RESUMEN
Skin becomes thin, dry, pale, and finely wrinkled with age. Retinoids are a large class of compounds that are important in modern therapy for dermatological treatment of wrinkled skin. Of the retinoids, retinol and vitamin A palmitate are thought to induce thickening of the epidermis and to be effective for treatment of skin diseases. Accordingly, α-lipoic acid or the reduced form, dihydrolipoate, are potent scavengers of hydroxyl radicals, superoxide radicals, peroxyl radicals, singlet oxygen, and nitric oxide with anti-inflammatory properties (1). Cosmetic ingredient stability prediction relies on kinetic quantitative chemical analysis of active components at different temperatures. Vitamin A palmitate and α-lipoic acid, are known to be unstable to light or heat (2). The aims of this study were to evaluate the stability of α-lipoic acid and vitamin A palmitate in the presence of vitamin E (acetate) and other antioxidants in lipophilic/hydrophilic medium (O/W emulsions) at pH 3.0, 5.0, and 7.0. The formulations that were investigated contained 0.12% (w/w) vitamin A palmitate, 0.4% (w/w) vitamin E acetate, and 0.5 % α-lipoic acid (formulation A), supplemented with ascorbyl palmitate, magnesium ascorbyl phosphate, and vitamin C (formulation B) or with butylhydroxytoluene (BHT, formulation C) or ascorbyl palmitate (formulation D). The chemical analyses of α-lipoic acid and vitamin A palmitate were carried out by HPLC. Formulations C and D at pH 7.0 were selected as the most stable for these components. The purpose of this paper is the selection of the most stable formulations for their application in in vivo studies.
Asunto(s)
Cosméticos , Emulsiones , Ácido Tióctico/química , Vitamina A/análogos & derivados , Cromatografía Líquida de Alta Presión , Diterpenos , Estabilidad de Medicamentos , Ésteres de Retinilo , Espectrofotometría Ultravioleta , Vitamina A/químicaRESUMEN
Shiga toxin-producing Escherichia coli (STEC) colonizes the human intestine, causing haemorrhagic colitis and haemolytic uraemic syndrome (HUS). Treatment options are limited to intravenous fluids in part because sublethal doses of some antibiotics have been shown to stimulate increased toxin release and enhance the risk of progression to HUS. Preventative antimicrobial agents, especially those that build on the natural antimicrobial action of the host defence, may provide a better option. In order to survive the acid stress of gastric passage, STEC is equipped with numerous acid resistance and DNA repair mechanisms. Inhibition of acid-induced DNA repair may offer a strategy to target survival of ingested STEC. We report here that brief pretreatment with a novel antimicrobial peptide, which was previously shown to inhibit bacterial DNA repair, significantly and profoundly reduces survival of acid-stressed O157â:âH7 and non-O157â:âH7 STEC seropathotypes that are highly associated with HUS. Reduction in survival rates of STEC range from 3 to 5 log. We also show that peptide/acid treatment results in little or no increase in toxin production, thereby reducing the risk of progression to HUS. This study identifies the peptide wrwycr as a potential new candidate for a preventative antimicrobial for STEC infection.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/farmacología , Escherichia coli O157/efectos de los fármacos , Escherichia coli/clasificación , Escherichia coli/efectos de los fármacos , Ácido Clorhídrico/farmacología , Toxinas Shiga/biosíntesis , Escherichia coli Shiga-Toxigénica/efectos de los fármacos , Animales , Péptidos Catiónicos Antimicrobianos/síntesis química , Péptidos Catiónicos Antimicrobianos/química , Chlorocebus aethiops , Escherichia coli/fisiología , Escherichia coli O157/fisiología , Síndrome Hemolítico-Urémico/microbiología , Humanos , Concentración de Iones de Hidrógeno , Pruebas de Sensibilidad Microbiana , Serotipificación , Células VeroRESUMEN
The stability of ascorbyl palmitate, sodium ascorbyl phosphate and magnesium ascorbyl phosphate in topical formulations was investigated by direct reverse phase high performance liquid chromatography after sample dilution with a suitable buffer - organic solvent mixture. Ascorbyl palmitate, sodium ascorbyl phosphate and magnesium ascorbyl phosphate are derivatives of ascorbic acid which differ in hydrolipophilic properties. They are widely used in cosmetic and pharmaceutical preparations. According to the results, ascorbyl esters showed significant differences: sodium ascorbyl phosphate and magnesium ascorbyl phosphate are more stable derivatives of vitamin C than ascorbyl palmitate and may be easily used in cosmetic products.
