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1.
Nanomaterials (Basel) ; 8(12)2018 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-30558254

RESUMO

Materials exposed to plasmas in magnetic confinement nuclear reactors will accumulate radiation-induced defects and energetically implanted gas atoms (from the plasma and transmutations), of which insoluble helium (He) is likely to be the most problematic. The large surface-area-to-volume ratio exhibited by nanoporous materials provides an unsaturable sink with the potential to continuously remove both point defects and He. This property enhances the possibilities for these materials to be tailored for high radiation-damage resistance. In order to explore the potential effect of this on the individual ligaments of nanoporous materials, we present results on the response of tungsten (W) nanoparticles (NPs) to 15 keV He ion irradiation. Tungsten foils and various sizes of NPs were ion irradiated concurrently and imaged in-situ via transmission electron microscopy at 750 °C. Helium bubbles were not observed in NPs with diameters less than 20 nm but did form in larger NPs and the foils. No dislocation loops or black spot damage were observed in any NPs up to 100 nm in diameter but were found to accumulate in the W foils. These results indicate that a nanoporous material, particularly one made up of ligaments with characteristic dimensions of 30 nm or less, is likely to exhibit significant resistance to He accumulation and structural damage and, therefore, be highly tolerant to radiation.

2.
Sci Rep ; 7(1): 7724, 2017 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-28798360

RESUMO

The self-organisation of void and gas bubbles in solids into superlattices is an intriguing nanoscale phenomenon. Despite the discovery of these lattices 45 years ago, the atomistics behind the ordering mechanisms responsible for the formation of these nanostructures are yet to be fully elucidated. Here we report on the direct observation via transmission electron microscopy of the formation of bubble lattices under He ion bombardment. By careful control of the irradiation conditions, it has been possible to engineer the bubble size and spacing of the superlattice leading to important conclusions about the significance of vacancy supply in determining the physical characteristics of the system. Furthermore, no bubble lattice alignment was observed in the <111> directions pointing to a key driving mechanism for the formation of these ordered nanostructures being the two-dimensional diffusion of self-interstitial atoms.

3.
Andrology ; 4(1): 41-5, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26754331

RESUMO

We compared a novel 5% testosterone (T) cream (AndroForte 5, Lawley Pharmaceuticals, Australia) with a 1% T gel (Testogel, Besins Healthcare, Australia). Using an open-label crossover design, subjects were randomized to one of two treatment sequences using either the T gel or T cream first in a 1 : 1 ratio. Each treatment period was 30 days with a 7-14 days washout period between them. On Days 1 and 30 of each treatment period blood was sampled at -15, -5 min, 0, 2, 4, 5, 6, 7, 8, 9, 10, 12 and 16 h post study drug administration. Sixteen men with established androgen deficiency aged between 29 and 73 years, who had undertaken a washout from prior testosterone therapy participated in the study. One subject failed to complete both arms and another was excluded post-completion because of a major protocol violation. Bioequivalence was established based on key pharmacokinetic (PK) variables: AUC, C(avg), C(max), T(max), % fluctuation (with and without baseline correction) for the two formulations of testosterone on Day 1 and Day 30. The ratio and 90% CI of AUC 0.99 (0.86-1.14), C(max) 1.02 (0.84-1.24) and C(avg) 0.99 (0.86-1.14) for T cream/T gel were within the predetermined bio-equivalence criteria of 80% to 125% at Day 30. There were no statistically significant differences between secondary biochemical markers: serum dihydrotestosterone (DHT), oestradiol (E2), sex hormone-binding globulin (SHBG), luteinizing hormone (LH) and (FSH). The two testosterone formulations were shown to be bioequivalent.


Assuntos
Androgênios/deficiência , Hipogonadismo/tratamento farmacológico , Creme para a Pele/uso terapêutico , Testosterona , Administração Cutânea , Adulto , Idoso , Estudos Cross-Over , Di-Hidrotestosterona/sangue , Estradiol/sangue , Hormônio Foliculoestimulante/sangue , Géis , Humanos , Hormônio Luteinizante/sangue , Masculino , Pessoa de Meia-Idade , Globulina de Ligação a Hormônio Sexual/metabolismo , Testosterona/administração & dosagem , Testosterona/farmacocinética , Testosterona/uso terapêutico , Equivalência Terapêutica
4.
Int J Data Min Bioinform ; 2(1): 78-93, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18399329

RESUMO

RNA plays a critical role in mediating every step of cellular information transfer from genes to functional proteins. Pseudoknots are functionally important and widely occurring structural motifs found in all types of RNA. Therefore predicting their structures is an important problem. In this paper, we present a new RNA pseudoknot structure prediction method based on term rewriting. The method is implemented using the Mfold RNA/DNA folding package and the term rewriting language Maude. In our method, RNA structures are treated as terms and rules are discovered for predicting pseudoknots. Our method was tested on 211 pseudoknots in PseudoBase and achieves an average accuracy of 74.085% compared to the experimentally determined structure. In fact, most pseudoknots discovered by our method achieve an accuracy of above 90%. These results indicate that term rewriting has a broad potential in RNA applications ranging from prediction of pseudoknots to discovery of higher level RNA structures involving complex RNA tertiary interactions.


