Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 13 de 13
Filtrar
1.
J Nanosci Nanotechnol ; 7(8): 2767-73, 2007 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-17685295

RESUMEN

This study describes the synthesis of a free-standing nanogold membrane by the spontaneous reduction of aqueous chloroaurate ions by the diamine molecule DAEE at a liquid-liquid interface. The free standing nanogold membrane, provides a biocompatible surface for the immobilization of proteins. F-Protease (F-Prot) was then bound to the nanogold membrane via interaction with the gold nanoparticles leading to a new class of biocatalyst. A highlight of the new biocatalyst wherein the enzyme is bound to the nanogold membrane is the ease with which separation from the reaction medium may be achieved by simple filtration. In relation to the free enzyme in solution, the F-Prot in the bioconjugate material exhibited a slightly higher biocatalytic activity and significantly enhanced pH and temperature stability. The F-Prot nanogold membrane bioconjugate material also exhibited excellent biocatalytic activity over ten successive reuse cycles.


Asunto(s)
Proteínas Fúngicas/química , Oro/química , Nanopartículas del Metal/química , Nanocompuestos/química , Péptido Hidrolasas/química , Catálisis , Cloro/química , Concentración de Iones de Hidrógeno , Iones , Microscopía Electrónica de Transmisión , Nanopartículas/química , Nanotecnología/métodos , Temperatura
2.
Curr Opin Microbiol ; 2(2): 175-80, 1999 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-10322168

RESUMEN

Both prokaryotes and eukaryotes exhibit a cold-shock response upon an abrupt temperature downshift. Cold-shock proteins are synthesized to overcome the deleterious effects of cold shock. CspA, the major cold-shock protein of Escherichia coli, has recently been studied with respect to its structure, function and regulation at the level of transcription, translation and mRNA stability. Homologues of CspA are present in a number of bacteria. Widespread distribution, ancient origin, involvement in the protein translational machinery of the cell and the existence of multiple families in many organisms suggest that these proteins are indispensable for survival during cold-shock acclimation and that they are probably also important for growth under optimal conditions.


Asunto(s)
Adaptación Biológica , Frío , Regulación de la Expresión Génica , Animales , Fenómenos Fisiológicos Bacterianos , Proteínas Bacterianas/metabolismo , Mamíferos/fisiología , Fenómenos Fisiológicos de las Plantas , Saccharomyces cerevisiae/fisiología , Factores de Transcripción/metabolismo
3.
Biochim Biophys Acta ; 1208(1): 189-92, 1994 Sep 21.
Artículo en Inglés | MEDLINE | ID: mdl-7916211

RESUMEN

Denatured tubulins form stable complexes with groEL upon dilution into refolding buffer. These complexes are retained on an immunoaffinity column which contains chemically immobilized antibodies to groEL. Tubulin remains bound to the immobilized groEL column after extensive washing and is released upon incubation with groES and ATP. Similar results were obtained with glutamine synthetase. These data suggest that groEL can function while it is attached to a solid support system.


Asunto(s)
Proteínas Bacterianas , Cromatografía de Afinidad , Proteínas de Choque Térmico , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Adenosina Trifosfato/farmacología , Animales , Chaperonina 10 , Chaperonina 60 , Pollos , Cricetinae , Escherichia coli/genética , Proteínas de Choque Térmico/farmacología , Técnicas Inmunológicas , Pliegue de Proteína , Proteínas Recombinantes/aislamiento & purificación , Tubulina (Proteína)/genética
4.
J Med Chem ; 30(2): 437-40, 1987 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-3806625

RESUMEN

Alkylation of adenine (5a) or 2-amino-6-chloropurine (5b) with excess trans-1,4-dichloro-2-butene (4), effected by K2CO3 in dimethyl sulfoxide or tetra-n-butylammonium fluoride in tetrahydrofuran, led in 90-95% regioselectivity to 9-alkylpurines 6a and 6b. The title compounds 2a and 2b were obtained by refluxing intermediates 6a and 6b in 0.1 M NaOH or HCl. Adenine derivative 2a is a substrate for adenosine deaminase whereas both 2a and 2b exhibit 50% inhibition of the growth of murine leukemia L 1210 cell culture at 1 mM concentration.