Asunto(s)
Ácido Ascórbico/análogos & derivados , Ácido Tióctico/química , Vitamina A/química , Vitamina E/química , Vitaminas/química , Antioxidantes/química , Ácido Ascórbico/química , Cromatografía Líquida de Alta Presión , Cosméticos/química , Estabilidad de Medicamentos , Emulsiones/química , CinéticaRESUMEN
The effectiveness of any cosmetic product containing a functional ingredient is determined by the skin delivery of the active molecule, which is influenced by the type of carrier and the molecule itself. Furthermore, the functional ingredient should be stable in the formulation. The purpose of this paper is to study the stability of lipoic acid in the presence of vitamins A (as palmitate) and E (as acetate) in semisolids for cosmetic use. The systems formulated were studied in regard to their aspect, pH, stability under centrifugation, and rheological behavior. The chemical analyses of lipoic acid and vitamins A and E were carried out by HPLC after studying the specificity of the method employed in each case. The quantitation of the active principles was performed by HPLC with C18 (5 microm) columns. The mobile phase was methanol for the vitamins, with spectrophotometric detection at 325 nm for vitamin A and 230 nm for vitamin E. The mobile phase for lipoic acid was methanol:water (80:20) and phosphoric acid at pH 3.0, with spectrophotometric detection at 332 nm. All systems were stable to centrifugation, and no significant modification of rheological behavior was observed in relation to the base emulsion used as control. The chemical studies performed indicated that although lipoic acid is not very stable in these formulations, the presence of vitamin A favors its chemical stability.
Asunto(s)
Cosméticos/química , Ácido Tióctico/química , Vitamina A/química , Vitamina E/química , Centrifugación , Estabilidad de Medicamentos , Emulsiones/química , Concentración de Iones de Hidrógeno , ViscosidadRESUMEN
A sensitive and simple high-performance liquid chromatographic (HPLC) method for the assay of 6,11-dihydro-2-methoxy-5H-benzo[a]carbazole (1) and 6,11-dihydro-2-methoxy-11-[2-(1-piperidinyl)]ethyl-5H-benzo[a]carbazole (2) was developed. The procedure is based on the use of the reversed-phase high-performance liquid chromatographic (RP-HPLC) method with UV detector. Each analysis required no longer than 11 min. A linear relationship between the concentration of both the drugs and the UV absorbance at 254 nm was obtained. This linearity was maintained over the concentration ranged from 5 to 80 microg/ml. The detection limits were found to be 1.6 and 0.7 ng for compounds 1 and 2. The quantitation limits were found to be 5.3 and 2.5 ng for compounds 1 and 2, respectively. For recovery studies, several determinations were carried out. Recovery values ranged from 98 to 102.1% for compound 1 and from 98.4 to 101.6% for compound 2. Method precision was also evaluated and RSD% found was less than 2%. This method was applied without any interference from degradation products.
Asunto(s)
Antifúngicos/análisis , Carbazoles/análisis , Piperidinas/análisis , Calibración , Cromatografía Líquida de Alta Presión/métodos , Estabilidad de Medicamentos , Sensibilidad y Especificidad , Soluciones , Espectrofotometría Ultravioleta/métodosRESUMEN
In the following report, thermal cycling coupled with random 10-mers as primers was used to construct randomly amplified shotgun libraries (RASLs). This approach allowed shotgun libraries to be constructed from nanogram quantities of input DNA. RASLs contained inserts from throughout a target genome in an unbiased fashion and did not appear to contain chimeric sequences. This protocol should be useful for shotgun sequencing the genomes of unculturable organisms and rapidly producing shotgun libraries from cosmids, fosmids, yeast artificial chromosomes (YACs), and bacterial artificial chromosomes (BACs).
