Polimorfismos en los genes de dihidrofolato-reductasa ( dhfr ) y dihidropteroato-sintasa ( dhps ) y modelado estructural del gen dhps en aislamientos colombianos de Toxoplasma gondii / Gene polymorphisms in the dihydrofolate reductase ( dhfr ) and dihydropteroate synthase ( dhps ) genes and structural modelling of the dhps gene in Colombian isolates of Toxoplasma gondii

Introducción. No existen reportes sobre las variaciones en la secuencia de los genes blanco de los medicamentos anti- Toxoplasma en aislamientos provenientes de Suramérica. Objetivo. Clonar y secuenciar los genes de la dihidrofolato-reductasa ( dhfr ) y la dihidropteroato-sintetasa ( dhps ) de la cepa de referencia RH y de dos aislamientos colombianos de Toxoplasma gondii. Materiales y métodos. Se obtuvieron dos aislamientos de T. gondii en líquido céfalorraquídeo de pacientes colombianos positivos para HIV con toxoplasmosis cerebral. Se extrajo el ADN de los genes dhfr y dhps y se amplificaron mediante reacción en cadena de la polimerasa (PCR). Los productos fueron clonados en el vector pGEM-T y secuenciados. Resultados. Se encontró un cambio de adenina por guanina (A « G) en la posición 235 del exón 2 del gen dhps , dos cambios de guanina por citocina (G « C) en las posiciones 259 y 260 y un cambio de timina por guanina (T « G) en la posición 371 del exón 4 del gen dhps. Por análisis bioinformático, en este último exón se identificó un polimorfismo no sinónimo en la región codificante, que podría llevar al cambio de una Glu (CAA o CAG) por una His (codificada por los codones AAU o AAC). Se calculó el modelo estructural de la enzima dihidropteroato-sintetasa (DHPS) de T. gondii y se identificaron las modificaciones en la estructura secundaria ocasionadas por las mutaciones. Conclusiones. La metodología estandarizada puede servir como base para la búsqueda de polimorfismos en muestras de pacientes con diferentes manifestaciones clínicas de toxoplasmosis y para establecer su posible relación con los cambios en la sensibilidad a los antifolatos y la reacción al tratamiento.

Introduction: There are no reports describing polymorphisms in target genes of anti- Toxoplasma drugs in South American isolates. Objective: This study sought to perform cloning and sequencing of the dihydrofolate reductase ( dhfr ) and dihydropteroate-synthase ( dhps ) genes of the reference Rh strain and two Colombian isolates of Toxoplasma gondii . Materials and methods: Two isolates were obtained from the cerebrospinal fluid of HIV-infected patients with cerebral toxoplasmosis. A DNA extraction technique and PCR assay for the dhfr and dhps genes were standardized, and the products of amplification were cloned into Escherichia coli and sequenced. Results: One polymorphism (A « G) was found at position 235 of exon 2 in the dhps gene. In addition, two polymorphisms (G « C) at positions 259 and 260 and one polymorphism (T « G) at position 371 within exon 4 of the dhps gene were detected. In this last exon, a bioinformatic analysis revealed a non-synonymous polymorphism in the coding region that could lead to the substitution of Glu (CAA or CAG) for His (encoded by codons AAU or AAC). A structural model of the T. gondii DHPS protein was calculated, and the results revealed modifications in secondary structure due to mutations. Conclusions: The methods described in this study can be used as a tool to search for polymorphisms in samples from patients with different clinical manifestations of toxoplasmosis and to examine their relationship with the therapeutic response.

Molecular docking of variant of quinolone resistance-determining region in the DNA gyrase subunit A to substrates in Klebsiella pneumonia / 中华传染病杂志

Objective To investigate the binding capacities of two variants of quinolone resistance-determining region in the DNA gyrase subunit A to substrates in Klebsiella pneumonia (K.pneumonia).Methods Tertiary structures of two variants (type Ⅰ and type FH) of quinolone resistance-determining region in the DNA gyrase subunit A in K.pneumonia were predicted by homology modeling referring to that of wild type.Then,DOCK module in ArgusLab 4.1 software was used to perform molecular docking of two variants and wild type to seven kinds of quinolones substrates,and calculate binding free energies (△G).Moreover,numbers and distances of interaction between amino acid residues of DNA gyrase subunit A and ciprofloxacin were calculated.Results Molecular docking showed that binding free energies of type Ⅰ and type FH to pipemidic acid,ciprofloxacin,gatifloxacin were-26.607 50,-29.530 39,-29.493 09 kJ/mol and-26.696 44,-28.972 83,-29.590 50 kJ/mol,respectively,which declined greater than those of wild type (-27.188 82,-30.872 00 and-30.244 04 kJ/mol,respectively) and showed drug resistance.While binding free energies of type Ⅰ and type FH to levofloxacin were-29.013 81 and-29.497 57kJ/mol,respectively,and that of wild type was-28.016 20 kJ/mol.The binding free energies of type Ⅰ and type FH to nalidixic acid,norfloxacin,ofloxacin increased or declined.Moreover,if distance was less than 5 angstroms,atom pairs formed between wild type of DNA gyrase subunit A and ciprofloxacin had 16 pairs,while type Ⅰ and type FH had 2 pairs and 4 pairs,respectively.If distance was less than 4 angstroms,atom pairs formed between wild type and ciprofloxacin had 8 pairs,while type Ⅰ and type FH had no atom pairs.Conclusion Decline of binding capacities of two variants of DNA gyrase subunit A in K.pneumonia to ciprofloxacin played a role in drug resistance.