Assuntos
Algoritmos , Modelos Químicos , Modelos Moleculares , RNA/química , RNA/ultraestrutura , Análise de Sequência de RNA/métodos , Sequência de Bases , Simulação por Computador , Dados de Sequência Molecular , Conformação de Ácido Nucleico
5.
AIDS Res Hum Retroviruses ; 20(2): 135-44, 2004 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-15018700

RESUMO

The retroviral integrase protein (IN) is essential for virus replication and, therefore, an attractive target for the development of inhibitors to treat human immunodeficiency virus (HIV) infection. Diverse classes of compounds that are active against this protein have been discovered using in vitro assays. Here we describe the synthesis of a novel compound, 3,8-dibromo-7-amino-4-hydroxy-2-naphthalenesulfonic acid (2BrNSA), which inhibits the in vitro activities of the full-length HIV-1 and avian sarcoma virus (ASV) integrases, and the isolated catalytic core fragment of the ASV protein (residues 52-207). The compound also inhibits retroviral reverse transcriptase in vitro, but the IC(50) for the HIV-1 enzyme is almost two orders of magnitude higher than for HIV-1 integrase. The inhibitor was found to be active in cell culture, preventing reporter gene transduction of HeLa cells by both ASV and HIV-1 vectors. Neither viral attachment nor uptake into cells appeared to be affected in these transfections, whereas accumulation of vector DNA and its joining to host DNA were both drastically reduced in the presence of the inhibitor. Propagation of two different strains of replication-competent HIV-1 in human peripheral blood mononuclear cells (PBMCs) was also reduced by the inhibitor, allowing survival of a substantial number of cells in the treated cultures. Based on these and other results we speculate that binding of 2BrNSA to integrase in infected cells interferes not only with its catalytic activity but also with critical interactions that are required for the formation or function of the reverse transcriptase complex. Its activity in cell culture suggests that this inhibitor may provide a valuable new lead for further development of drugs that target early steps in the HIV life cycle.


Assuntos
Inibidores de Integrase de HIV/farmacologia , HIV-1/efeitos dos fármacos , HIV-1/enzimologia , Naftalenos/farmacologia , Vírus do Sarcoma Aviário/efeitos dos fármacos , Vírus do Sarcoma Aviário/enzimologia , Vírus do Sarcoma Aviário/genética , Sequência de Bases , DNA Viral/genética , Inibidores de Integrase de HIV/química , HIV-1/genética , Células HeLa , Humanos , Técnicas In Vitro , Inibidores de Integrase/química , Inibidores de Integrase/farmacologia , Naftalenos/química , Transdução Genética , Replicação Viral/efeitos dos fármacos
6.
Med Sci Monit ; 9(8): BR289-301, 2003 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12942025

RESUMO

BACKGROUND: Electrostatic steering of ligands into active/binding sites is important for enzymatic catalysis and enhancing the affinity of metal-binding proteins. The net charge center of a protein, which describes the distribution of the charged residues in the protein subunit, has a specific location. As active sites in biological molecules are also located specifically, the biological implications of the model were examined by analyzing the relative locations of the net charge centers and the active/binding sites. MATERIAL/METHODS: Spatial structures from a set of 11 proteins with experimentally defined electrostatic steering residues and definitely known active site residues were analyzed. RESULTS: In 9 of 11 proteins, 70-100% of the active site residues belong to the residue cluster around the net charge center. On average, the charge center cluster identifies 78% of the active sites and 80% of the steering residues with few false positives. In several cases, comparison of proteins from different sources has shown that the differences in association rates correlate quantitatively with the relative locations of the net charge centers. CONCLUSIONS: In most of the proteins studied the net charge center is located close to the active site. Therefore, the active site in a novel enzyme or the binding site in a protein can be predicted solely from the three-dimensional structure of the protein. The residues forming clusters around the net charge center are often organized into continuous hydrophilic sequences, important for rapid and correct formation of the active site region during protein folding.


