químicos , Modelos Moleculares , Proteínas/química , Proteínas/ultraestrutura , Análise de Sequência de Proteína/métodos , Difração de Raios X/métodos , Simulação por Computador , Conformação Proteica , Estrutura Terciária de Proteína , Espalhamento a Baixo Ângulo , Alinhamento de Sequência/métodos , Homologia de Sequência de Aminoácidos
6.
Leuk Lymphoma ; 47(9): 1915-20, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17065006

RESUMO

Evidence from clinical and laboratory studies suggests that angiogenesis is important in the progression of solid tumours and hematologic malignancies. We have shown that the naturally occurring tetrapeptide Acetyl-Ser-Asp-Lys-Pro (AcSDKP) is a potent angiogenic factor normally present at nanomolar concentrations in the blood. A murine leukemia model was used to assess whether there was a correlation between levels of endogenous AcSDKP and the development of disease. Levels of AcSDKP in the plasma and bone marrow (BM) cells from mice bearing an acute myeloid leukemia (AML) were five- to ten-fold greater than those in non-leukemic mice. Furthermore, a strong correlation between the concentration of endogenous AcSDKP and the progression of AML was demonstrated. These results are consistent with the marked increase in BM vascularity observed in leukemic mice. The physiologic relevance of these findings awaits further studies and the contribution of AcSDKP to the pathogenesis of leukemia is under investigation.


Assuntos
Medula Óssea/metabolismo , Inibidores do Crescimento/sangue , Leucemia Experimental/sangue , Leucemia Mieloide Aguda/sangue , Neovascularização Patológica/patologia , Oligopeptídeos/sangue , Animais , Medula Óssea/patologia , Progressão da Doença , Inibidores do Crescimento/farmacologia , Técnicas Imunoenzimáticas , Leucemia Experimental/patologia , Leucemia Mieloide Aguda/patologia , Masculino , Camundongos , Camundongos Endogâmicos CBA , Oligopeptídeos/farmacologia , Baço/citologia
7.
J Mol Biol ; 359(4): 940-9, 2006 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-16697012

RESUMO

Human multidrug resistance protein 1 (MRP1) is a membrane protein that belongs to the ATP-binding cassette (ABC) superfamily of transport proteins. MRP1 contributes to chemotherapy failure by exporting a wide range of anti-cancer drugs when over expressed in the plasma membrane of cells. Here, we report the first high-resolution crystal structure of human MRP1-NBD1. Drug efflux requires energy resulting from hydrolysis of ATP by nucleotide binding domains (NBDs). Contrary to the prokaryotic NBDs, the extremely low intrinsic ATPase activity of isolated MRP1-NBDs allowed us to obtain the structure of wild-type NBD1 in complex with Mg2+/ATP. The structure shows that MRP1-NBD1 adopts a canonical fold, but reveals an unexpected non-productive conformation of the catalytic site, providing an explanation for the low intrinsic ATPase activity of NBD1 and new hypotheses on the cooperativity of ATPase activity between NBD1 and NBD2 upon heterodimer formation.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/química , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Trifosfato de Adenosina/metabolismo , Magnésio/metabolismo , Nucleotídeos/metabolismo , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/metabolismo , Sequência de Aminoácidos , Ácido Aspártico/metabolismo , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Dimerização , Histidina/metabolismo , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Homologia Estrutural de Proteína
8.
Biochem J ; 391(Pt 3): 481-90, 2005 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-16014004

RESUMO

MRP1 (multidrug-resistance-associated protein 1; also known as ABCC1) is a member of the human ABC (ATP-binding cassette) transporter superfamily that confers cell resistance to chemotherapeutic agents. Considering the structural and functional similarities to the other ABC proteins, the interaction between its two NBDs (nucleotide-binding domains), NBD1 (N-terminal NBD) and NBD2 (C-terminal NBD), is proposed to be essential for the regulation of the ATP-binding/ATP-hydrolysis cycle of MRP1. We were interested in the ability of recombinant NBD1 and NBD2 to interact with each other and to influence ATPase activity. We purified NBD1 (Asn642-Ser871) and NBD2 (Ser1286-Val1531) as soluble monomers under native conditions. We measured extremely low intrinsic ATPase activity of NBD1 (10(-5) s(-1)) and NBD2 (6x10(-6) s(-1)) and no increase in the ATP-hydrolysis rate could be detected in an NBD1+NBD2 mixture, with concentrations up to 200 microM. Despite the fact that both monomers bind ATP, no stable NBD1.NBD2 heterodimer could be isolated by gel-filtration chromatography or native-PAGE, but we observed some significant modifications of the heteronuclear single-quantum correlation NMR spectrum of 15N-NBD1 in the presence of NBD2. This apparent NBD1.NBD2 interaction only occurred in the presence of Mg2+ and ATP. Partial sequential assignment of the NBD1 backbone resonances shows that residue Gly771 of the LSGGQ sequence is involved in NBD1.NBD2 complex formation. This is the first NMR observation of a direct interaction between the ABC signature and the opposite NBD. Our study also reveals that the NBD1.NBD2 heterodimer of MRP1 is a transient complex. This labile interaction is not sufficient to induce an ATPase co-operativity of the NBDs and suggests that other structures are required for the ATPase activation mechanism.