Asunto(s)
Biblioteca de Genes , Reacción en Cadena de la Polimerasa/métodos , Cromosomas Artificiales Bacterianos , Cromosomas Artificiales de Levadura , Colifagos/genética , Cósmidos , Cartilla de ADNRESUMEN
Perceived control is a personality characteristic that contributes to well-being, but few studies have attempted to integrate the functions of perceived control with those of other determinants of health. This research tested two hypotheses about the functions of perceived control: (a) individual differences in perceived control would account for socioeconomic differences in self-rated health status; (b) performance of health-related behaviors would account for the health benefits of perceived control. Using data from adult, nonproxy respondents in the National Population Health Survey of Canada (1995; n = 11, 110), confirmatory factor analysis supported a measurement model of self-rated health status composed of two correlated factors: physical health (i.e., chronic conditions. restricted activities, self-rated general health, physical functional capacity) and mental health (i.e., distress, depression). Structural equation modeling supported the first hypothesis, but not the second, regarding perceived control as a determinant of self-rated physical and mental health. Health-related behaviors partially mediated age differences in self-rated health, but different behaviors functioned in this way for men than for women. The findings suggest that psychological process, that of perceiving control over life events, underlies social inequality in health. Health-related behaviors appear not to serve as the primary mechanism through which perceived control influences health.
Asunto(s)
Actitud Frente a la Salud , Conductas Relacionadas con la Salud , Conocimientos, Actitudes y Práctica en Salud , Estado de Salud , Renta/estadística & datos numéricos , Control Interno-Externo , Salud Mental , Apoyo Social , Adolescente , Adulto , Factores de Edad , Anciano , Anciano de 80 o más Años , Canadá , Niño , Estudios Transversales , Escolaridad , Análisis Factorial , Femenino , Encuestas Epidemiológicas , Humanos , Masculino , Persona de Mediana Edad , Modelos Psicológicos , Factores Sexuales , Factores SocioeconómicosRESUMEN
Silybine (SBN), isosilybine (ISBN), silycristine (SCN), silydianine (SDN), and taxifoline (TXF) are the main active flavonoids commonly found in the dried fruits of Silybum marianum, Gaertner (Compositae). Concentrations of these compounds, except TXF, are usually expressed together as silymarin content. This paper describes a simple dissolution test developed to estimate silymarin (Sl) in pharmaceutical formulations. Five commercial products were tested using this new method (including tablets, sugar tablets, and capsules): two from Argentina, one from Brazil, one from Spain, and one from Italy. Results demonstrated that, provided the dosage form disintegrates, amounts dissolved range from 50 to 90% of the labeled value. Products were analyzed by high performance liquid chromatography (HPLC) and UV spectrophotometry.
Asunto(s)
Antioxidantes/análisis , Química Farmacéutica/métodos , Silimarina/análisis , Cápsulas , Cromatografía Líquida de Alta Presión , Modelos Lineales , ComprimidosRESUMEN
Two recently synthesized, trisubstituted dihydrobenzo(a)carbazoles were investigated regarding their anti-HIV and antitumoral activity. The compounds showed some activity against melanoma, renal cancer and breast cancer cell lines.