Assuntos
Conformação Proteica , Proteínas/química , Sequência de Aminoácidos , Sítios de Ligação , Modelos Moleculares , Proteínas/genética , Eletricidade Estática
7.
Acta Crystallogr D Biol Crystallogr ; 57(Pt 11): 1545-51, 2001 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-11679718

RESUMO

Tcl1 and Mtcp1, members of the Tcl1 family, are implicated in T-cell prolymphocytic leukemia. The crystal structure of a dimer of murine Tcl1 has been determined at 2.5 A resolution with an R factor of 0.225. Murine Tcl1, human Tcl1 and Mtcp1 share very similar subunit structures, with RMS differences of 0.6 and 1.4 A for C(alpha) atoms, respectively, while the sequences share 50 and 36% identity, respectively. These structures fold into an eight-stranded beta-barrel of unique topology and high internal symmetry of 1.1-1.3 A for the two halves of human and murine Tcl1 and 1.7 A for Mtcp1, despite the low 12-13% sequence identity. The molecular surfaces of all three structures showed a common planar region which is likely to be involved in protein-protein interactions.


Assuntos
Proteínas de Ligação a DNA/química , GTP Fosfo-Hidrolases/química , Proteínas Proto-Oncogênicas , Fatores de Transcrição/química , Sequência de Aminoácidos , Animais , Cristalização , Cristalografia por Raios X , Proteínas de Ligação a DNA/fisiologia , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/fisiologia
8.
Biochemistry ; 40(25): 7464-73, 2001 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-11412099

RESUMO

The eukaryotic cyclic nucleotide-gated (CNG) ion channels are a family of large membrane proteins activated by cytoplasmic cGMP or cAMP. Their cyclic nucleotide-binding domain is structurally homologous with that of the catabolite gene-activator protein (CAP), a soluble Escherichia coli transcription factor. Differences in ligand activation among sensory channels suggest differences in the underlying molecular mechanisms of signal readout. To study the structural, functional, and conformational consequences of nucleotide binding, we fused the cyclic nucleotide-binding domain from the bovine retinal rod CNG channel alpha subunit (Bralpha) to the DNA-binding domain from CAP. The chimera forms a soluble dimer that binds both cGMP and cAMP with association constants of 3.7 x 10(4) M(-1) for [(3)H]cGMP and 3.1 x 10(4) M(-1) for [(3)H]cAMP. The binding of cAMP, but not cGMP, exposes a chymotrypsin cleavage site in the chimera at a position similar to the site in the CAP exposed by cAMP binding. At high cAMP concentrations, a biphasic pattern of cleavage is seen, suggesting that the low-affinity cAMP binding sites are also occupied. Cyclic AMP promotes specific binding to a DNA fragment encoding the lac operator region; the K(d) for the protein-DNA binding is approximately 200 nM, which is 2-fold higher than the K(d) for CAP under identical conditions. A 7 A crystal structure shows that the overall secondary and tertiary structure of Bralpha/CAP is the same as that of CAP with two cAMP molecules bound per dimer. The biochemical characterization of the chimera suggests it will be a useful system for testing hypotheses about channel activation, providing further insight into channel function.


Assuntos
Proteína Receptora de AMP Cíclico/genética , Proteínas de Ligação a DNA/genética , Canais Iônicos/genética , Canais Iônicos/metabolismo , Proteínas Recombinantes de Fusão/fisiologia , Segmento Externo da Célula Bastonete/fisiologia , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bovinos , Quimotripsina/metabolismo , Cristalografia por Raios X , AMP Cíclico/metabolismo , Proteína Receptora de AMP Cíclico/química , Proteína Receptora de AMP Cíclico/metabolismo , Canais de Cátion Regulados por Nucleotídeos Cíclicos , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Dimerização , Escherichia coli/genética , Vetores Genéticos/síntese química , Hidrólise , Dados de Sequência Molecular , Ligação Proteica/genética , Conformação Proteica , Estrutura Terciária de Proteína/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Solubilidade , Relação Estrutura-Atividade
9.
Proteins ; 43(4): 353-64, 2001 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-11340652

RESUMO

Electrostatic interactions are important for protein folding. At low resolution, the electrostatic field of the whole molecule can be described in terms of charge center(s). To study electrostatic effects, the centers of positive and negative charge were calculated for 20 small proteins of known structure, for which hydrogen exchange cores had been determined experimentally. Two observations seem to be important. First, in all 20 proteins studied 30-100% of the residues forming hydrogen exchange core(s) were clustered around the charge centers. Moreover, in each protein more than half of the core sequences are located near the centers of charge. Second, the general architecture of all-alpha proteins from the set seems to be stabilized by interactions of residues surrounding the charge centers. In most of the alpha-beta proteins, either or both of the centers are located near a pair of consecutive strands, and this is even more characteristic for alpha/Beta and all-beta structures. Consecutive strands are very probable sites of early folding events. These two points lead to the conclusion that charge centers, defined solely from the structure of the folded protein may indicate the location of a protein's hydrogen exchange/folding core. In addition, almost all the proteins contain well-conserved continuous hydrophobic sequences of three or more residues located in the vicinity of the charge centers. These hydrophobic sequences may be primary nucleation sites for protein folding. The results suggest the following scheme for the order of events in folding: local hydrophobic nucleation, electrostatic collapse of the core, global hydrophobic collapse, and slow annealing to the native state. This analysis emphasizes the importance of treating electrostatics during protein-folding simulations.


