RESUMEN
OBJECTIVE: To identify copy number variant (CNV) causes of periventricular nodular heterotopia (PNH) in patients for whom FLNA sequencing is negative. METHODS: Screening of 35 patients from 33 pedigrees on an Affymetrix 6.0 microarray led to the identification of one individual bearing a CNV that disrupted FLNA. FLNA-disrupting CNVs were also isolated in 2 other individuals by multiplex ligation probe amplification. These 3 cases were further characterized by high-resolution oligo array comparative genomic hybridization (CGH), and the precise junctional breakpoints of the rearrangements were identified by PCR amplification and sequencing. RESULTS: We report 3 cases of PNH caused by nonrecurrent genomic rearrangements that disrupt one copy of FLNA. The first individual carried a 113-kb deletion that removes all but the first exon of FLNA. A second patient harbored a complex rearrangement including a deletion of the 3' end of FLNA accompanied by a partial duplication event. A third patient bore a 39-kb deletion encompassing all of FLNA and the neighboring gene EMD. High-resolution oligo array CGH of the FLNA locus suggests distinct molecular mechanisms for each of these rearrangements, and implicates nearby low copy repeats in their pathogenesis. CONCLUSIONS: These results demonstrate that FLNA is prone to pathogenic rearrangements, and highlight the importance of screening for CNVs in individuals with PNH lacking FLNA point mutations.
Asunto(s)
Proteínas Contráctiles/genética , Reordenamiento Génico/genética , Proteínas de Microfilamentos/genética , Heterotopia Nodular Periventricular/genética , Mutación Puntual/genética , Adulto , Anticonvulsivantes/uso terapéutico , Puntos de Rotura del Cromosoma , ADN/genética , Variaciones en el Número de Copia de ADN , Resistencia a Medicamentos , Exones/genética , Femenino , Filaminas , Humanos , Lactante , Recién Nacido , Análisis por Micromatrices , Persona de Mediana Edad , Hibridación de Ácido Nucleico , Linaje , Reacción en Cadena de la Polimerasa , Embarazo , Reacción en Cadena en Tiempo Real de la Polimerasa , Convulsiones/etiología , Convulsiones/genéticaRESUMEN
A convenient and reliable method to prepare procaterol HCl oral dosage form at an extremely low dosage (25 microg/cap) is presented in this paper. Procaterol HCl was mixed with the film-forming agent hydroxypropyl methylcellulose in an aqueous solution, which was then spray-coated on sugar spheres (Nu-pareil PG 20/25) to produce procaterol HCl pellets. The IR spectra of coated and noncoated pellets indicated that procaterol HCl was coated on the sugar spheres successfully with a weight increment less than 1%. Most of the coated pellets were able to pass through an 18-mesh screen with no agglomeration. The average weights of coated pellets filled inside of capsules were monitored during the filling process. A simple liquid chromatographic method was developed and validated for the assay and uniformity test of procaterol HCl in different dosage forms. The results of assay and content uniformity test for both in-house product and a commercial product, i.e., Meptin-mini tablet, were satisfied. The data of f(2) function and ANOVA analysis for the dissolution profiles of both procaterol HCl products suggested that they are pharmaceutical equivalent. In an in vivo study (n = 24), a single dose of 75 microg procaterol HCl was administrated to each volunteer and the plasma concentration of procaterol was determined by a LC/MS/MS method, developed by the same authors. There were no significant differences (p > 0.05) in the data of AUC(0-->16 h), AUC(0-->infinity), C(max), and MRT for both preparations. It is confirmed that the pellets capsule produced in this study is bioequivalent with Meptin-mini tablet.
Asunto(s)
Agonistas Adrenérgicos beta/farmacocinética , Procaterol/farmacocinética , Tecnología Farmacéutica , Administración Oral , Agonistas Adrenérgicos beta/administración & dosificación , Agonistas Adrenérgicos beta/química , Adulto , Cápsulas , Cromatografía Líquida de Alta Presión , Estudios Cruzados , Humanos , Masculino , Procaterol/administración & dosificación , Procaterol/química , Control de Calidad , Solubilidad , Comprimidos , Equivalencia TerapéuticaRESUMEN
To reach their proper targets, axons rely upon the actions of highly conserved families of attractive and repulsive guidance molecules, including the netrins, Slits, semaphorins and ephrins. These guidance systems are used to generate an astonishingly varied set of neuronal circuits. Here we consider the mechanisms by which a few guidance systems can be used to generate diverse outcomes. Recent studies have revealed extensive transcriptional and post-transcriptional regulation of guidance cues and their receptors, as well as combinatorial mechanisms that integrate information from different families of guidance cues.