Assuntos
Adenosina Trifosfatases/metabolismo , Glicina/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/química , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Adenosina Trifosfatases/química , Sequência de Aminoácidos , Dimerização , Regulação da Expressão Gênica , Glicina/genética , Células HL-60 , Humanos , Espectroscopia de Ressonância Magnética , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes
9.
Bull Acad Natl Med ; 189(5): 789-95; discussion 795-6, 2005 May.
Artigo em Francês | MEDLINE | ID: mdl-16433451

RESUMO

Cystic Fibrosis is the most common lethal genetic disease among Caucasian population. Despite considerable efforts, no significant progress has been so far achieved by gene therapies approaches. On the basis of a surprising clinical observation, we have developed an approach using anti-cancer drugs promoting the over expression of ABC transporters closely related to the deficient protein CFTR, which seem able to share functions with it and to restore the missing function(s).


Assuntos
Colchicina/uso terapêutico , Fibrose Cística/tratamento farmacológico , Supressores da Gota/uso terapêutico , Transportadores de Cassetes de Ligação de ATP/metabolismo , Colchicina/farmacologia , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Supressores da Gota/farmacologia , Humanos
10.
Biochemistry ; 42(50): 14903-12, 2003 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-14674766

RESUMO

Ras1p and Ras2p, from Saccharomyces cerevisiae, are GTP-binding proteins that are essential elements in the signaling cascade leading to the activation of adenylyl cyclase. To overcome proteolytic activities that have hampered biochemical studies of Ras1p so far, its gene was genetically modified after which full-length Ras1p could be obtained. The interaction of farnesylated and unprenylated Ras1p with guanine nucleotides, guanine nucleotide exchange factors, GTPase activating proteins, and adenylyl cyclase was compared to Ras2p and human Ha-Ras interactions. Farnesylation of Ras proteins was demonstrated to be a prerequisite for membrane-bound guanine nucleotide exchange factor dependent formation of Ras-GTP complexes, and for efficient Ras-mediated adenylyl cyclase activation. To relate observed functional deviations with sequence differences between Ras1p and Ras2p, which reside almost exclusively within the hypervariable region, truncated versions and chimaeras of the Ras proteins were made. The characteristics of these constructs point to the presence of the hypervariable region of yeast Ras proteins for an efficient activation of adenylyl cyclase. The importance of the latter was confirmed as inhibition of the activation of adenylyl cyclase by an isolated farnesylated hypervariable region of Ras2p could be shown. This strongly suggests that the hypervariable region of Ras proteins can interact directly with adenylyl cyclase.


Assuntos
Adenilil Ciclases/fisiologia , Variação Genética , Proteínas Recombinantes de Fusão/síntese química , Proteínas de Saccharomyces cerevisiae/síntese química , Transdução de Sinais/genética , Proteínas ras/síntese química , Inibidores de Adenilil Ciclases , Adenilil Ciclases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/fisiologia , Membrana Celular/metabolismo , Ativação Enzimática/genética , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/fisiologia , Genes ras , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Humanos , Proteína Oncogênica p21(ras)/química , Proteína Oncogênica p21(ras)/genética , Ligação Proteica/genética , Prenilação de Proteína/genética , Estrutura Terciária de Proteína/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Ativadoras de ras GTPase/fisiologia , Fatores ras de Troca de Nucleotídeo Guanina/fisiologia , Proteínas ras/química , Proteínas ras/genética , Proteínas ras/isolamento & purificação , Proteínas ras/metabolismo , ras-GRF1/metabolismo , ras-GRF1/fisiologia
11.
Biochem J ; 376(Pt 3): 749-56, 2003 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-12954082