Assuntos
Dobramento de Proteína , Proteínas/química , Motivos de Aminoácidos , Sequência Conservada , Modelos Químicos , Modelos Teóricos , Conformação Proteica , Eletricidade Estática
10.
Proteins ; 43(4): 455-64, 2001 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-11340661

RESUMO

Emergence of drug-resistant mutants of HIV-1 protease is an ongoing problem in the fight against AIDS. The mechanisms governing resistance are both complex and varied. We have determined crystal structures of HIV-1 protease mutants, D30N, K45I, N88D, and L90M complexed with peptide inhibitor analogues of CA-p2 and p2-NC cleavage sites in the Gag-pol precursor in order to study the structural mechanisms underlying resistance. The structures were determined at 1.55-1.9-A resolution and compared with the wild-type structure. The conformational disorder seen for most of the hydrophobic side-chains around the inhibitor binding site indicates flexibility of binding. Eight water molecules are conserved in all 9 structures; their location suggests that they are important for catalysis as well as structural stability. Structural differences among the mutants were analyzed in relation to the observed changes in protease activity and stability. Mutant L90M shows steric contacts with the catalytic Asp25 that could destabilize the catalytic loop at the dimer interface, leading to its observed decreased dimer stability and activity. Mutant K45I reduces the mobility of the flap and the inhibitor and contributes to an enhancement in structural stability and activity. The side-chain variations at residue 30 relative to wild-type are the largest in D30N and the changes are consistent with the altered activity observed with peptide substrates. Polar interactions in D30N are maintained, in agreement with the observed urea sensitivity. The side-chains of D30N and N88D are linked through a water molecule suggesting correlated changes at the two sites, as seen with clinical inhibitors. Structural changes seen in N88D are small; however, water molecules that mediate interactions between Asn88 and Thr74/Thr31/Asp30 in other complexes are missing in N88D.


Assuntos
Resistência Microbiana a Medicamentos/genética , Protease de HIV/química , Protease de HIV/metabolismo , HIV-1/efeitos dos fármacos , HIV-1/enzimologia , Substituição de Aminoácidos , Sítios de Ligação , Catálise , Cristalografia por Raios X , Estabilidade Enzimática , Protease de HIV/genética , Inibidores da Protease de HIV/farmacologia , Humanos , Modelos Moleculares , Conformação Molecular , Mutação , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Relação Estrutura-Atividade , Termodinâmica
11.
Acta Crystallogr D Biol Crystallogr ; 57(Pt 5): 763-5, 2001 May.
Artigo em Inglês | MEDLINE | ID: mdl-11320330

RESUMO

The crystal structure of human Mtcp1 was determined at 2 A resolution after the X-ray diffraction limit was improved by post-crystallization soaking in 2.0 M ammonium sulfate for 1-5 months. The effects of varying the ammonium sulfate concentration and addition of polyethylene glycol to the soaking solution were examined in order to understand the phenomenon and to reduce the soaking time. Soaking the crystal for one week in a solution of 1.5 M ammonium sulfate and 2% PEG 3400 gave the desired improvement in diffraction quality. Therefore, different soaking conditions should be explored when crystals show disordered and low-resolution diffraction.


Assuntos
Proteínas Proto-Oncogênicas/química , Cristalização , Cristalografia por Raios X , Humanos , Conformação Proteica , Proteínas Recombinantes/química
12.
J Natl Med Assoc ; 93(3 Suppl): 6S-7S, 2001 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-12653393

RESUMO

Pharmaceutical development and medical research continues at a fevered pitch. Historically, however, African Americans and other minorities have not been adequately represented in the studies determining a drug's safety and efficacy in humans. A history of misuse in the medical research systems (most notably the Tuskeegee study of syphillis in a population of illiterate, poor black men) have left many blacks wary of the health care system. However, attempts to address the health disparities faced by African Americans must include processes for including wider representation of blacks--as patients as well as investigators--in clinical trials.