Asunto(s)
Comunicación Celular/genética , Diferenciación Celular/genética , Regulación del Desarrollo de la Expresión Génica/fisiología , Conos de Crecimiento/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Sistema Nervioso/embriología , Vías Nerviosas/embriología , Animales , Conos de Crecimiento/ultraestructura , Humanos , Factores de Crecimiento Nervioso/genética , Sistema Nervioso/citología , Sistema Nervioso/metabolismo , Vías Nerviosas/citología , Vías Nerviosas/metabolismo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismoRESUMEN
Robo receptors interact with ligands of the Slit family. The nematode C. elegans has one Robo receptor (SAX-3) and one Slit protein (SLT-1), which direct ventral axon guidance and guidance at the midline. In larvae, slt-1 expression in dorsal muscles repels axons to promote ventral guidance. SLT-1 acts through the SAX-3 receptor, in parallel with the ventral attractant UNC-6 (Netrin). Removing both UNC-6 and SLT-1 eliminates all ventral guidance information for some axons, revealing an underlying longitudinal guidance pathway. In the embryo, slt-1 is expressed at high levels in anterior epidermis. Embryonic expression of SLT-1 provides anterior-posterior guidance information to migrating CAN neurons. Surprisingly, slt-1 mutants do not exhibit the nerve ring and epithelial defects of sax-3 mutants, suggesting that SAX-3 has both Slit-dependent and Slit-independent functions in development.
Asunto(s)
Axones/fisiología , Proteínas de Caenorhabditis elegans , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismo , Animales , Caenorhabditis elegans , Movimiento Celular , Regulación del Desarrollo de la Expresión Génica , Proteínas Fluorescentes Verdes , Proteínas del Helminto/genética , Proteínas del Helminto/metabolismo , Indicadores y Reactivos/metabolismo , Proteínas Luminiscentes/genética , Músculos/inervación , Músculos/fisiología , Mutagénesis/fisiología , Proteínas del Tejido Nervioso/química , Netrinas , Neuronas/fisiología , Neuronas/ultraestructura , Estructura Terciaria de Proteína , Proteínas RoundaboutRESUMEN
Rabbit muscle creatine kinase (CK) was modified by 5,5'-dithio-bis(2-nitrobenzoic acid) accompanied by 3 M guanidine hydrochloride denaturation to produce a partially folded state with modified thiol groups. The partially folded CK was in a monomeric state detected by size exclusion chromatography, native-polyacrylamide gel electrophoresis, circular dichroism, and intrinsic fluorescence studies. After dithiothreitol (DTT) treatment, about 70% CK activity was regained with a two-phase kinetic course. Rate constants calculated for regaining of activity and refolding were compared with those for CK modified with various treatments to show that refolding and recovery of activity were synchronized. To further characterize the partially folded CK state and its folding pathway, the molecular chaperone GroEL was used to evaluate whether it can bind with partly folded CK during refolding, and 1-anilinonaphthalene-8-sulfonate was used to detect the hydrophobic surface of the monomeric state of CK. The monomeric state of CK did not bind with GroEL, although it had a larger area of hydrophobic surface relative to the native state. These results may provide different evidence for the structural requirement of GroEL recognition to the substrate protein compared with previously reported results that GroEL bound with substrate proteins mainly through hydrophobic surface. The present study provides data for a monomeric intermediate trapped by the modification of the SH groups during the refolding of CK. Schemes are given for explaining both the partial folding CK pathway and the refolding pathway.