Asunto(s)
Antineoplásicos/síntesis química , Carbazoles/síntesis química , Sustancias Intercalantes/síntesis química , Fármacos Anti-VIH/síntesis química , Fármacos Anti-VIH/farmacología , Antineoplásicos/farmacología , Carbazoles/farmacología , Cromatografía en Capa Delgada , Replicación del ADN/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Sustancias Intercalantes/farmacología , Espectroscopía de Resonancia Magnética , Músculo Liso Vascular , Espectrofotometría Infrarroja , Células Tumorales CultivadasRESUMEN
Site-specific recombination is involved in processes ranging from resolution of bacterial chromosome dimers to adeno-associated viral integration and is a versatile tool for mammalian genetics. The bacteriophage lambda-encoded site-specific recombinase integrase (Int) is one of the best studied site-specific recombinases and mediates recombination via four distinct pathways. We have characterized a mutant version of lambda Int, IntT236I; this mutant can perform the bent-L pathway only, whereas the corresponding IntT236A mutant can perform bent-L, excision and integration pathways. Experiments with both IntT236I and IntT236A show that the hydroxyl group of threonine is necessary for wild-type recombination. Substitution of the threonine by serine leads to nearly complete rescue of the mutant phenotypes. In addition, our data show that the IntT236I mutant is defective partially due to obstructive steric interactions. Comparisons of crystal structures reveal that the threonine at residue 236 may play an important role in stabilizing recombination intermediates through solvent-mediated protein-DNA interactions at the core-binding sites and that the hydroxyl group is important for effective cleavage and Holliday junction formation. Our data also indicate that Int contacts the core sites differently in intermediates assembled in excisive versus bent-L recombination.
Asunto(s)
Bacteriófago lambda/enzimología , Integrasas/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Simulación por Computador , Secuencia Conservada , Electroforesis en Gel de Poliacrilamida , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Conformación Proteica , Relación Estructura-Actividad , Treonina/metabolismoRESUMEN
A simple and accurate liquid chromatographic method was developed to estimate cyproterone acetate (CA) in pharmaceuticals. The drug was chromatographed on a reversed-phase C18 column. Eluents were monitored at a wavelength of 254 nm utilizing a mixture (60:40) of acetonitrile and water. Solution concentrations were measured on a weight basis to avoid the use of an internal standard. The method was statistically validated for linearity, accuracy, precision, and selectivity. Due to its simplicity and accuracy, we believe that the method can be used for routine quality control analysis. No specific sample preparation is required except for the use of a column guard and a suitable prefilter attached to the syringe.
Asunto(s)
Antagonistas de Andrógenos/química , Acetato de Ciproterona/química , Cromatografía Líquida de Alta Presión/métodos , ComprimidosRESUMEN
A wide variety of tools have been used to dissect biochemical pathways, inhibitors being chief among them. Combinatorial approaches have made the search for inhibitors much more efficient. We have applied such an approach to identify hexapeptides which inhibit different steps in a site-specific recombination reaction mediated by the bacteriophage lambda integrase protein. Integrase's mechanism is still incompletely understood, in large part because several pathway intermediates remain hard to isolate. Integrase-catalyzed recombination is very efficient, but if blocked, it is highly reversible to substrates; this combination makes some intermediates exceedingly transient. We have used synthetic peptide combinatorial libraries to screen for hexapeptides that affect the recombination pathway at different stages, and have identified two families of peptides: one probably blocks DNA cleavage, the other may stabilize the Holliday junction intermediates. These peptides do not resemble parts of integrase or any of the other helper functions in the pathway. The deconvolution of hexapeptide libraries based both on inhibition of an enzymatic reaction as well as on accumulation of reaction intermediates is a novel approach to finding useful tools for dissecting a biochemical pathway.
Asunto(s)
Bacteriófago lambda/enzimología , Técnicas Químicas Combinatorias , Inhibidores de Integrasa/aislamiento & purificación , Inhibidores de Integrasa/farmacología , Integrasas/metabolismo , Biblioteca de Péptidos , Recombinación Genética/efectos de los fármacos , Alanina/genética , Alanina/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Bacteriófago lambda/genética , Catálisis/efectos de los fármacos , ADN/química , ADN/genética , ADN/metabolismo , Endopeptidasa K/metabolismo , Concentración 50 Inhibidora , Inhibidores de Integrasa/química , Integrasas/genética , Cinética , Conformación de Ácido Nucleico/efectos de los fármacos , Péptidos/química , Péptidos/genética , Péptidos/aislamiento & purificación , Péptidos/farmacología , Unión Proteica , Recombinación Genética/genéticaRESUMEN
The study of biochemical pathways requires the isolation and characterization of each and every intermediate in the pathway. For the site-specific recombination reactions catalyzed by the bacteriophage lambda tyrosine recombinase integrase (Int), this has been difficult because of the high level of efficiency of the reaction, the highly reversible nature of certain reaction steps, and the lack of requirements for high-energy cofactors or metals. By screening synthetic peptide combinatorial libraries, we have identified two related hexapeptides, KWWCRW and KWWWRW, that block the strand-cleavage activity of Int but not the assembly of higher-order intermediates. Although the peptides bind DNA, their inhibitory activity appears to be more specifically targeted to the Int-substrate complex, insofar as inhibition is resistant to high levels of non-specific competitor DNA and the peptides have higher levels of affinity for the Int-DNA substrate complex than for DNA alone. The peptides inhibit the four pathways of Int-mediated recombination with different potencies, suggesting that the interactions of the Int enzyme with its DNA substrates differs among pathways. The KWWCRW and KWWWRW peptides also inhibit vaccinia virus topoisomerase, a type IB enzyme, which is mechanistically and structurally related to Int. The peptides differentially affect the forward and reverse DNA transesterification steps of the vaccinia topoisomerase. They block formation of the covalent vaccinia topoisomerase-DNA intermediate, but have no apparent effect on DNA religation by preformed covalent complexes. The peptides also inhibit Escherichia coli topoisomerase I, a type IA enzyme. Finally, the peptides inhibit the bacteriophage T4 type II topoisomerase and several restriction enzymes with 2000-fold lower potency than they inhibit integrase in the bent-L pathway.
Asunto(s)
ADN-Topoisomerasas de Tipo I/metabolismo , ADN/metabolismo , Inhibidores de Integrasa/farmacología , Integrasas/metabolismo , Péptidos/farmacología , Inhibidores de Topoisomerasa I , Secuencia de Aminoácidos , Sitios de Ligazón Microbiológica/genética , Proteínas Bacterianas/metabolismo , Bacteriófago T4/enzimología , Bacteriófago lambda/enzimología , Secuencia de Bases , Catálisis/efectos de los fármacos , ADN/química , ADN/genética , Enzimas de Restricción del ADN/antagonistas & inhibidores , Enzimas de Restricción del ADN/metabolismo , ADN Superhelicoidal/química , ADN Superhelicoidal/genética , ADN Superhelicoidal/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/farmacología , Escherichia coli/enzimología , Concentración 50 Inhibidora , Inhibidores de Integrasa/química , Factores de Integración del Huésped , Cinética , Conformación de Ácido Nucleico/efectos de los fármacos , Concentración Osmolar , Péptidos/química , Unión Proteica/efectos de los fármacos , Recombinación Genética/efectos de los fármacos , Recombinación Genética/genética , Especificidad por Sustrato , Vaccinia/enzimologíaAsunto(s)
Antifúngicos/síntesis química , Candida/efectos de los fármacos , Carbazoles/síntesis química , Triazoles/farmacología , Antifúngicos/farmacología , Antifúngicos/toxicidad , Carbazoles/farmacología , Carbazoles/toxicidad , Fluconazol/farmacología , Pruebas de Sensibilidad Microbiana , Mutágenos/toxicidad , Salmonella typhimurium/efectos de los fármacos , Salmonella typhimurium/genéticaRESUMEN
Bacteriophage lambda integrase (Int) catalyzes at least four site-specific recombination pathways between pairs of attachment (att) sites. Protein-protein contacts between monomers of Int are presumed to be important for these site-specific recombination events for several reasons: Int binds to the att sites cooperatively, catalytic Int mutants can complement each other for strand cleavage, and crystal structures for two other recombinases in the Int family (Cre from phage P1 and Int from Haemophilus influenzae phage HP1) show extensive protein-protein contacts between monomers. We have begun to investigate interactions between Int monomers by three approaches. First, using a genetic assay, we show that regions of protein-protein interactions occur throughout Int, including in the amino-terminal domain. This domain was previously thought to be important only for high-affinity protein-DNA interactions. Second, we have found that an amino-terminal His tag reduces cooperative binding to DNA. This disruption in cooperativity decreases the stable interaction of Int with core sites, where catalysis occurs. Third, using protein-protein cross-linking to investigate the multimerization of Int during recombination, we show that Int predominantly forms dimers, trimers, and tetramers. Moreover, we show that the cysteine at position 25 is present at or near the interface between monomers that is involved in the formation of dimers and tetramers. Our evidence indicates that the amino-terminal domain of Int is involved in protein-protein interactions that are likely to be important for recombination.
Asunto(s)
Sitios de Ligazón Microbiológica , Bacteriófago lambda/genética , Integrasas/genética , Integrasas/metabolismo , Recombinación Genética , Bacteriófago lambda/enzimología , Western Blotting , ADN/metabolismo , ADN Bacteriano/metabolismo , ADN Viral/metabolismo , Dimerización , Integrasas/química , Unión Proteica , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Integration host factor (IHF) is a bacterial protein that binds and severely bends a specific DNA target. IHF binding sites are approximately 30 to 35 bp long and are apparently divided into two domains. While the 3' domain is conserved, the 5' domain is degenerate but is typically AT rich. As a result of physical constraints that IHF must impose on DNA in order to bind, it is believed that this 5' domain must possess structural characteristics conducive for both binding and bending with little regard for specific contacts between the protein and the DNA. We have examined the sequence requirements of the 5' binding domain of the IHF binding target. Using a SELEX procedure, we randomized and selected variants of a natural IHF site. We then analyzed these variants to determine how the 5' binding domain affects the structure, affinity, and function of an IHF-DNA complex in a native system. Despite finding individual sequences that varied over 100-fold in affinity for IHF, we found no apparent correlation between affinity and function.
Asunto(s)
Proteínas Bacterianas/metabolismo , ADN/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos/genética , Bacteriófago lambda/genética , Composición de Base , Secuencia de Bases , Unión Competitiva , Clonación Molecular , Secuencia de Consenso/genética , ADN/química , ADN/genética , Huella de ADN , Proteínas de Unión al ADN/metabolismo , Biblioteca de Genes , Concentración 50 Inhibidora , Factores de Integración del Huésped , Ligandos , Conformación de Ácido Nucleico , Unión Proteica , Recombinación Genética/genética , Análisis de Secuencia de ADNRESUMEN
Bacteriophage lambda uses site-specific recombination to move its DNA into and out of the Escherichia coli genome. The recombination event is mediated by the phage-encoded integrase (Int) at short DNA sequences known as attachment ( att ) sites. Int catalyzes recombination via at least four distinct pathways, distinguishable by their requirements for accessory proteins and by the sequence of their substrates. The simplest recombination reaction catalyzed by Int does not require any accessory proteins and takes place between two attL sites. This reaction proceeds through an intermediate known as the straight-L bimolecular complex (SL-BMC), a stable complex which contains two attL sites synapsed by Int. We have investigated the orientation of the two substrates in the SL-BMC with respect to each other using two independent direct methods, a ligation assay and visualization by atomic force microscopy (AFM). Both show that the two DNA substrates in the complex are arranged in a tetrahedral or nearly square planar alignment skewed towards parallel. The DNA molecules in the complex are bent.
Asunto(s)
Bacteriófago lambda/genética , ADN Viral , Recombinación Genética , Sitios de Ligazón Microbiológica , ADN Viral/ultraestructura , Microscopía de Fuerza AtómicaRESUMEN
A simple and accurate liquid chromatographic method was developed for estimation of estradiol valerate and medroxyprogesterone acetate in pharmaceuticals. Drugs were chromatographed on a reverse phase C18 column, using a mixture (30:70) of ammonium nitrate buffer and acetonitrile and eluants monitored at a wavelength of 280 nm. Solution concentrations were measured on a weight basis to avoid the use of an internal standard. The method was statistically validated for its linearity, accuracy, precision and selectivity. Due to its simplicity and accuracy, the authors believe that the method may be used for routine quality control analysis. It does not require any specific sample preparation except the use of a column guard before the analytical column and suitable prefilter attached to the syringe prior to injection.