Assuntos
Afro-Americanos/estatística & dados numéricos , Pesquisa Biomédica , Seleção de Pacientes , Preconceito , Ensaios Clínicos como Assunto , Grupo com Ancestrais do Continente Europeu/estatística & dados numéricos , Feminino , Humanos , Masculino , Estados Unidos
13.
Am J Physiol Heart Circ Physiol ; 279(4): H1982-8, 2000 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-11009488

RESUMO

Nitric oxide (NO) has concentration-dependent biphasic myocardial contractile effects. We tested the hypothesis, in isolated rat hearts, that NO cardiostimulation is primarily non-cGMP dependent. Infusion of 3-morpholinosydnonimine (SIN-1, 10(-5) M), which may participate in S-nitrosylation (S-NO) via peroxynitrite formation, increased the rate of left ventricular pressure rise (+dP/dt; 19 +/- 4%, P < 0.001, n = 11) without increasing effluent cGMP or cAMP. Superoxide dismutase (SOD; 150 U/ml) blocked SIN-1 cardiostimulation and led to cGMP elaboration. Sodium nitroprusside (10(-10)-10(-7) M), an iron nitrosyl compound, did not augment +dP/dt but increased cGMP approximately eightfold (P < 0.001), whereas diethylamine/NO (DEA/NO; 10(-7) M), a spontaneous NO. donor, increased +dP/dt (5 +/- 2%, P < 0.05, n = 6) without augmenting cGMP. SIN-1 and DEA/NO +dP/dt increase persisted despite guanylyl cyclase inhibition with 1H-(1,2,4)oxadiazolo-(4,3,-a)quinoxalin-1-one (10(-5) M, P < 0.05 for both donors), suggesting a cGMP-independent mechanism. Glutathione (5 x 10(-4) M, n = 15) prevented SIN-1 cardiostimulation, suggesting S-NO formation. SIN-1 also produced SOD-inhibitable cardiostimulation in vivo in mice. Thus peroxynitrite and NO donors can stimulate myocardial contractility independently of guanylyl cyclase activation, suggesting a role for S-NO reactions in NO/peroxynitrite-positive inotropic effects in intact hearts.


Assuntos
GMP Cíclico/fisiologia , Molsidomina/análogos & derivados , Contração Miocárdica/efeitos dos fármacos , Nitratos/farmacologia , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico/metabolismo , Animais , GMP Cíclico/metabolismo , Dietilaminas/farmacologia , Combinação de Medicamentos , Inibidores Enzimáticos/farmacologia , Glutationa/farmacologia , Técnicas In Vitro , Masculino , Molsidomina/antagonistas & inibidores , Molsidomina/farmacologia , Óxido Nítrico/farmacologia , Nitroprussiato/farmacologia , Nucleotídeos Cíclicos/metabolismo , Oxidiazóis/farmacologia , Oxirredução , Quinoxalinas/farmacologia , Ratos , Ratos Wistar , Superóxido Dismutase/farmacologia
14.
Eur J Biochem ; 267(20): 6287-95, 2000 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-11012683

RESUMO

Human T-cell leukemia virus type-1 (HTLV-1) is associated with a number of human diseases. Based on the therapeutic success of human immunodeficiency virus type 1 (HIV-1) PR inhibitors, the proteinase (PR) of HTLV-1 is a potential target for chemotherapy. To facilitate the design of potent inhibitors, the subsite specificity of HTLV-1 PR was characterized and compared to that of HIV-1 PR. Two sets of substrates were used that contained single amino-acid substitutions in peptides representing naturally occurring cleavage sites in HIV-1 and HTLV-1. The original HIV-1 matrix/capsid cleavage site substrate and most of its substituted peptides were not hydrolyzed by the HTLV-1 enzyme, except for those with hydrophobic residues at the P4 and P2 positions. On the other hand, most of the peptides representing the HTLV-1 capsid/nucleocapsid cleavage site were substrates of both enzymes. A large difference in the specificity of HTLV-1 and HIV-1 proteinases was demonstrated by kinetic measurements, particularly with regard to the S4 and S2 subsites, whereas the S1 subsite appeared to be more conserved. A molecular model of the HTLV-1 PR in complex with this substrate was built, based on the crystal structure of the S9 mutant of Rous sarcoma virus PR, in order to understand the molecular basis of the enzyme specificity. Based on the kinetics of shortened analogs of the HTLV-1 substrate and on analysis of the modeled complex of HTLV-1 PR with substrate, the substrate binding site of the HTLV-1 PR appeared to be more extended than that of HIV-1 PR. Kinetic results also suggested that the cleavage site between the capsid and nucleocapsid protein of HTLV-1 is evolutionarily optimized for rapid hydrolysis.