Asunto(s)
Creatina Quinasa/metabolismo , Músculos/enzimología , Naftalenosulfonatos de Anilina , Animales , Chaperonina 60/química , Chaperonina 60/metabolismo , Dicroismo Circular , Creatina Quinasa/química , Dinitrobencenos , Electroforesis en Gel de Poliacrilamida , Guanidina , Cinética , Desnaturalización Proteica , Pliegue de Proteína , Conejos , Espectrometría de Fluorescencia , Espectrofotometría UltravioletaRESUMEN
To investigate a novel branch of the shikimate biosynthesis pathway operating in the formation of 3-amino-5-hydroxybenzoic acid (AHBA), the unique biosynthetic precursor of rifamycin and related ansamycins, a series of target-directed mutations and heterologous gene expressions were investigated in Amycolatopsis mediterranei and Streptomyces coelicolor. The genes involved in AHBA formation were inactivated individually, and the resulting mutants were further examined by incubating the cell-free extracts with known intermediates of the pathway and analyzing for AHBA formation. The rifL, -M, and -N genes were shown to be involved in the step(s) from either phosphoenolpyruvate/d-erythrose 4-phosphate or other precursors to 3,4-dideoxy-4-amino-d-arabino-heptulosonate 7-phosphate. The gene products of the rifH, -G, and -J genes resemble enzymes involved in the shikimate biosynthesis pathway (August, P. R., Tang, L., Yoon, Y. J., Ning, S., Müller, R., Yu, T.-W., Taylor, M., Hoffmann, D., Kim, C.-G., Zhang, X., Hutchinson, C. R., and Floss, H. G. (1998) Chem. Biol. 5, 69-79). Mutants of the rifH and -J genes produced rifamycin B at 1% and 10%, respectively, of the yields of the wild type; inactivation of the rifG gene did not affect rifamycin production significantly. Finally, coexpressing the rifG-N and -J genes in S. coelicolor YU105 under the control of the act promoter led to significant production of AHBA in the fermented cultures, confirming that seven of these genes are indeed necessary and sufficient for AHBA formation. The effects of deletion of individual genes from the heterologous expression cassette on AHBA formation duplicated the effects of the genomic rifG-N and -J mutations on rifamycin production, indicating that all these genes encode proteins with catalytic rather than regulatory functions in AHBA formation for rifamycin biosynthesis by A. mediterranei.
Asunto(s)
Actinomycetales/enzimología , Actinomycetales/genética , Aminobenzoatos/metabolismo , Análisis Mutacional de ADN/métodos , Genes Bacterianos , Familia de Multigenes , Rifamicinas/biosíntesis , Secuencia de Aminoácidos , Secuencia de Bases , Cartilla de ADN , Genes Reguladores , Hidroxibenzoatos , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Plásmidos , Regiones Promotoras Genéticas , Mapeo Restrictivo , Rifabutina/metabolismoRESUMEN
The role of two thioesterase genes in the premature release of polyketide synthase intermediates during rifamycin biosynthesis in the Amycolatopsis mediterranei S699 strain was investigated. Creation of an in-frame deletion in the rifR gene led to a 30 approximately 60% decrease in the production of both rifamycin B by the S699 strain or a series of tetra- to decaketide shunt products of polyketide chain assembly by the rifF strain. Since a similar percentage decrease was seen in both genetic backgrounds, we conclude that the RifR thioesterase 2 is not involved in premature release of the carbon chain assembly intermediates. Similarly, fusion of the Saccharopolyspora erythraea DEBS3 thioesterase I domain to the C-terminus of the RifE PKS subunit did not result in a noticeable increase in the amount of the undecaketide intermediate formed nor in the amounts of the tetra- to decaketide shunt products. Hence, premature release of the carbon chain assembly intermediates is an unusual property of the Rif PKS itself.
Asunto(s)
Actinomycetales/metabolismo , Antibacterianos/biosíntesis , Esterasas/metabolismo , Extensión de la Cadena Peptídica de Translación , Rifamicinas/biosíntesis , Actinomycetales/genética , Secuencia de Aminoácidos , Secuencia de Bases , Genes Bacterianos , Datos de Secuencia Molecular , Complejos Multienzimáticos/genética , MutaciónRESUMEN
Epidermal nevus syndrome is seldom encountered, and its association with hypermelanosis and the chronic syndrome of inappropriate antidiuretic hormone secretion (SIADH) has never been reported. A male neonate who developed intractable seizures and hyponatremia soon after birth is reported. He had alopecic patches on the scalp at birth. Large areas of skin hyperpigmentation, and epidermal nevi developed gradually. The clinical picture of hypotonic hyponatremia, high urine osmolality, elevated urine sodium, and euvolemia was compatible with SIADH. The seizures did not correlate with the hyponatremia, and no other cause for the seizures could be identified. The hyponatremia became chronic and was treated with a direct supply of sodium chloride. The development of the patient was markedly delayed at the last visit when he was 1 year of age. It is suggested that hypermelanosis and chronic SIADH may also be a variant presentation of epidermal nevus syndrome.
