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1.
Int J Eat Disord ; 30(2): 187-92, 2001 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-11449452

RESUMO

OBJECTIVE: The main aim of this study was to assess the level of agreement between the Eating Disorders Examination (EDE) and its self-report version (EDE-Q) on key items in a clinic sample of patients with bulimia nervosa. A second objective was to assess the concordance between self-reported and objective body weight in the sample. METHOD: Sixty females who met DSM-IV criteria for bulimia nervosa (purging type) participated. Fifty-seven of them completed both the EDE and the EDE-Q. Self-reported weight was obtained during a telephone screening interview. Objective weight was subsequently measured at an assessment about a week later. RESULTS: The EDE generated higher scores than the EDE-Q for the frequency of objective binge and vomiting episodes. The two methods produced equivalent results for subjective binge episodes, laxative and diuretic misuse, and concerns about shape and weight. The self-report method underestimated body weight. DISCUSSION: These findings suggest that some core features of eating disorders are more accurately assessed using the EDE interview.


Assuntos
Imagem Corporal , Bulimia/classificação , Adulto , Peso Corporal , Bulimia/complicações , Bulimia/psicologia , Diuréticos/efeitos adversos , Comportamento Alimentar , Feminino , Humanos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Inquéritos e Questionários/normas , Vômito
2.
J Biol Chem ; 275(50): 39012-7, 2000 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-10960471

RESUMO

The formyl peptide receptor (FPR) is a chemotactic G protein-coupled receptor found on the surface of phagocytes. We have previously shown that the formyl peptide binding site maps to the membrane-spanning region (Miettinen, H. M., Mills, J. S., Gripentrog, J. M., Dratz, E. A., Granger, B. L., and Jesaitis, A. J. (1997) J. Immunol. 159, 4045-4054). Recent reports have indicated that non-formylated peptides, such as MMWLL can also activate this receptor (Chen, J., Bernstein, H. S., Chen, M., Wang, L., Ishi, M., Turck, C. W., and Coughlin, S. R. (1995) J. Biol. Chem. 270, 23398-23401.) Here we show that the selectivity for the binding of different NH(2)-terminal analogs of MMWLL or MLF can be markedly altered by mutating Asp-106 to asparagine or Arg-201 to alanine. Both D106N and R201A produced a similar change in ligand specificity, including an enhanced ability to bind the HIV-1 peptide DP178. In contrast, the mutation R205A exhibited altered specificity at the COOH terminus of fMLF, with R205A binding fMLF-O-butyl > fMLF-O-methyl > fMLF, whereas wt FPR bound fMLF > fMLF-O-methyl approximately fMLF-O-butyl. These data, taken together with our previous finding that the leucine side chain of fMLF is probably bound to FPR near FPR (93)VRK(95) (Mills, J. S., Miettinen, H. M., Barnidge, D., Vlases, M. J., Wimer-Mackin, S., Dratz, E. A., and Jesaitis, A. J. (1998) J. Biol. Chem. 273, 10428-10435.), indicate that the most likely positioning of fMLF in the binding pocket of FPR is approximately parallel to the fifth transmembrane helix with the formamide group of fMLF hydrogen-bonded to both Asp-106 and Arg-201, the leucine side chain pointing toward the second transmembrane region, and the COOH-terminal carboxyl group of fMLF ion-paired with Arg-205.


Assuntos
Receptores Imunológicos/química , Receptores de Peptídeos/química , Alanina/química , Animais , Fármacos Anti-HIV/farmacologia , Arginina/química , Asparagina/química , Ácido Aspártico/química , Sítios de Ligação , Células CHO , Quimiotaxia/efeitos dos fármacos , Cricetinae , Relação Dose-Resposta a Droga , Enfuvirtida , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Proteína gp41 do Envelope de HIV/farmacologia , Humanos , Cinética , Leucina/química , Ligantes , Metionina/química , Modelos Químicos , Mutagênese Sítio-Dirigida , Mutação , Biossíntese Peptídica , Fragmentos de Peptídeos/farmacologia , Fenilalanina/química , Ligação Proteica , Receptores de Formil Peptídeo , Receptores Imunológicos/metabolismo , Receptores de Peptídeos/metabolismo , Sódio/farmacologia , Triptofano/química
3.
Antiviral Res ; 48(3): 187-96, 2000 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-11164505