Assuntos
Ácido Aspártico Endopeptidases/química , Ácido Aspártico Endopeptidases/metabolismo , Protease de HIV/química , Protease de HIV/metabolismo , Sequência de Aminoácidos , Vírus do Sarcoma Aviário/enzimologia , Humanos , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Inibidores de Proteases/farmacologia , Estrutura Secundária de Proteína , Alinhamento de Sequência , Especificidade por Substrato
15.
J Clin Invest ; 106(5): 697-703, 2000 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-10974023

RESUMO

The cardiac beta-adrenergic pathway potently stimulates myocardial performance, thereby providing a mechanism for myocardial contractile reserve. beta-Adrenergic activation also increases cardiac nitric oxide (NO) production, which attenuates positive inotropy, suggesting a possible negative feedback mechanism. Recently, in vitro studies suggest that stimulation of the beta(3)-adrenoceptor results in a negative inotropic effect through NO signaling. In this study, using mice with homozygous beta(3)-adrenoceptor deletion mutations, we tested the hypothesis that the beta(3)-adrenoceptor is responsible for beta-adrenergic activation of NO. Although resting indices of myocardial contraction were similar, beta-adrenergic-stimulated inotropy was increased in beta(3)(-/-) mice, and similar hyper-responsiveness was seen in mice lacking endothelial NO synthase (NOS3). NOS inhibition augmented isoproterenol-stimulated inotropy in wild-type (WT), but not in beta(3)(-/-) mice. Moreover, isoproterenol increased myocardial cGMP in WT, but not beta(3)(-/-), mice. NOS3 protein abundance was not changed in beta(3)(-/-) mice, and cardiac beta(3)-adrenoceptor mRNA was detected in both NOS3(-/-) and WT mice. These findings indicate that the beta(3)-adrenergic subtype participates in NO-mediated negative feedback over beta-adrenergic stimulation.


Assuntos
Contração Miocárdica/fisiologia , Óxido Nítrico/metabolismo , Receptores Adrenérgicos beta/metabolismo , Agonistas Adrenérgicos beta/farmacologia , Animais , Retroalimentação , Isoproterenol/farmacologia , Camundongos , Camundongos Mutantes , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo II , Óxido Nítrico Sintase Tipo III , Receptores Adrenérgicos beta/genética , Receptores Adrenérgicos beta 3 , Sistema Nervoso Simpático/fisiologia
16.
Protein Eng ; 13(6): 431-6, 2000 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-10877854

RESUMO

The processing of precursor proteins (Gag and Gag-pol) by the viral protease is absolutely required in order to generate infectious particles. This prompted us to consider novel strategies that target viral maturation. Towards this end, we have engineered an HIV-1 virion associated protein, Vpr, to contain protease cleavage signal sequences from Gag and Gag-pol precursor proteins. We previously reported that virus particles derived from HIV-1 proviral DNA, encoding chimeric Vpr, showed a lack of infectivity, depending on the fusion partner. As an extension of that work, the potential of chimeric Vpr as a substrate for HIV-1 protease was tested utilizing an epitope-based assay. Chimeric Vpr molecules were modified such that the Flag epitope is removed following cleavage, thus allowing us to determine the efficiency of protease cleavage. Following incubation with the protease, the resultant products were analyzed by radioimmunoprecipitation using antibodies directed against the Flag epitope. Densitometric analysis of the autoradiograms showed processing to be both rapid and specific. Further, the analysis of virus particles containing chimeric Vpr by immunoblot showed reactivities to antibodies against the Flag epitope similar to the data observed in vitro. These results suggest that the pseudosubstrate approach may provide another avenue for developing antiviral agents.


Assuntos
Antivirais/farmacologia , Produtos do Gene vpr/biossíntese , Produtos do Gene vpr/farmacologia , Protease de HIV/metabolismo , HIV-1/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Compostos de Aminobifenil/farmacologia , Densitometria , Desenho de Drogas , Epitopos/genética , Epitopos/metabolismo , Produtos do Gene gag/genética , Produtos do Gene vpr/genética , HIV-1/genética , HIV-1/crescimento & desenvolvimento , HIV-1/metabolismo , Immunoblotting , Oligopeptídeos , Peptídeos/genética , Plasmídeos/genética , Precursores de Proteínas/genética , Ensaio de Radioimunoprecipitação , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacologia , Produtos do Gene gag do Vírus da Imunodeficiência Humana , Produtos do Gene pol do Vírus da Imunodeficiência Humana , Produtos do Gene vpr do Vírus da Imunodeficiência Humana
17.
Crit Care Med ; 28(5): 1263-8, 2000 May.
Artigo em Inglês | MEDLINE | ID: mdl-10834663

RESUMO

OBJECTIVE: The nitric oxide synthase inhibitor L-N(G)-methylarginine hydrochloride (L-NMMA HC1 546C88) causes reductions in cardiac output (CO), a potential limitation to clinical application. This drop in CO exceeds that from phenylephrine at matched systemic arterial pressure. We tested the hypothesis that the greater fall in CO attributable to L-NMMA primarily reflects a difference in venoconstriction between agents, such that phenylephrine produces larger increases in preload (an independent determinant of CO). DESIGN: Random infusion of phenylephrine or L-NMMA. SETTING: An animal research laboratory. SUBJECTS: Eight healthy, conscious, male dogs. INTERVENTIONS: L-N(G)-methylarginine hydrochloride (20 mg/kg for 1 hr) and phenylephrine (0.5 to 3 microg/kg/min) were administered into eight dogs chronically instrumented to measure left ventricular pressure and dimension. Data were measured at a constant heart rate (140 beats/min) to render CO proportional to stroke dimension. MEASUREMENTS AND MAIN RESULTS: At a matched increase in afterload (effective arterial elastance), L-NMMA increased preload (end-diastolic dimension) to a lesser degree (3.8%+/-1.5%, p < .05) than phenylephrine (9.6%+/-1.6%, p < .05 vs. L-NMMA). Neither L-NMMA nor phenylephrine affected the slope of the end-systolic pressure dimension relationship, although L-NMMA shifted the relationship rightward (1.7+/-0.7 mm, p < .05), consistent with a mild negative inotropic effect. L-NMMA decreased the stroke dimension to a greater extent than phenylephrine (-24.1%+/-6.8% and -10.6%+/-3.4%, respectively, p < .05). CONCLUSIONS: Differential CO responses to phenylephrine and L-NMMA were primarily attributable to changes in preload. Variable venular vs. arteriolar constrictor effects must be considered when evaluating the integrated cardiovascular response to a vasoactive agent.


Assuntos
Pressão Sanguínea/efeitos dos fármacos , Débito Cardíaco/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Função Ventricular Esquerda/efeitos dos fármacos , ômega-N-Metilarginina/farmacologia , Animais , Débito Cardíaco/fisiologia , Cães , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/fisiopatologia , Humanos , Masculino , Fenilefrina/farmacologia , Choque Séptico/fisiopatologia , Resistência Vascular/efeitos dos fármacos , Resistência Vascular/fisiologia , Vasoconstritores/farmacologia
18.
Diabetes ; 48(9): 1698-705, 1999 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-10480597

RESUMO

Mutations in human glucokinase are implicated in the development of diabetes and hypoglycemia. Human glucokinase shares 54% identical amino acid residues with human brain hexokinase I. This similarity was used to model the structure of glucokinase by analogy to the crystal structure of brain hexokinase. Glucokinase was modeled with both its substrates, glucose and MgATP, to understand the effect of mutations. The glucose is predicted to form hydrogen bond interactions with the side chains of glucokinase residues Thr 168, Lys 169, Asn 204, Asp 205, Asn 231, and Glu 290, similar to those observed for brain hexokinase I. The magnesium ion is coordinated by the carboxylates of Asp 78 and Asp 205 and the gamma-phosphate of ATP. ATP is predicted to form hydrogen bond interactions with residues Gly 81, Thr 82, Asn 83, Arg 85, Lys 169, Thr 228, Lys 296, Thr 332, and Ser 336. Mutations of residues close to the predicted ATP binding site produced dramatic changes in the Km for ATP, the catalytic rate, and a loss of cooperativity, which confirmed our model. Mutations of residues in the glucose binding site dramatically reduced the catalytic activity, as did a mutation that was predicted to disrupt an alpha-helix. Other mutations located far from the active site gave smaller changes in kinetic parameters. In the absence of a crystal structure for glucokinase, our models help rationalize the potential effects of mutations in diabetes and hypoglycemia, and the models may also facilitate the discovery of pharmacological glucokinase activators and inhibitors.