Asunto(s)
Epilepsia/etiología , Hiponatremia/etiología , Síndrome de Secreción Inadecuada de ADH/complicaciones , Melanosis/patología , Nevo Intradérmico/patología , Neoplasias Cutáneas/patología , Encéfalo/patología , Enfermedad Crónica , Diagnóstico Diferencial , Humanos , Síndrome de Secreción Inadecuada de ADH/congénito , Recién Nacido , Masculino , Melanosis/congénito , Nevo Intradérmico/congénito , Neoplasias Cutáneas/congénito , SíndromeRESUMEN
The application of a simple lag-time assay for antioxidant capacity using myoglobin and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) or ABTS has been studied for its general application conditions. In the presence of an antioxidant, the ABTS(*+) radical-cation-forming chromogenic reaction, catalyzed by myoglobin and initiated by hydrogen peroxide (H(2)O(2)), has a lag period, and its duration is linearly correlated to the concentration of that antioxidant. The high linearity between the lag time and the antioxidant concentration remained unchanged regardless of the assay conditions. It was also found that the linearity was better for antioxidants at lower concentrations. The change of assay condition could significantly affect the relative antioxidant value of a chemical to the standard (ascorbic acid), although not to a large extent. Most of antioxidants investigated were found suitable to be assayed using this method. Some antioxidants, e.g., genistein, however, were not, probably due to their low reactivity toward ferrylmyoglobin or ABTS(*+). In conclusion, the lag-time assay is a reliable method for measuring the antioxidant capacity, provided caution is taken for antioxidants that mainly act through lowering the rate of the chromogenic reaction.
Asunto(s)
Antioxidantes/análisis , Indicadores y Reactivos/química , Mioglobina/química , Ácidos Sulfónicos/química , Benzotiazoles , Dimetilsulfóxido/química , Quelantes del Hierro/químicaRESUMEN
There is currently intense interest in unravelling the modus operandi of type I modular polyketide synthases in order to lay the ground work for their use in the combinatorial biosynthesis of new bioactive molecules. Much of our knowledge is derived from studies on 6-deoxyerythronolide B (DEBS), the enzyme assembling the polyketide backbone of erythromycin. Work on the rifamycin polyketide synthase has revealed a number of features that differ from those seen with DEBS.
Asunto(s)
Familia de Multigenes , Rifamicinas/biosíntesis , Modelos Químicos , Complejos Multienzimáticos/metabolismoRESUMEN
The biosynthesis of ansamycin antibiotics, including rifamycin B, involves the synthesis of an aromatic precursor, 3-amino-5-hydroxybenzoic acid (AHBA), which serves as starter for the assembly of the antibiotics' polyketide backbone. The terminal enzyme of AHBA formation, AHBA synthase, is a dimeric, pyridoxal 5'-phosphate (PLP) dependent enzyme with pronounced sequence homology to a number of PLP enzymes involved in the biosynthesis of antibiotic sugar moieties. The structure of AHBA synthase from Amycolatopsis mediterranei has been determined to 2.0 A resolution, with bound cofactor, PLP, and in a complex with PLP and an inhibitor (gabaculine). The overall fold of AHBA synthase is similar to that of the aspartate aminotransferase family of PLP-dependent enzymes, with a large domain containing a seven-stranded beta-sheet surrounded by alpha-helices and a smaller domain consisting of a four-stranded antiparallel beta-sheet and four alpha-helices. The uninhibited form of the enzyme shows the cofactor covalently linked to Lys188 in an internal aldimine linkage. On binding the inhibitor, gabaculine, the internal aldimine linkage is broken, and a covalent bond is observed between the cofactor and inhibitor. The active site is composed of residues from two subunits of AHBA synthase, indicating that AHBA synthase is active as a dimer.