Asunto(s)
Adenina/análogos & derivados , Antibióticos Antineoplásicos/síntesis química , Guanina/análogos & derivados , Adenina/síntesis química , Adenina/farmacología , Adenina/uso terapéutico , Adenosina/análogos & derivados , Adenosina/farmacología , Inhibidores de la Adenosina Desaminasa , Animales , Guanina/síntesis química , Guanina/farmacología , Guanina/uso terapéutico , Indicadores y Reactivos , Leucemia L1210/tratamiento farmacológico , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Ratones , Espectrofotometría Ultravioleta , Relación Estructura-Actividad
5.
J Med Chem ; 34(1): 421-9, 1991 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-1992143

RESUMEN

A series of unsaturated analogues of nucleosides were prepared and their cytotoxic, antitumor, and antiviral activities were investigated. Alkylation of cytosine with (E)-1,4-dichloro-2-butene gave chloro derivative 2f, which was hydrolyzed to alcohol 2h. Cytosine, adenine, 2-amino-6-chloropurine, thymine, and (Z)-1,4-chloro-2-butene gave compounds 4c-f, which, after hydrolysis, afforded alcohols 4a, 4b, 4g, and 4h. Alkenes 4d and 4e were cyclized to heterocycles 12 and 13. Alkylation of 2,6-diaminopurine with 1,4-dichloro-2-butyne led to chloro derivative 6a, which was hydrolyzed to alcohol 6b. Allenic isomerization of 6b gave compound 5c. Chloro derivatives 2e-g, 4c-f, 5d, and 6c-e as well as pyrimidine oxacyclopentenes 9c and 9d are slow-acting inhibitors of murine leukemia L1210 of IC50 10-100 microM. The most active were analogues 4c, 4d, 4e, and 6e (IC50 10-20 microM). The corresponding hydroxy derivatives were less active of inactive. Inhibition of macromolecular synthesis with compounds 4c, 4d, 6e, 9c, and 9d follows the order: DNA greater than RNA greater than or equal to protein. Cytotoxic effects of 4c, 6e, and 9d are not reversed with any of the four basic ribonucleosides or 2'-deoxyribonucleosides. Inhibitory activity of cytosine derivative 9c is reversed with uridine and 2'-deoxyuridine but not with the corresponding cytosine nucleosides. Zone assays in several tumor cell lines show that active compounds are cytotoxic agents with little selectivity for tumor cells. Analogue 6c showed 16.7% ILS in leukemia P388/o implanted ip in mice at 510 and 1020 mg/kg, respectively. Cytallene (5b) and 6'beta-hydroxyaristeromycin (10) exhibited significant activity against Friend and Rauscher murine leukemia viruses. The rest of the hydroxy derivatives, with the exception of 4a, were moderately effective or inactive as antiviral agents. None of the chloro derivatives or oxacyclopentenes exhibited an antiviral effect at noncytotoxic concentrations. Z-Olefin 4b and 2-aminoadenallene (5c) are substrates for adenosine deaminase.


Asunto(s)
Antimetabolitos Antineoplásicos/síntesis química , Antivirales/síntesis química , Nucleósidos/síntesis química , Adenosina Desaminasa/metabolismo , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Virus de la Leucemia Murina de Friend/efectos de los fármacos , VIH-1/efectos de los fármacos , VIH-2/efectos de los fármacos , Humanos , Indicadores y Reactivos , Leucemia L1210 , Ratones , Estructura Molecular , Nucleósidos/química , Nucleósidos/farmacología , Virus Rauscher/efectos de los fármacos , Relación Estructura-Actividad
6.
Nucleic Acids Symp Ser ; (18): 25-8, 1987.
Artículo en Inglés | MEDLINE | ID: mdl-3697137

RESUMEN

Reaction of adenine (1a) or cytosine (1b) with excess 1,4-dichloro-2-butyne catalyzed by K2CO3 in (CH3)2SO gave the 4-chloro-2-butynyl derivatives 2a and 2b. The latter were converted to the 4-hydroxy-2-butynyl compounds 3a and 3b by refluxing in 0.1 M HCl. Isomerization of 3a in 0.1 M NaOH at 100 degrees C for 1 h gave an equilibrium mixture of 3a and allene 4a. Pure 4a was obtained by column chromatography. Similarly, compound 3b was transformed/0.1 M NaOH, 20% aq. dioxane, 9 h, 100 degrees C/ to a mixture of 3b and 4b from which pure 4b was obtained by chromatography and crystallization. By contrast, reflux of 3a or 3b in 1 M NaOH in 50% aq. dioxane for 1 h afforded cyclized products - dihydrofuryl derivatives 5a and 5b. Hydrogenation of 4a and 5a gave 9-(4-hydroxybutyl)adenine (6a) and 9-(tetrahydro-2-furyl)adenine (7a), respectively. Scope and limitations of allenic isomerization in nucleic acid base series, spectroscopy and biological activity of the obtained products will be discussed.