Modelación por homología de la proteína Luxs de Porphyromonas gingivalis cepa W83 / Modelling by homology of Luxs protein in Porphyromonas gingivalis strain W83

Antecedentes: En las proteínas no se logra siempre su cristalización, de buen tamaño y de buena calidad para someterla a difracción de rayos X. De tal manera que se abre un campo para el desarrollo de estudios teóricos moleculares y proteínicos, que permiten la representación de las moléculas en tres dimensiones, proporcionando una información espacial para estudiar la interacción entre ligandos y receptores macromoleculares. Materialesy Métodos: Estudio In silico, a partir del análisis de secuencias primarias de seis diferentes proteínas LuxS cristalizadas de diversas bacterias, se seleccionó la proteína 1J6X del Helicobacter pylori, por su similaridad con la secuencia de la proteína LuxS en Porphyromonas gingivalis (P. gingivalis) cepa W83, para producir un modelo por homología de esta proteína, utilizando los programas Sybyl y MOE. Se realizó un acoplamiento con el ligando natural para evaluar la reproducibilidad del modelo en un ambiente biológico. Resultados: Se desarrolló el modelado de la proteína LuxS de P. gingivalis cepa W83, que permite el acercamiento a una estructura que se propone, por la interacción entre la proteína y su ligando natural. El modelo generado con recursos computacionales logró una correcta estructura molecular que aceptó la realización de diversos cálculos. El acoplamiento demostró una cavidad donde se logran diversas posiciones del ligando con buenos resultados. Conclusiones: Se obtuvo un modelo 3D para la proteína LuxS en la P. gingivalis cepa W83 validado por diferentes métodos computacionales con una adecuada reproducibilidad biológica por medio del acoplamiento molecular.

Background: Crystallization is not always achieved for all proteins in a good size and a good quality for X-ray diffraction. So that condition opens a field for the development of theoretical molecular and protein studies allowing the representation of the molecules in 3D, providing spatial information to study the interaction between ligands and macromolecular receptors. Materials and Methods: In silico study from primary sequence analysis of six different proteins LuxS crystallized of several bacteria. 1J6X protein of Helicobacter pylori was selected for its similarity with the LuxS protein sequence in Porphyromonas gingivalis (P. gingivalis) strain W83 to produce a homology model of this protein, using the Sybyl and MOE software. A docking was performed to assess the reproducibility of the model in a biological environment. Results: The LuxS protein modelling of P. gingivalis strain W83 was developed, which allows the approach to a proposed structure for the interaction between the protein and its natural ligand. The model generated with computational resources achieved the correct position and biological behavior by means of developed calculations. The docking showed a cavity in which the ligand adopted several positions with good results. Conclusions: A LuxS protein model was obtained, validated by different methods. This generated a 3D model for LuxS protein in P. gingivalis strain W83 with biological reproducibility by means of molecular docking.

A supramolecular proposal of lignin structure and its relation with the wood properties

In spite of the great importance of cellulose the lignin is considered the second most abundant substance of the wood. However, little attention has been given it, mainly to wood properties. The lignin as well as other structural compounds (cellulose and hemicelluloses), has obviously an important role on the wood properties, probably due its composition and existent bonds. In general lignins have β-O-4 (Alkyl Aril Ether) as majoritary bond. This bond in a continued structure form big molecules with spiral conformation as virtual model. Based on this idea, lignins that have high/low β-O-4 content may have differentiated spiraled structures,suggesting different behaviors on the wood properties,which shows that the lignins (Guaicyl:Syringyl (GS)) of angiosperms, for example, which have higher β-O-4 content would present higher spiral conformation than gymnosperms lignins(HG). On the other hand HG lignins have chance of being more anchored on the matrix compound than GS lignins. In this context, the β-O-4 bonds of lignins possibly affect the wood properties, therefore, it is considered relevant for wood technology science discussion.

Apesar da grande importância da celulose a lignina é considerada a segunda substância mais abundante da madeira. Entretanto, pouca atenção tem sido dada a ela, principalmente com relação às propriedades da madeira. A lignina assim como outras substâncias (celulose e hemicelulose), tem obviamente um papel importante sobre as propriedades da madeira, provavelmente devido a sua composição e a existências de ligações. Geralmente as ligninas possuem majoritariamente ligaçõesβ-O-4 (Éter Alquil-Arílico), esta ligação em uma estrutura contínua forma grandes moléculas com conformação em espiral, como visto em modelo virtual. Com base nesta idéia, ligninas que possuem alto/baixo teor de β-O-4, podem ter estruturas espiraladas diferenciadas, sugerindo comportamentos diferentes sobre as propriedades da madeira. Isto mostra que as ligninas de angiospermas ((Guaicílica:Siringilica) (GS)), que possuem mais alto teor de β-O-4, por exemplo, apresentariam uma conformação mais espiralar do que as ligninas de gimnospermas (HG). Por outro lado, as ligninas HG possuem mais chances de serem ancoradas sobre a substância matriz do que as ligninas GS. Neste contexto, ligações β-O-4 das ligninas afetam as propriedades da madeira, portanto, isto pode ser considerado relevante para discussão em ciência e tecnologia da madeira.