RESUMO

Antisense phosphorothioate oligonucleotides (ODN1 0x5 OMe) directed against the E1 start region of human papillomavirus 11 (HPV11) can inhibit papillomavirus induced growth of implanted human foreskin in a mouse xenograft model. Administration of a mismatch control oligonucleotide (ODN9 0x5 OMe), in which guanine was replaced with adenine in the same model, had no effect on papilloma induced growth. However, the apparent antiviral activity of ODN1 0x5 OMe was also shown in a lethal mouse cytomegalovirus (CMV) model, in which the oligonucleotides are not expected to have antisense activity. To understand the mechanisms of action of these oligonucleotides, a mismatch oligonucleotide (ODN61 0x5 OMe) was prepared which retained the CpG motifs of ODN1 0x5 OMe. This was tested in the mouse xenograft model and shown to have moderate inhibitory activity. As a definitive experiment, a comparison was made between the efficacy of the active oligonucleotide ODN1 0x5 OMe against two papilloma viruses HPV11 and HPV40. Both these viruses cause benign genital warts, but differ by four bases in their E1 sequence that was the target for ODN1 0x5 OMe. Papillomavirus induced growth in the mouse xenograft model was inhibited by ODN1 0x5 OMe in both cases, suggesting that oligonucleotide molecules have a non-specific antiviral activity that is not directly related to their antisense sequence.


Assuntos
Proteínas de Ligação a DNA/efeitos dos fármacos , Oligonucleotídeos Antissenso/farmacologia , Papillomaviridae/efeitos dos fármacos , Proteínas Virais/efeitos dos fármacos , Animais , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Feminino , Infecções por Herpesviridae/virologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Muromegalovirus/efeitos dos fármacos , Infecções por Papillomavirus/virologia , Transplante de Pele , Transplante Heterólogo , Infecções Tumorais por Vírus/virologia , Proteínas Virais/genética
4.
J Biol Chem ; 273(17): 10428-35, 1998 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-9553101

RESUMO

A novel fluorescent photoaffinity cross-linking probe, formyl-Met-p-benzoyl-L-phenylalanine-Phe-Tyr-Lys-epsilon-N-fluorescei n (fMBpaFYK-fl), was synthesized and used to identify binding site residues in recombinant human phagocyte chemoattractant formyl peptide receptor (FPR). After photoactivation, fluorescein-labeled membranes from Chinese hamster ovary cells were solubilized in octylglucoside and separated by tandem anion exchange and gel filtration chromatography. A single peak of fluorescence was observed in extracts of FPR-expressing cells that was absent in extracts from wild type controls. Photolabeled Chinese hamster ovary membranes were cleaved with CNBr, and the fluorescent fragments were isolated on an antifluorescein immunoaffinity matrix. Matrix-assisted laser desorption ionization mass spectrometry identified a major species with mass = 1754, consistent with the CNBr fragment of fMBpaFYK-fl cross-linked to Val-Arg-Lys-Ala-Hse (an expected CNBr fragment of FPR, residues 83-87). This peptide was further cleaved with trypsin, repurified by antifluorescein immunoaffinity, and subjected to matrix-assisted laser desorption ionization mass spectrometry. A tryptic fragment with mass = 1582 was observed, which is the mass of fMBpaFYK-fl cross-linked to Val-Arg-Lys (FPR residues 83-85), an expected trypsin cleavage product of Val-Arg-Lys-Ala-Hse. Residues 83-85 lie within the putative second transmembrane-spanning region of FPR near the extracellular surface. A 3D model of FPR is presented, which accounts for intramembrane, site-directed mutagenesis results (Miettinen, H. M., Mills, J., Gripentrog, J., Dratz, E. A., Granger, B. L., and Jesaitis, A. J. (1997) J. Immunol. 159, 4045-4054) and the photochemical cross-linking data.