RESUMO

Multidrug-resistance-associated protein 1 (MRP1/ABCC1) is a human ATP-binding cassette transporter that confers cell resistance to antitumour drugs. Its NBDs (nucleotide-binding domains) bind/hydrolyse ATP, a key step in the activation of MRP1 function. To relate its intrinsic functional features to the mechanism of action of the full-size transporter, we expressed the N-terminal NBD1 domain (Asn(642) to Ser(871)) in Escherichia coli. NBD1 was highly purified under native conditions and was characterized as a soluble monomer. (15)N-labelling allowed recording of the first two-dimensional NMR spectra of this domain. The NMR study showed that NBD1 was folded, and that Trp(653) was a key residue in the NBD1-ATP interaction. Thus, interaction of NBD1 with ATP/ADP was studied by intrinsic tryptophan fluorescence. The affinity for ATP and ADP were in the same range (K (d(ATP))=118 microM and K (d(ADP))=139 microM). Binding of nucleotides did not influence the monomeric state of NBD1. The ATPase activity of NBD1 was magnesium-dependent and very low [V (max) and K (m) values of 5x10(-5) pmol of ATP x (pmol NBD1)(-1) x s(-1) and 833 microM ATP respectively]. The present study suggests that NBD1 has a low contribution to the ATPase activity of full-length MRP1 and/or that this activity requires NBD1-NBD2 heterodimer formation.


Assuntos
Trifosfato de Adenosina/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/química , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Triptofano/metabolismo , Difosfato de Adenosina/metabolismo , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Células HL-60 , Humanos , Magnésio/fisiologia , Dados de Sequência Molecular , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Ressonância Magnética Nuclear Biomolecular , Estrutura Terciária de Proteína , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Espectrometria de Fluorescência , Triptofano/fisiologia
12.
Biophys Chem ; 105(1): 29-43, 2003 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-12932577

RESUMO

Fumagillin is a potent anti-angiogenic drug used in cancer treatments. It is also one of the few molecules active against the Enterocytozoon and Encephalitozoon parasites responsible for various clinical syndromes in HIV-infected or immunosuppressive treated patients. Its toxicity, however, makes desirable the design of more specific molecules. The fumagillin target, as anti-angiogenic agent, is the methionine aminopeptidase, an ubiquitous metallo-enzyme responsible for the removing of the N-terminal methionine in nascent proteins. By analogy, it has been proposed that this enzyme could also be the target in the parasites. As a first approach to verify this and to determine if it would be possible to design a more specific derivative, we have built a homology model of the E. cuniculi aminopeptidase. The charges of the two cobalt ions present in the active site and of the side-chains involved in their binding were computed using ab-initio methods. A preliminary comparison of the interactions of the fumagillin and of a related compound, the TNP-470, with both the human and the parasitic enzymes strongly support the hypothesis that the parasitic aminopeptidase is indeed the target of the fumagillin. It also suggests that the TNP-470 interact identically with both enzymes while there could be small differences in case of the fumagillin.


Assuntos
Aminopeptidases/química , Aminopeptidases/metabolismo , Cobalto/química , Microsporídios não Classificados/enzimologia , Sequência de Aminoácidos , Aminopeptidases/antagonistas & inibidores , Inibidores da Angiogênese/química , Inibidores da Angiogênese/metabolismo , Inibidores da Angiogênese/farmacologia , Antiprotozoários/química , Antiprotozoários/metabolismo , Antiprotozoários/farmacologia , Sítios de Ligação , Cicloexanos , Desenho de Fármacos , Ácidos Graxos Insaturados/química , Ácidos Graxos Insaturados/metabolismo , Ácidos Graxos Insaturados/farmacologia , Humanos , Metionil Aminopeptidases , Modelos Moleculares , Dados de Sequência Molecular , O-(Cloroacetilcarbamoil)fumagilol , Ligação Proteica , Alinhamento de Sequência , Sesquiterpenos/química , Sesquiterpenos/metabolismo , Sesquiterpenos/farmacologia , Eletricidade Estática
13.
Biotechnol Prog ; 19(3): 727-33, 2003.
Artigo em Inglês | MEDLINE | ID: mdl-12790631