Asunto(s)
Hidroliasas/química , Actinobacteria/enzimología , Sitios de Unión , Simulación por Computador , Cristalización , Cristalografía por Rayos X , Ácidos Ciclohexanocarboxílicos/química , Ácidos Ciclohexanocarboxílicos/metabolismo , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Hidroliasas/antagonistas & inhibidores , Hidroliasas/metabolismo , Modelos Moleculares , Unión Proteica , Pliegue de Proteína , Fosfato de Piridoxal/química , Fosfato de Piridoxal/metabolismoRESUMEN
The assembly of the polyketide backbone of rifamycin B on the type I rifamycin polyketide synthase (PKS), encoded by the rifA-rifE genes, is terminated by the product of the rifF gene, an amide synthase that releases the completed undecaketide as its macrocyclic lactam. Inactivation of rifF gives a rifamycin B nonproducing mutant that still accumulates a series of linear polyketides ranging from the tetra- to a decaketide, also detected in the wild type, demonstrating that the PKS operates in a processive manner. Disruptions of the rifD module 8 and rifE module 9 and module 10 genes also result in accumulation of such linear polyketides as a consequence of premature termination of polyketide assembly. Whereas the tetraketide carries an unmodified aromatic chromophore, the penta- through decaketides have undergone oxidative cyclization to the naphthoquinone, suggesting that this modification occurs during, not after, PKS assembly. The structure of one of the accumulated compounds together with (18)O experiments suggests that this oxidative cyclization produces an 8-hydroxy-7, 8-dihydronaphthoquinone structure that, after the stage of proansamycin X, is dehydrogenated to an 8-hydroxynaphthoquinone.
Asunto(s)
Actinomycetales/enzimología , Actinomycetales/genética , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/metabolismo , Rifamicinas/biosíntesis , Secuencia de Aminoácidos , Secuencia de Bases , Datos de Secuencia Molecular , Estructura Molecular , Mutagénesis , Mapeo Restrictivo , Rifamicinas/metabolismoRESUMEN
Phthalate esters are among the most extensively used industrial chemicals and are widely distributed in the environment. Di-(2-ethylhexyl)phthalate (DEHP) and its hydrolysis product mono-(2-ethylhexyl)phthalate (MEHP) have been examined for genotoxic activity on previous occasions. Only MEHP was found to cause chromosome damage in CHO cells but was without effect in the sister chromatid exchange and hypoxanthine guanine phosphoribosyl assay. DEHP was found to be a weak direct acting mutagen in Salmonella typhimurium strain TA100, the mutagenic activity of which could be abolished by rat liver microsomes (S9 mix). The clastogenicity and weak mutagenicity suggest a possible contributory role for these compounds in the observed carcinogenicity of the phthalates, which have been thought predominantly to be linked to cancer pathology through proliferation of hepatic peroxisomes. The present study showed that these compounds could produce DNA damage in human blood cells in the Comet assay and also, that rat liver microsomes could abolish the effect of DEHP. Thus in the intact animal, no response may be observed.
Asunto(s)
Dietilhexil Ftalato/análogos & derivados , Dietilhexil Ftalato/farmacología , Pruebas de Mutagenicidad , Animales , Células CHO , Cricetinae , Daño del ADN , Dietilhexil Ftalato/farmacocinética , Humanos , Hipoxantina Fosforribosiltransferasa/genética , Microsomas Hepáticos/metabolismo , Mutágenos/farmacología , Ratas , Salmonella typhimurium/efectos de los fármacos , Intercambio de Cromátides Hermanas/efectos de los fármacosRESUMEN
Creatine kinase with its thiol groups modified by 5, 5'-dithio-bis(2-nitrobenzoic acid) has been shown to be partially folded in a monomeric state using fluorescence, circular dichroism, proteolysis, and size exclusion chromatography studies. In the presence of DTT, the partially folded modified creatine kinase can be reactivated and refolded following a biphasic course, suggesting the existence of a monomeric intermediate during the refolding of CK. The results provide evidence for our previously suggested model of the refolding pathway of urea-denatured creatine kinase.
Asunto(s)
Creatina Quinasa/química , Ácido Ditionitrobenzoico/farmacología , Pliegue de Proteína , Animales , Dicroismo Circular , Ditiotreitol/química , Activación Enzimática , Cinética , Músculo Esquelético/enzimología , Conejos , Espectrometría de Fluorescencia , Compuestos de Sulfhidrilo/químicaRESUMEN
In previous studies, N-(N'-acetyl-L-propyl)-N-nitrosoglycine (APNG) has been shown to be a potent mutagen in a variety of genotoxicity assays and a carcinogen in a limited cancer study. APNG decomposes to a carboxymethyldiazonium ion, which can also be generated from potassium diazoacetate (KDA). KDA is particularly interesting because it is a stable nitrosated derivative of glycine, one of the most common dietary amino acids. KDA has been shown to produce more O6 carboxymethyl- and O6 methyl-adducts than APNG, so it was anticipated that it might also be a potent genotoxic agent. Thus in the present study KDA has been investigated in the single cell gel electrophoresis (Comet) assay, which primarily measures DNA strand breakage. Since KDA has been shown to be formed in the gut, the genotoxic effects of KDA were investigated in vitro in human adenocarcinoma colon Caco-2 cells, and in rat primary colon cells and compared to responses from human peripheral lymphocytes. KDA induced DNA damage in the three cell types, confirming that KDA is genotoxic in a range of mammalian cells.
Asunto(s)
Compuestos Azo/farmacología , Colon/efectos de los fármacos , Daño del ADN , Glicina/análogos & derivados , Linfocitos/ultraestructura , Pruebas de Mutagenicidad , Adulto , Animales , Células CACO-2 , Células Cultivadas , Relación Dosis-Respuesta a Droga , Electroforesis , Femenino , Glicina/farmacología , Humanos , Linfocitos/efectos de los fármacos , RatasRESUMEN
The single cell gel electrophoresis assay (Comet assay) was used to measure DNA damage in peripheral lymphocytes from a group of individuals from The Gambia in order to determine whether such damage could be associated with increased exposure to aflatoxin in this population. Responses obtained were correlated to responses previously obtained [1] in a cross-sectional study in the same individuals of various cytogenetic alterations [chromosomal aberrations, micronuclei (crest positive and negative staining), and sister chromatid exchanges], and aflatoxin-albumin adducts. Analysis of variance methods were used to assess the effects of smoking, GSTM1 genotype, sex, age, and smoking status. A comparison was also made between The Gambian individuals and a group of healthy, non-smoking volunteers in the United Kingdom where aflatoxin exposure would be expected to be low. From the earlier study [1], it was determined that the levels of the sister chromatid exchanges and micronuclei were higher in The Gambian group than in a European group where aflatoxin exposure was lower, but that there were no correlations between the adduct levels and the cytogenetic abnormalities at the individual level. In the present study, DNA damage as measured in the Comet assay was not significantly higher than in the healthy United Kingdom volunteers. In addition, there were no associations between cytogenetic damage, GSTM1 genotype, age, sex, lifestyle factors (smoking and aflatoxin exposure), and Comet response at the individual level. Comet response was higher in females than males in The Gambia if one outlier was excluded from analysis and not taking into account other sources of variability. It would appear that DNA damage as measured in the Comet assay in peripheral blood lymphocytes is not a sensitive genotoxic marker of aflatoxin exposure in this population.
Asunto(s)
Aflatoxinas/efectos adversos , Aflatoxinas/farmacología , Daño del ADN , Pruebas de Mutagenicidad , Mutágenos/efectos adversos , Mutágenos/farmacología , Adolescente , Adulto , Anciano , Femenino , Gambia , Humanos , Masculino , Persona de Mediana Edad , FumarRESUMEN
The single recombinant expressing the Streptomyces coelicolor minimal whiE (spore pigment) polyketide synthase (PKS) is uniquely capable of generating a large array of well more than 30 polyketides, many of which, so far, are novel to this recombinant. The characterized polyketides represent a diverse set of molecules that differ in size (chain length) and shape (cyclization pattern). This combinatorial biosynthetic library is, by far, the largest and most complex of its kind described to date and indicates that the minimal whiE PKS does not independently control polyketide chain length nor dictate the first cyclization event. Rather, the minimal PKS enzyme complex must rely on the stabilizing effects of additional subunits (i.e., the cyclase whiE-ORFVI) to ensure that the chain reaches the full 24 carbons and cyclizes correctly. This dramatic loss of control implies that the growing polyketide chain does not remain enzyme bound, resulting in the spontaneous cyclization of the methyl terminus. Among the six characterized dodecaketides, four different first-ring cyclization regiochemistries are represented, including C7/C12, C8/C13, C10/C15, and C13/C15. The dodecaketide TW93h possesses a unique 2,4-dioxaadamantane ring system and represents a new structural class of polyketides with no related structures isolated from natural or engineered organisms, thus supporting the claim that engineered biosynthesis is capable of producing novel chemotypes.