Assuntos
N-Formilmetionina Leucil-Fenilalanina/metabolismo , Neutrófilos/metabolismo , Receptores Imunológicos/metabolismo , Receptores de Peptídeos/metabolismo , Marcadores de Afinidade , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células CHO , Membrana Celular/metabolismo , Cricetinae , Corantes Fluorescentes , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Receptores de Formil Peptídeo , Receptores Imunológicos/química , Receptores Imunológicos/genética , Receptores de Peptídeos/química , Receptores de Peptídeos/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
5.
J Gen Virol ; 79 ( Pt 5): 1121-31, 1998 May.
Artigo em Inglês | MEDLINE | ID: mdl-9603327

RESUMO

Hepadnaviruses have a complex replication cycle which includes reverse transcription of the pregenomic RNA. The initial step in this process in hepatitis B virus (HBV) requires the viral polymerase to engage a highly stable region of secondary structure within the pregenomic RNA termed the epsilon stem-loop. While reverse transcriptases belonging to the retrovirus family use a specific cellular tRNA as primer, HBV polymerase utilizes a tyrosine residue located within its own N terminus. Therefore, the first deoxyribonucleotide is covalently coupled to HBV polymerase prior to extension of the DNA strand by conventional reverse transcription. We have expressed HBV polymerase in a baculovirus and following purification have found it to be active with respect to protein-priming and reverse transcription of copurified RNA. Importantly, we found both of these processes to be critically dependent on the presence of the epsilon stem-loop. The metal ion preferences of HBV polymerase were also investigated for both the protein-priming and reverse transcription activities of this enzyme. Reverse transcription was dependent on magnesium, with an optimal concentration of 5 mM. However, protein-priming was strongly favoured by manganese ions and was optimal at a concentration of 1 mM. Thus, using manganese as sole source of metal ions our activity assay is restricted to the protein-priming event and will allow the search for novel antivirals specifically blocking this unique mechanism.


Assuntos
Produtos do Gene pol/metabolismo , Vírus da Hepatite B/enzimologia , Metais , RNA Viral/química , Animais , Cátions Bivalentes , Linhagem Celular , Produtos do Gene pol/genética , Humanos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Spodoptera , Relação Estrutura-Atividade
7.
J Immunol ; 159(8): 4045-54, 1997 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-9378994

RESUMO

We propose that the N-formyl-Met-Leu-Phe binding site in the human neutrophil formyl peptide receptor (FPR) lies in the predicted transmembrane region. We examined the expression, binding, and G protein coupling of 28 mutated forms of FPR in stably transfected Chinese hamster ovary cells. The amino acids we mutated are: 1) predicted to be oriented toward the interhelical space; 2) analogous to those required for ligand binding in various other G protein-coupled receptors; 3) divergent from lipoxin A4 receptor, a low affinity receptor for formylated peptides; and 4) either highly conserved or divergent in other G protein-coupled receptors. Some mutations resulted in intracellular retention, suggesting that the receptors were misfolded. Most mutated receptors that were transported to the plasmalemma bound f-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein with affinities similar to the wild-type receptor (Kd = 6 nM). However, mutations L78A (helix II), D106N, L109A (helix III), T157A (helix IV), R201A, I204Y, and R205A (helix V), W254A and Y257A (helix VI), and F291A (helix VII) resulted in reduced affinities (Kd = 30-128 nM). Of these mutations, D106N, R201A, and R205A also appeared to affect G protein coupling, suggesting that these residues may also be involved in signal transduction and/or are essential for proper folding of the molecule. Some of the FPR residues that appeared to be involved in binding of formylated peptides were located at sites analogous to those identified in ligand binding to certain other G protein-coupled receptors. It is thus possible that several G protein-coupled receptors have a common placement of ligand-binding amino acids.