RESUMO

By coupling the toxic restriction endoribonuclease RegB, from the bacteriophage T4, to the prokaryotic T7 and the eukaryotic GAL1 promoters, we constructed a two-function plasmid called pTOXR-1. This plasmid is a zero-background cloning vector. It allows an efficient positive selection of recombinant plasmids without the need to completely digest, dephosphorylate, or purify the vector prior to the ligation step. The pTOXR-1 positive selection system requires no special Escherichia coli strains, no special culture media, and no addition of inducer to the selective plates. In addition, since this vector carries all signals required for both prokaryotic and eukaryotic expression, it allows the overproduction of heterologous proteins, fused to a polyhistidine tag, in the bacterium E. coli and in the yeast Saccharomyces cerevisiae from a single plasmid. Hence, this vector may be a useful time-saving tool for one-step cloning and versatile protein expression in bacteria and yeast.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clonagem Molecular/métodos , Escherichia coli/metabolismo , Plasmídeos/genética , Engenharia de Proteínas/métodos , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Recombinantes/biossíntese , Saccharomyces cerevisiae/metabolismo , Proteínas de Bactérias/biossíntese , Escherichia coli/genética , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Regulação Fúngica da Expressão Gênica/fisiologia , Regiões Promotoras Genéticas , Proteínas Recombinantes/genética , Proteínas Ribossômicas/biossíntese , Proteínas Ribossômicas/genética , Saccharomyces cerevisiae/genética , Seleção Genética
14.
JOP ; 4(2): 92-103, 2003 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-12629266

RESUMO

Human lithostathine was initially isolated from pancreatic stones in patients with alcoholic calcifying chronic pancreatitis. It is secreted into the pancreatic juice where it was believed to inhibit stone formation. The N-terminal undecapeptide was assumed to play an important role in the mechanism, by adsorption to the crystal surface. Later, the role of lithostathine in calcite formation and growth was questioned, together with the associated mechanism of action. In particular, although lithostathine adsorbs on calcite crystal, this property does not now seem to be specific. Moreover, the N-terminal undecapeptide is not likely to have, by itself, the function of the entire protein. The different aspects of this controversy are reviewed and discussed, particularly in the light of recent structural biology. Comparative biological data now available allow us to draw an interesting parallel between lithostathine and other related proteins. Finally, lithostathine might affect stone formation and may also have another function which could be investigated in the other proteins belonging to the same structural family.


Assuntos
Proteínas de Ligação ao Cálcio/fisiologia , Proteínas do Tecido Nervoso , Sequência de Aminoácidos/fisiologia , Animais , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/metabolismo , Humanos , Litostatina , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína/fisiologia , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade
16.
Blood ; 101(8): 3014-20, 2003 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-12480715

RESUMO

The tetrapeptide acetyl-Ser-Asp-Lys-Pro (AcSDKP), purified from bone marrow and constitutively synthesized in vivo, belongs to the family of negative regulators of hematopoiesis. It protects the stem cell compartment from the toxicity of anticancer drugs and irradiation and consequently contributes to a reduction in marrow failure. This current work provides experimental evidence for another novel biologic function of AcSDKP. We report that AcSDKP is a mediator of angiogenesis, as measured by its ability to modulate endothelial cell function in vitro and angiogenesis in vivo. AcSDKP at nanomolar concentrations stimulates in vitro endothelial cell migration and differentiation into capillary-like structures on Matrigel as well as enhances the secretion of an active form of matrix metalloproteinase-1 (MMP-1). In vivo, AcSDKP promotes a significant angiogenic response in the chicken embryo chorioallantoic membrane (CAM) and in the abdominal muscle of the rat. Moreover, it induces the formation of blood vessels in Matrigel plugs implanted subcutaneously in the rat. This is the first report demonstrating the ability of AcSDKP to interact directly with endothelial cells and to elicit an angiogenic response in vitro and in vivo.


Assuntos
Neovascularização Fisiológica/efeitos dos fármacos , Oligopeptídeos/farmacologia , Parede Abdominal/irrigação sanguínea , Adenocarcinoma/patologia , Alantoide/irrigação sanguínea , Animais , Diferenciação Celular/efeitos dos fármacos , Fusão Celular , Linhagem Celular Transformada , Movimento Celular/efeitos dos fármacos , Células Cultivadas/efeitos dos fármacos , Embrião de Galinha , Córion/irrigação sanguínea , Colágeno , Combinação de Medicamentos , Implantes de Medicamento , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Indução Enzimática/efeitos dos fármacos , Humanos , Laminina , Neoplasias Pulmonares/patologia , Metaloproteinase 1 da Matriz/biossíntese , Metaloproteinase 1 da Matriz/metabolismo , Oligopeptídeos/administração & dosagem , Oligopeptídeos/síntese química , Proteoglicanas , Ratos , Ratos Wistar , Células Tumorais Cultivadas , Veias Umbilicais/citologia
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