Asunto(s)
Cetonas/síntesis química , Complejos Multienzimáticos/metabolismo , Streptomyces/enzimología , Cetonas/química , Espectroscopía de Resonancia Magnética , Estructura Molecular , Complejos Multienzimáticos/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Espectrometría de Masa Bombardeada por Átomos Veloces , Espectrometría de Masa de Ion SecundarioRESUMEN
Over 200 agents/factors have been examined in the single cell gel electrophoresis assay, more commonly known as the Comet assay, performed either in vitro or in vivo in a variety of species. Unequivocal carcinogenicity data are available for 119 of them, amongst which unequivocal Comet assay data exist for 95 agents. Of these 95 agents the prevalence of carcinogens was 88% (84/95). The carcinogens that were Comet positive (sensitivity) formed 88% (74/84), the non-carcinogens that were Comet negative (specificity) formed 64% (7/11). This simple analysis of the Comet assay has not taken account of the difference between in vitro and in vivo responses, species differences or organ and tissue differences. Also, limitations as to the conduct of the assay have not been examined in any depth. Thus, at the present time the Comet assay has high sensitivity for carcinogens, but its specificity is uncertain because few non-carcinogens have been tested.
Asunto(s)
Pruebas de Carcinogenicidad/métodos , Carcinógenos/toxicidad , Electroforesis/métodos , Exposición a Riesgos Ambientales/análisis , Pruebas de Mutagenicidad/métodos , Animales , Humanos , Reproducibilidad de los ResultadosRESUMEN
The flavonoids, silymarin, myricetin, quercitin, kaempferol, rutin and kaempferol-3-rutinoside have been examined in combination with the food mutagens, 3-amino-1-methyl-5H-pyrido (4,3-b)indole (Trp-P-2) and 2-amino-3-methylimidazo-(4,5-f) quinoline (IQ), in the Comet assay in human lymphocytes from donor A and human sperm from donor B. These compounds alone have been shown to produce positive responses in the Comet assay, as have the food mutagens. However, in combination with the food mutagens, the flavonoids produced antigenotoxic effects since DNA damage was reduced in the Comet assay in lymphocytes and sperm. The assays were performed in the absence of metabolic activation, since when quercetin and kaempferol were examined in blood with metabolic activation, there was little or no difference in response to that obtained in its absence. In the blood, there was an exacerbation or synergy of response at the lowest doses of the flavonoids. In the sperm, with silymarin, myricetin and quercitin, antigenotoxic effects only were observed, but with kaempferol, in general, there were no protective effects. The food mutagen, 2-amino-1-methyl-6-phenylimadazo (4,5-b)pyridine (PhIP), was also examined in addition to Trp-P-2 and IQ in combination with silymarin and myricetin in donors A and C in human lymphocytes only. Similar exacerbation of effects were found at low doses of these flavonoids with antigenotoxic effects at high doses. This was confirmed in the Ames test. There were slightly different profiles in lymphocytes and sperm, but antigenotoxic effects were observed over a similar dose range. This would suggest that effects occur in somatic and germ cells on a one-to-one ratio. These results have implications for man in terms of risk assessment and in the modulation of isolated food constituents.
Asunto(s)
Antimutagênicos/farmacología , Flavonoides/farmacología , Linfocitos/efectos de los fármacos , Mutágenos/toxicidad , Espermatozoides/efectos de los fármacos , Adulto , Carbolinas/toxicidad , Electroforesis/métodos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Quinolinas/toxicidadRESUMEN
There are two forms of diabetes, insulin-dependent Diabetes mellitus (IDDM) and non-insulin dependent Diabetes mellitus (NIDDM). There is evidence to suggest that reactive oxygen is involved in the pathogenicity and complications arising from IDDM, but there is little to suggest a role of oxidative stress in the pathogenesis of NIDDM. In order to investigate this hypothesis further, peripheral blood samples were taken from control individuals and IDDM and NIDDM patients and examined for antioxidant capacity and in the Comet assay for DNA strand breakage. The individuals answered a questionnaire to provide information relating to lifestyle factors in case such factors might have a confounding effect. There were 20 controls, 22 IDDM patients and 23 NIDDM patients. No differences could be detected in control and diabetic patient groups in terms of creatinine levels and antioxidant capacity. DNA damage in the Comet assay was at a lower level than in the control in the IDDM patients and a slightly lower level in the NIDDM patients, which might indicate that these cells are handling more oxidative damage on a regular basis. As expected, there were differences in glycosylated haemoglobin (HbA(1C)) levels. The confounding factors (smoking, drinking and vitamin intakes, etc.) appeared to have no effect.