Asunto(s)
Nucleósidos/síntesis química , Adenina , Citosina , Hidrocarburos Clorados , Indicadores y Reactivos
7.
Mol Microbiol ; 33(5): 1004-14, 1999 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-10476034

RESUMEN

The CspA family of Escherichia coli comprises nine homologous proteins, CspA to CspI. CspA, the major cold shock protein, binds RNA with low sequence specificity and low binding affinity. This is considered to be important for its proposed function as an RNA chaperone to prevent the formation of secondary structures in RNA molecules, thus facilitating translation at low temperature. The cellular functions of other Csp proteins are yet to be fully elucidated, and their sequence specific binding capabilities have not been identified. As a step towards identification of the target genes of Csp proteins, we investigated the RNA binding specificities of CspB, CspC and CspE by an in vitro selection approach (SELEX). In the present study, we show that these proteins are able to bind preferentially to specific RNA/single-stranded DNA sequences. The consensus sequences for CspB, CspC and CspE are U/T stretches, AGGGAGGGA and AU/AT-rich regions, especially AAAUUU, respectively. CspE and CspB have Kd values in the range 0.23-0.9 x 10(-6) M, while CspC has 10-fold lower binding affinity. Consistent with our recent findings of transcriptional regulation of cspA by CspE, we have identified a motif identical to the CspE consensus. This motif is the putative CspE-mediated transcription pause recognition site in a 5'-untranslated region of the cspA mRNA.


Asunto(s)
Proteínas Bacterianas , Proteínas Portadoras/metabolismo , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Proteínas de Choque Térmico/metabolismo , ARN Bacteriano/metabolismo , Secuencia de Bases , Sitios de Unión , Unión Competitiva , Secuencia Conservada , ADN/metabolismo , ADN Bacteriano/metabolismo , ADN de Cadena Simple/metabolismo , Escherichia coli/genética , Amplificación de Genes , Técnicas Genéticas , Datos de Secuencia Molecular , Proteínas de Unión al ARN , Secuencias Repetitivas de Ácidos Nucleicos , Selección Genética , Especificidad por Sustrato
8.
J Bacteriol ; 183(4): 1205-14, 2001 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-11157932

RESUMEN

Nine homologous proteins, CspA to CspI, constitute the CspA family of Escherichia coli. Recent studies are aimed at elucidating the individual cellular functions of these proteins. Two members of this family, CspC and CspE, are constitutively produced at 37 degrees C. In the present study, these two proteins were evaluated for their cellular role(s). The expression of three stress proteins, OsmY, Dps, and UspA, is significantly affected by the overexpression and deletion of CspC and CspE. RpoS is a regulatory element for osmY and dps. Further analysis showed a larger amount and greater stability of the rpoS mRNA as well as a higher level of RpoS itself with the overexpression of CspC and CspE. This suggests that CspC and CspE upregulate the expression of OsmY and Dps by regulating the expression of RpoS itself. Indeed, this upregulation is lost in the Delta rpoS strain. Other RpoS-controlled proteins such as ProP and KatG, are also upregulated by the overexpression of CspC. The present study suggests that CspC and CspE are the important elements involved in the regulation of the expression of RpoS, a global stress response regulator, and UspA, a protein responding to numerous stresses. In the light of these observations, it seems plausible that CspC and CspE function as regulatory elements for the expression of stress proteins in the complex stress response network of E. coli.


Asunto(s)
Proteínas de Escherichia coli , Escherichia coli/fisiología , Regulación Bacteriana de la Expresión Génica , Proteínas de Choque Térmico/metabolismo , Proteínas de Unión Periplasmáticas , Simportadores , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Portadoras/genética , Proteínas de Unión al ADN/genética , Eliminación de Gen , Proteínas de Choque Térmico/biosíntesis , Presión Osmótica , Peroxidasas/genética , Estabilidad del ARN , ARN Bacteriano/metabolismo , ARN Mensajero/metabolismo , Factor sigma/biosíntesis
9.
Arch Microbiol ; 166(6): 414-7, 1996 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-9082920