Assuntos
N-Formilmetionina Leucil-Fenilalanina/metabolismo , Mapeamento de Peptídeos , Receptores Imunológicos/metabolismo , Receptores de Peptídeos/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Sítios de Ligação/imunologia , Células CHO , Membrana Celular/química , Membrana Celular/metabolismo , Cricetinae , Proteínas de Ligação ao GTP/metabolismo , Humanos , Ligantes , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica/genética , Ligação Proteica/imunologia , Estrutura Terciária de Proteína , Receptores de Formil Peptídeo , Receptores Imunológicos/biossíntese , Receptores Imunológicos/química , Receptores Imunológicos/genética , Receptores de Peptídeos/biossíntese , Receptores de Peptídeos/química , Receptores de Peptídeos/genética
8.
J Gen Virol ; 78 ( Pt 9): 2153-7, 1997 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-9292001

RESUMO

Varicella-zoster virus (VZV) genes 33 and 33.5 are predicted to encode the VZV proteinase and its substrate (the assembly protein) respectively. These genes were expressed in insect cells using recombinant baculovirus and it was confirmed that gene 33 encodes a proteinase capable of autoproteolytic processing at two positions. When VZV gene 33.5 was co-expressed with the VZV proteinase, processing of the VZV33.5 gene product was observed. A polyclonal antiserum to the VZV assembly protein domain highlighted a set of proteins in VZV infected HEL cells identical to those identified in insect cells expressing VZV genes 33 and 33.5. To facilitate further characterization of the VZV proteinase the enzyme was purified by affinity chromatography from an E. coli expression system and in vitro activity was observed.


Assuntos
Herpesvirus Humano 3/enzimologia , Serina Endopeptidases/metabolismo , Proteínas Virais/metabolismo , Animais , Baculoviridae/genética , Linhagem Celular , Escherichia coli/genética , Genes Virais , Herpesvirus Humano 3/genética , Precursores de Proteínas/biossíntese , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Serina Endopeptidases/biossíntese , Serina Endopeptidases/genética , Serina Endopeptidases/isolamento & purificação , Spodoptera , Proteínas Virais/biossíntese , Proteínas Virais/genética , Proteínas Estruturais Virais/genética
9.
J Virol ; 71(5): 3886-94, 1997 May.
Artigo em Inglês | MEDLINE | ID: mdl-9094665

RESUMO

The herpes simplex virus VP16 protein functions as a potent transcriptional activator and targets DNA sites with the consensus TAATGARAT present in all the viral immediate-early gene promoters. To do so, VP16 directs assembly of a multiprotein complex involving two cellular proteins, host cell factor (HCF) and the Oct-1 DNA-binding transcription factor. To investigate the importance of specific protein-protein interactions to formation of this VP16-induced complex (VIC), we used oligopeptides to prevent VIC assembly. Linear and cyclic peptides corresponding to a region of VP16 previously implicated in complex formation were potent inhibitors of VIC assembly. To further characterize the protein interactions involved, we cloned a human cDNA encoding the minimal VP16 interaction domain of HCF, containing amino acids 1 to 380 [HCF (1-380)]. The REHAYS-based peptides active in preventing VIC assembly were found to specifically block binding of VP16 to HCF (1-380), without affecting VP16-Oct-1 binding. The inhibitory activity of these VP16 peptides was strictly sequence specific for the EHAY residues. Site-directed mutagenesis of the HCF (1-380) domain revealed residues E102 and K105 to be critical determinants in support of VIC formation. Alteration of a single residue in HCF, K105, was shown to virtually abolish complex assembly. Interestingly however, none of the HCF mutants that were impaired in their ability to support complex formation exhibited defects in direct VP16 binding, supporting loss of function at a higher order in complex assembly.


Assuntos
Proteínas de Ligação a DNA , Proteína Vmw65 do Vírus do Herpes Simples/fisiologia , Proteínas/fisiologia , Sequência de Aminoácidos , Proteínas de Homeodomínio/metabolismo , Fator C1 de Célula Hospedeira , Humanos , Dados de Sequência Molecular , Mutação , Fator 1 de Transcrição de Octâmero , Relação Estrutura-Atividade , Fatores de Transcrição/metabolismo
10.
Biochim Biophys Acta ; 1339(1): 113-25, 1997 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-9165106

RESUMO

Genes encoding a number of mutants of HIV-1 proteinase were sub-cloned and expressed in E. coli. The proteinases containing mutations of single residues (e.g., G48V, V82F, I84V and L90M) were purified and their catalytic efficiencies relative to that of wild-type proteinase were examined using a polyprotein (recombinant HIV-1 gag) substrate and several series of synthetic peptides based on the -Hydrophobic * Hydrophobic-, -Aromatic * Pro- and pseudo-symmetrical types of cleavage junction. The L90M proteinase showed only small changes, whereas the activity of the other mutant enzymes was compromised more severely, particularly towards substrates of the -Aromatic * Pro- and pseudo-symmetrical types. The susceptibility of the mutants and the wild-type proteinase to inhibition by eleven different compounds was compared. The L90M proteinase again showed only marginal changes in its susceptibility to all except one of the inhibitors examined. The K(i) values determined for one inhibitor (Ro31-8959) showed that its potency towards the V82F, L90M, I84V and G48V mutant proteinases respectively was 2-, 3-, 17- and 27-fold less than against the wild-type proteinase. Several of the other inhibitors examined form a systematic series with Ro31-8959. The inhibition constants derived with these and a number of other inhibitors, including ABT-538 and L-735,524, are used in conjunction with the data on enzymic efficiency to assess whether each mutation in the proteinase confers an advantage for viral replication in the presence of any given inhibitor.


Assuntos
Ácido Aspártico Endopeptidases/genética , HIV-1/enzimologia , Fármacos Anti-HIV/farmacologia , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Ácido Aspártico Endopeptidases/metabolismo , Clonagem Molecular , Escherichia coli , Produtos do Gene gag/metabolismo , Inibidores da Protease de HIV/farmacologia , Mutação , Ritonavir/farmacologia , Saquinavir/farmacologia
11.
J Virol ; 70(6): 4136-41, 1996 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-8648756

RESUMO

After the U53 gene encoding the proteinase from human herpesvirus 6 (HHV-6) was sequenced, it was expressed in Escherichia coli, and the activity of the purified, recombinant HHV-6 proteinase was characterized quantitatively by using synthetic peptide substrates mimicking the release and maturation cleavage sites in the polyprotein precursors of HHV-6, human cytomegalovirus (CMV), murine CMV, and Epstein-Barr virus. Despite sharing 40% identity with other betaherpesvirus proteinases such as human CMV proteinase, the one-chain HHV-6 enzyme was distinguished from these two-chain proteinases by the absence of an internal autocatalytic cleavage site.


Assuntos
Endopeptidases/metabolismo , Herpesvirus Humano 6/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Endopeptidases/genética , Endopeptidases/isolamento & purificação , Humanos , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Coelhos , Proteínas Recombinantes/metabolismo
13.
J Biol Chem ; 269(7): 4787-93, 1994 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-8106448

RESUMO

Genes were constructed to encode single-chain tethered human immunodeficiency virus HIV-1/HIV-1 and HIV-2/HIV-2 homodimeric proteinases and two HIV-1/HIV-2 heterodimers which differed in the nature of the interface strands. All four constructs under the control of a heat-inducible promoter were expressed in E. coli and the resultant proteinases were purified therefrom. Kinetic parameters (Km, kcat and kcat/Km) were derived for the interaction of the tethered homo and heterodimeric proteinases with two distinct substrates at a variety of pH values. All four enzymes were comparably active toward one substrate. With the second substrate at pH 4.7, the kcat/Km value was best for the HIV-1/1 tethered homodimer, 15-fold lower for the two heterodimeric proteinases, and was reduced by an additional 6-fold for the HIV-2/2 homodimer. From the Ki values determined for the interactions of the four tethered dimer proteinases with a systematic series of synthetic inhibitors, a parallel trend was observed. Whereas several inhibitors were equipotent against all four enzymes, two were discriminatory in that they inhibited strongly the HIV-1/1 homodimer and the two heterodimeric proteinases but had little effect on the HIV -2/2 tethered homodimer (or its untethered wild-type counterpart from HIV-2). The significance of these findings for active site interaction with HIV-proteinases is considered.


Assuntos
Ácido Aspártico Endopeptidases/metabolismo , Inibidores da Protease de HIV/farmacologia , Protease de HIV/metabolismo , HIV-1/enzimologia , HIV-2/enzimologia , Sequência de Aminoácidos , Ácido Aspártico Endopeptidases/biossíntese , Clonagem Molecular , Escherichia coli , Protease de HIV/biossíntese , Inibidores da Protease de HIV/metabolismo , Cinética , Substâncias Macromoleculares , Dados de Sequência Molecular , Plasmídeos , Reação em Cadeia da Polimerase , Multimerização Proteica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , Especificidade por Substrato
15.
J Biol Chem ; 268(31): 23611-5, 1993 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-8226888

RESUMO

Activation of cGMP phosphodiesterase (PDE) by the rod G-protein transducin is a key event in visual signal transduction in vertebrate photoreceptor cells. Interaction between the GTP-bound form of the alpha-subunit of transducin (alpha t*) and the PDE inhibitory gamma-subunit (P gamma) is a major component of PDE activation. The central polycationic region of P gamma, P gamma-24-45, has been implicated as one of the sites involved in alpha t*.P gamma interaction. Here we determine the site on alpha t* that interacts with P gamma-24-45 using a photo-cross-linking approach. The synthetic peptides Cys(ACM)Tyr-P gamma-24-45-Cys (where ACM indicates acetamidomethyl group) and Cys-P gamma-24-45 were labeled with 4-(N-maleimido)benzophenone at the COOH and NH2 termini, respectively, and then cross-linked to alpha t. When the photoprobe was attached to the COOH terminus of the peptide, a specific high yield cross-linked product (80%) was formed between the peptide and alpha t GTP gamma S (guanosine 5'-O-(thiotriphosphate)). A lower yield of cross-linking (35%) was seen between the peptide and alpha t GDP. The site of cross-linking between Cys(ACM)Tyr-P gamma-24-45-Cys and alpha t GTP gamma S was localized to within alpha t-306-310 using a variety of chemical and proteolytic cleavages of the cross-linked product, analysis of the fragments with SDS-polyacrylamide gel electrophoresis, and matrix-assisted laser desorption ionization mass spectrometry.


Assuntos
3',5'-GMP Cíclico Fosfodiesterases/metabolismo , Transducina/metabolismo , 3',5'-GMP Cíclico Fosfodiesterases/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Bovinos , Reagentes de Ligações Cruzadas , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Substâncias Macromoleculares , Espectrometria de Massas , Dados de Sequência Molecular , Peptídeos/química , Segmento Externo da Célula Bastonete/química , Transducina/química
16.
FEBS Lett ; 314(3): 449-54, 1992 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-1468583

RESUMO

The wild-type -Phe*Pro- bond located at the N-terminus of the mature aspartic proteinase of HIV-1 was replaced by -Ile-Pro- or -Val-Pro-. By this means, processing at this cleavage junction was prevented and so, extended or precursor forms of HIV-proteinase were generated. These constructs were expressed in Escherichia coli, purified therefrom, and their specificity, activity at different pH values and susceptibility to the potent inhibitor, Ro31-8959, was assessed. A hitherto unobserved cleavage junction (at approximately Ala-Phe*Leu-Gln approximately) in the frame-shift region of the gag-pol viral genome was identified and confirmed by demonstrating cleavage of a synthetic peptide corresponding to this region. The implications for viral replication of self-processing at neural pH by proteinase whilst still present (in a precursor form) as a component of the polyprotein are considered; such reactions, however, are still blocked even at pH values as high as 8.0 by Ro31-8959.