Assuntos
Trifosfato de Adenosina/química , Glucoquinase/química , Glucose/química , Hiperglicemia/genética , Hipoglicemia/genética , Modelos Moleculares , Sequência de Aminoácidos , Sítios de Ligação , Humanos , Dados de Sequência Molecular , Mutação , Conformação Proteica
19.
Circ Res ; 85(5): 437-45, 1999 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-10473673

RESUMO

Allopurinol, an inhibitor of xanthine oxidase, increases myofilament calcium responsiveness and blunts calcium cycling in isolated cardiac muscle. We sought to extend these observations to conscious dogs with and without pacing-induced heart failure and tested the prediction that allopurinol would have a positive inotropic effect without increasing energy expenditure, thereby increasing mechanical efficiency. In control dogs (n=10), allopurinol (200 mg IV) caused a small positive inotropic effect; (dP/dt)(max) increased from 3103+/-162 to 3373+/-225 mm Hg/s (+8.3+/-3.2%; P=0.01), but preload-recruitable stroke work and ventricular elastance did not change. In heart failure (n=5), this effect was larger; (dP/dt)(max) rose from 1602+/-190 to 1988+/-251 mm Hg/s (+24.4+/-8.7%; P=0.03), preload-recruitable stroke work increased from 55.8+/-9.1 to 84. 9+/-12.2 mm Hg (+28.1+/-5.3%; P=0.02), and ventricular elastance rose from 6.0+/-1.6 to 10.5+/-2.2 mm Hg/mm (P=0.03). Allopurinol did not affect myocardial lusitropic properties either in control or heart failure dogs. In heart failure dogs, but not controls, allopurinol decreased myocardial oxygen consumption (-49+/-4.6%; P=0. 002) and substantially increased mechanical efficiency (stroke work/myocardial oxygen consumption; +122+/-42%; P=0.04). Moreover, xanthine oxidase activity was approximately 4-fold increased in failing versus control dog hearts (387+/-125 versus 78+/-72 pmol/min. mg(-1); P=0.04) but was not detectable in plasma. These data indicate that allopurinol possesses unique inotropic properties, increasing myocardial contractility while simultaneously reducing cardiac energy requirements. The resultant boost in myocardial contractile efficiency may prove beneficial in the treatment of congestive heart failure.


Assuntos
Alopurinol/farmacologia , Cardiotônicos/farmacologia , Inibidores Enzimáticos/farmacologia , Insuficiência Cardíaca/tratamento farmacológico , Contração Miocárdica/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Função Ventricular Esquerda/efeitos dos fármacos , Alopurinol/administração & dosagem , Alopurinol/uso terapêutico , Animais , Estimulação Cardíaca Artificial , Cardiotônicos/administração & dosagem , Cardiotônicos/uso terapêutico , Estado de Consciência , Diástole/efeitos dos fármacos , Progressão da Doença , Cães , Avaliação Pré-Clínica de Medicamentos , Metabolismo Energético , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/uso terapêutico , Insuficiência Cardíaca/metabolismo , Frequência Cardíaca/efeitos dos fármacos , Injeções Intravenosas , Masculino , Proteínas Musculares/antagonistas & inibidores , Miocárdio/enzimologia , Estresse Oxidativo , Xantina Oxidase/antagonistas & inibidores
20.
Eur J Biochem ; 263(1): 238-45, 1999 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-10429209

RESUMO

Mutants of HIV-1 protease that are commonly selected on exposure to different drugs, V82S, G48V, N88D and L90M, showed reduced catalytic activity compared to the wild-type protease on cleavage site peptides, CA-p2, p6pol-PR and PR-RT, critical for viral maturation. Mutant V82S is the least active (2-20% of wild-type protease), mutants N88D, R8Q, and L90M exhibit activities ranging from 20 to 40% and G48V from 50 to 80% of the wild-type activity. In contrast, D30N is variable in its activity on different substrates (10-110% of wild-type), with the PR-RT site being the most affected. Mutants K45I and M46L, usually selected in combination with other mutations, showed activities that are similar to (60-110%) or greater than (110-530%) wild-type, respectively. No direct relationship was observed between catalytic activity, inhibition, and structural stability. The mutants D30N and V82S were similar to wild-type protease in their stability toward urea denaturation, while R8Q, G48V, and L90M showed 1.5 to 2.7-fold decreased stability, and N88D and K45I showed 1.6 to 1.7-fold increased stability. The crystal structures of R8Q, K45I and L90M mutants complexed with a CA-p2 analog inhibitor were determined at 2.0, 1.55 and 1.88 A resolution, respectively, and compared to the wild-type structure. The intersubunit hydrophobic contacts observed in the crystal structures are in good agreement with the relative structural stability of the mutant proteases. All these results suggest that viral resistance does not arise by a single mechanism.


Assuntos
Protease de HIV/química , Protease de HIV/genética , HIV-1/efeitos dos fármacos , HIV-1/enzimologia , Domínio Catalítico/genética , Cristalografia por Raios X , Resistência Microbiana a Medicamentos/genética , Estabilidade Enzimática , Protease de HIV/metabolismo , Inibidores da Protease de HIV/farmacologia , HIV-1/genética , Humanos , Cinética , Modelos Moleculares , Mutação Puntual , Conformação Proteica
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