RESUMEN

In view of the functional similarities between subtilisin Carlsberg and the alkaline protease from Conidiobolus coronatus, the biochemical and structural properties of the two enzymes were compared. In spite of their similar biochemical properties, e.g., pH optima, heat stability, molecular mass, pI, esterase activity, and inhibition by diisopropyl fluorophosphate and phenylmethlysulfonylfluoride, the proteases were structurally dissimilar as revealed by (1) their amino acid compositions, (2) their inhibition by subtilisin inhibitor, (3) their immunological response to specific anti-Conidiobolus protease antibody, and (4) their tryptic peptide maps. Our results demonstrate that although they are functionally analogous, the Conidiobolus protease is structurally distinct from subtilisin Carlsberg. The Conidiobolus protease was also different from other bacterial and animal proteases (e.g. pronase, protease K, trypsin, and chymotrypsin) as evidenced by their lack of response to anti-Conidiobolus protease antibody in double diffusion and in neutralization assays. The Conidiobolus serine protease fails to obey the general rule that proteins with similar functions have similar primary sequences and, thus, are evolutionarily related. Our results strengthen the concept of convergent evolution for serine proteases and provide basis for research in evolutionary relationships among fungal, bacterial, and animal proteases.


Asunto(s)
Entomophthora/enzimología , Serina Endopeptidasas/aislamiento & purificación , Aminoácidos/análisis , Anticuerpos Antifúngicos/inmunología , Quimotripsina/análisis , Endopeptidasa K/análisis , Fluoruros/farmacología , Calor , Concentración de Iones de Hidrógeno , Pruebas de Neutralización , Mapeo Peptídico , Fosfatos/farmacología , Filogenia , Pronasa/análisis , Serina Endopeptidasas/clasificación , Serina Endopeptidasas/inmunología , Inhibidores de Serina Proteinasa/metabolismo , Subtilisinas/antagonistas & inhibidores , Subtilisinas/química , Tripsina/análisis
10.
J Mol Microbiol Biotechnol ; 3(3): 461-5, 2001 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-11361079

RESUMEN

In the present study we report that 4,5-dihydroxy-2-cyclopentan-1-one (DHCP), which is derived from heat-treatment of uronic acid or its derivatives, has antibacterial activity against Escherichia coli. The compound causes complete growth inhibition at 350 microM concentration. We have cloned a gene from E. coli, which confers DHCP resistance when present in multicopy. The putative protein encoded by this gene (dep- DHCP efflux protein) is a transmembrane efflux protein with a high homology to other antibiotic-efflux proteins including those for chloramphenicol, bicyclomycin and tetracycline. However, the Dep protein does not confer cross-resistance to any of the antibiotics tested.


Asunto(s)
Antibacterianos/farmacología , Proteínas Bacterianas/genética , Ciclopentanos/farmacología , Farmacorresistencia Microbiana/genética , Proteínas de Escherichia coli , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Proteínas de Transporte de Membrana , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Portadoras/química , Clonación Molecular , Escherichia coli/crecimiento & desarrollo , Biblioteca Genómica , Cinética , Proteínas de la Membrana/química , Datos de Secuencia Molecular , Biblioteca de Péptidos , Conformación Proteica , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Programas Informáticos
11.
Biochemistry ; 40(38): 11525-32, 2001 Sep 25.
Artículo en Inglés | MEDLINE | ID: mdl-11560501

RESUMEN

We present here the first report of a hydrophilic peptidic inhibitor, ATBI, from an extremophilic Bacillus sp. exhibiting a two-step inhibition mechanism against the aspartic proteases, pepsin and F-prot from Aspergillus saitoi. Kinetic analysis shows that these proteases are competitively inhibited by ATBI. The progress curves are time-dependent and consistent with slow-tight binding inhibition: E + I right arrow over left arrow (k(3), k(4)) EI right arrow over left arrow (k(5), k(6)) EI. The K(i) values for the first reversible complex (EI) of ATBI with pepsin and F-prot were (17 +/- 0.5) x 10(-9) M and (3.2 +/- 0.6) x 10(-6) M, whereas the overall inhibition constant K(i) values were (55 +/- 0.5) x 10(-12) M and (5.2 +/- 0.6) x 10(-8) M, respectively. The rate constant k(5) revealed a faster isomerization of EI for F-prot [(2.3 +/- 0.4) x 10(-3) s(-1)] than pepsin [(7.7 +/- 0.3) x 10(-4) s(-1)]. However, ATBI dissociated from the tight enzyme-inhibitor complex (EI) of F-prot faster [(3.8 +/- 0.5) x 10(-5) s(-1)] than pepsin [(2.5 +/- 0.4) x 10(-6) s(-1)]. Comparative analysis of the kinetic parameters with pepstatin, the known inhibitor of pepsin, revealed a higher value of k(5)/k(6) for ATBI. The binding of the inhibitor with the aspartic proteases and the subsequent conformational changes induced were monitored by exploiting the intrinsic tryptophanyl fluorescence. The rate constants derived from the fluorescence data were in agreement with those obtained from the kinetic analysis; therefore, the induced conformational changes were correlated to the isomerization of EI to EI. Chemical modification of the Asp or Glu by WRK and Lys residues by TNBS abolished the antiproteolytic activity and revealed the involvement of two carboxyl groups and one amine group of ATBI in the enzymatic inactivation.