Assuntos
Precursores Enzimáticos/metabolismo , Protease de HIV/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Cromatografia de Afinidade , Clonagem Molecular , DNA de Cadeia Simples , Eletroforese em Gel de Poliacrilamida , Precursores Enzimáticos/genética , Escherichia coli , Protease de HIV/genética , Dados de Sequência Molecular
17.
J Biol Chem ; 267(35): 25067-72, 1992 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-1334079

RESUMO

In photoreceptor cells of vertebrates light activates a series of protein-protein interactions resulting in activation of a cGMP-phosphodiesterase (PDE). Interaction between the GTP-bound form of rod G-protein alpha-subunit (alpha t) and PDE inhibitory gamma-subunit (P gamma) is a key event for effector enzyme activation. This interaction has been studied using P gamma labeled with the fluorescent probe, lucifer yellow vinyl sulfone, at Cys-68 (P gamma LY) and sites of interaction on alpha t and P gamma have been investigated. Addition of alpha tGTP gamma S to P gamma LY produced a 3.2-fold increase in the fluorescence of P gamma LY. The Kd for alpha tGTP gamma S.P gamma LY interaction was 36 nM. Addition of 1 microM alpha tGDP had no effect, but in the presence of A1F4-, alpha tGDP increased P gamma LY fluorescence by 85%. When P gamma LY was reconstituted with P alpha beta to form fluorescent holo-PDE, alpha tGTP gamma S increased the fluorescence of holo-PDE with a K0.5 = 0.7 microM. Also, alpha tGTP gamma S stimulated the activity of this PDE over an identical range of concentrations with a similar K0.5 (0.6 microM). alpha tGTP gamma S enhanced the fluorescence of a COOH-terminal P gamma fragment, P gamma LY-46-87, as well (Kd = 1.5 microM). We demonstrate that an alpha t peptide, alpha t-293-314, which activated PDE (Rarick, H. M., Artemyev, N. O., and Hamm, H. E. (1992) Science 256, 1031-1033), mediates PDE activation by interacting with the P gamma-46-87 region. Peptide alpha t-293-314 bound to P gamma LY (K0.5 = 1.2 microM) as well as to the carboxyl-terminal P gamma fragment, P gamma LY-46-87 (K0.5 = 1.7 microM) as measured by fluorescence increase, while other alpha t peptides had no effect. A peptide from the P gamma central region, P gamma-24-46, blocked the interaction between alpha tGTP gamma S and P gamma LY. The Kd for alpha tGTP gamma S.P gamma-24-46 interaction was 0.7 microM. On the other hand, P gamma-24-46 had no effect on alpha t-293-314 interaction with P gamma LY. Our data suggest that there are at least two distinct sites of interaction between alpha tGTP gamma S and P gamma. The interaction between alpha t-293-314 and P gamma-46-87 is important for PDE activation.(ABSTRACT TRUNCATED AT 400 WORDS)


Assuntos
3',5'-GMP Cíclico Fosfodiesterases/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Peptídeos/farmacologia , Células Fotorreceptoras/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Ligação Competitiva , Bovinos , Membrana Celular/metabolismo , Ativação Enzimática , Nucleotídeos de Guanina/farmacologia , Cinética , Substâncias Macromoleculares , Dados de Sequência Molecular , Peptídeos/síntese química
18.
J Neurochem ; 59(2): 708-14, 1992 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-1629740

RESUMO

Substantial amounts of phospholipase A2 activity were detected in bovine brain cytosol. The major phospholipase A2 activity was present in the precipitate at 40% saturation with solid ammonium sulfate. After the desaltate of the precipitate was loaded onto an Ultrogel AcA 54 gel filtration column, almost all the activity eluted in the void volume when chromatographed without 1 M KCl. However, when buffer with 1 M KCl was used as the eluent, two active peaks were obtained. One peak (peak I) eluted in the void volume, and the other (peak II) eluted with an apparent molecular mass of 39 kDa as compared with standards. The former was active with diacylglycero-3-phosphoethanolamine, whereas the latter was active with both diacylglycero-3-phosphoethanolamine and 1-alk-1'-enyl-2-acylglycero-3-phosphoethanolamine (plasmenylethanolamine). The apparent molecular mass of peak I was estimated to be 110 kDa as compared with standards on an Ultrogel AcA 34 gel filtration column. Both peaks were purified further with a hydrophobic chromatography column (AffiGel 10 coupled with plasmenylethanolamine) and then by high-resolution liquid chromatography on an MA7Q column. The phospholipase A2 obtained from peak II migrated as one main band with a 40-kDa molecular mass and two minor bands with 14- and 25-kDa molecular masses. Phospholipase A2 obtained from peak I eluted as a single peak on high-resolution liquid chromatography but contained two bands with apparent molecular masses of 100 and 110 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.(ABSTRACT TRUNCATED AT 250 WORDS)


Assuntos
Encéfalo/enzimologia , Cálcio/fisiologia , Citosol/enzimologia , Fosfolipases A/isolamento & purificação , Animais , Encéfalo/citologia , Bovinos , Cromatografia Líquida , Eletroforese em Gel de Poliacrilamida , Fluorometria , Concentração de Íons de Hidrogênio , Fosfolipases A2
19.
Antiviral Res ; 16(4): 295-305, 1991 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-1810306

RESUMO

Ro 31-8959 inhibits the spread of HIV infection and the production of cytopathic effects in cultures of acutely infected cells. IC50 values for these effects are in the range 0.5-6.0 nM and IC90 values are in the range 6.0-30.0 nM. This inhibitor is effective even when added to cultures at a late stage of infection, after syncytia have started to form. Virus antigen, virus particles and virus cytopathic effects can largely be cleared from cultures treated with compound from 3 days until 6 days post infection. In chronically-infected cells, inhibition of virus maturation can be detected after 24 hours' treatment with 10 nM Ro 31-8959. In addition, a significant reduction of the proteolytic processing of p56 to p24 can be demonstrated in these cells with compound at picomolar concentrations. These properties indicate that Ro 31-8959 is highly effective against HIV with the potential to inhibit acute, established acute and chronic infections.


Assuntos
Antivirais/farmacologia , Infecções por HIV/tratamento farmacológico , Inibidores da Protease de HIV , Fusão Celular/efeitos dos fármacos , Células Cultivadas , Antígenos HIV/análise , Protease de HIV/farmacologia , Humanos , Técnicas In Vitro , Saquinavir , Fatores de Tempo , Replicação Viral/efeitos dos fármacos
20.
FEBS Lett ; 283(2): 180-4, 1991 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-2044756

RESUMO

Mutations were introduced into the P2 and P1 positions of the junctions, (a) linking reverse transcriptase (RT) and integrase (IN) (-Leu*Phe-) and (b) between the p51 and RNase H domain (-Phe*Tyr-) within p66 of RT in the HIV-1 pol polyprotein. Processing by HIV proteinase (PR) in cis was monitored upon expression of these constructs in E. coli. Whereas the presence of Leu or Phe in P1 permitted rapid cleavage at either junction, substitution of a beta-branched (Ile) hydrophobic residue essentially abolished hydrolysis. By contrast, placement of a beta-branched (Val) residue in the P2 position flanking such -Hydrophobic*Hydrophobic- junctions resulted in effective cleavage of the scissile peptide bond. Gly in P2, however, abrogated cleavage. The significance of these findings in terms of PR specificity, polyprotein processing and the generation of homodimeric (p51/p51) RT for crystallisation purposes is discussed.


Assuntos
Produtos do Gene pol/genética , Protease de HIV/metabolismo , HIV-1/genética , Mutagênese Sítio-Dirigida , Sequência de Bases , Clonagem Molecular , Escherichia coli/genética , Produtos do Gene pol/metabolismo , HIV-1/metabolismo , Hidrólise , Dados de Sequência Molecular , Plasmídeos , Proteínas Recombinantes/metabolismo , Especificidade por Substrato
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