Asunto(s)
Ácido Aspártico Endopeptidasas/química , Ácido Aspártico Endopeptidasas/metabolismo , Bacillus/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/farmacología , Inhibidores de Proteasas/química , Aspergillus/enzimología , Bacillus/crecimiento & desarrollo , Proteínas Bacterianas/aislamiento & purificación , Cinética , Análisis de los Mínimos Cuadrados , Modelos Químicos , Pepsina A/antagonistas & inhibidores , Pepsina A/química , Pepstatinas/química , Pepstatinas/farmacología , Inhibidores de Proteasas/farmacología
12.
Mol Microbiol ; 31(5): 1429-41, 1999 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-10200963

RESUMEN

Escherichia coli contains nine members of the CspA protein family from CspA to Cspl. To elucidate the cellular function of CspE, we constructed a delta cspE strain. CspE is highly produced at 37 degrees C. The synthesis level of CspE transiently increased during the growth lag period after dilution of stationary-phase cells into the fresh medium at 37 degrees C. This is consistent with the delta cspE phenotype of the longer growth lag period after dilution. The protein synthesis patterns of the delta cspE strain and the wild-type strain were compared using two-dimensional gel electrophoresis. In the delta cspE strain, the synthesis of a number of proteins at 37 degrees C was found to be altered and cspA was derepressed. The derepression of cspA in the delta cspE strain was at the level of transcription in a promoter-independent fashion but was not caused by stabilization of the cspA mRNA, which was shown to be a major cause of CspA induction after cold shock. In vitro transcription assays demonstrated that both CspE and CspA enhanced transcription pause at the region immediately downstream of the cold box, a putative repressor binding site on the cspA mRNA. In a cell-free protein synthesis system using S-30 cell extracts, CspA production was specifically inhibited by the addition of CspE. These results indicate that CspE functions as a negative regulator for cspA expression at 37 degrees C, probably by interacting with the transcription elongation complex at the cspA cold box region.


Asunto(s)
Proteínas Bacterianas/genética , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Proteínas de Choque Térmico/fisiología , Proteínas Bacterianas/metabolismo , Southern Blotting , División Celular , Sistema Libre de Células , ARN Polimerasas Dirigidas por ADN/metabolismo , Densitometría , Escherichia coli/citología , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Modelos Genéticos , Mutagénesis , Regiones Promotoras Genéticas , Temperatura , Factores de Tiempo , Transcripción Genética , beta-Galactosidasa/metabolismo
13.
Proc Natl Acad Sci U S A ; 85(16): 6127-31, 1988 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-3261865

RESUMEN

Although several antiretroviral compounds are already known, almost no acyclic nucleoside derivatives lacking an oxacyclopentane have been reported to exert significant inhibition against human immunodeficiency virus type 1 (HIV-1) in vitro. We found two unsaturated acyclic nucleoside derivatives, adenallene [9-(4'-hydroxy-1',2'-butadienyl)adenine] and cytallene [1-(4'-hydroxy-1',2'-butadienyl)cytosine], that protect various CD4+ T-cell lines from the infectivity and cytopathic effect of HIV-1. These compounds inhibit the expression of HIV-1 gag-encoded protein and suppress viral DNA synthesis at concentrations that do not affect functions of normal T cells in vitro. They also inhibit the in vitro infectivity of another human retrovirus, HIV-2. Further in vitro analyses of the anti-HIV-1 activity revealed that the presence of two cumulated double bonds between the 1' and 2' carbons and between the 2' and 3' carbons confers antiretroviral activity in certain pyrimidine or purine derivatives containing a four-carbon chain. We have also found that the 4'-hydroxyl group is critical for the in vitro anti-HIV activity of adenallene. Our observations may provide structure-activity relationships for acyclic nucleoside analogues and may be of value in developing a new class of experimental drugs for the therapy of HIV-related diseases.


Asunto(s)
Adenina/análogos & derivados , Antivirales/farmacología , Citosina/análogos & derivados , VIH/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Adenina/farmacología , Efecto Citopatogénico Viral/efectos de los fármacos , Citosina/farmacología , ADN Viral/biosíntesis , Productos del Gen gag , Activación de Linfocitos/efectos de los fármacos , Proteínas de los Retroviridae/biosíntesis , Relación Estructura-Actividad
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA