Importância de polimorfismos de genes reguladores de citocinas em transplantes de células progenitoras hematopoiéticas / Importance of regulatory cytokine gene polymorphisms in hematopoietic stem cell transplantation

A compatibilidade genética HLA entre doador e receptor é um fator importante para o sucesso do transplante de células progenitoras hematopoiéticas (TCPH). No entanto, outros genes não-HLA estão sendo investigados em relação ao seu papel na incidência e gravidade da doença do enxerto contra o hospedeiro e na sobrevida, por modularem a intensidade da inflamação e os danos teciduais. Estes genes, não-HLA, incluem os genes de citocinas com polimorfismos dentro das seqüências 5' ou 3' regulatórias dos genes. Os polimorfismos ou microssatélites podem alterar a ligação dos fatores de transcrição aos sítios dentro dos genes promotores e a quantidade de citocina produzida. Este estudo revisa o papel potencial destes polimorfismos genéticos relativos às citocinas em prever o curso do TCPH.

HLA genetic matching of donor and recipient is an important requirement for optimizing outcome following hematopoietic stem cell transplantation (HSCT). However, other non-HLA genes are being investigated for their role in graft-versus-host disease incidence and severity and in survival, by modulating the intensity of inflammation and tissue injury. These non-HLA-encoded genes include cytokine genes with polymorphisms within the 5' or 3' regulatory sequences of the genes. The polymorphisms or microsatellites may alter the transcription factor binding sites within the gene promoters and the amount of cytokine produced. This chapter summarizes the potential role of these genetic polymorphisms regarding the cytokines in predicting outcome of HSCT.

Morphological and molecular differentiation of the pectinase producing fungi Penicillium expansum and Penicillium griseoroseum

Two species from the genus Penicillium, Penicillium expansum and P. griseoroseum (Brasilian isolates) were characterized morphologic and molecularlly. Morphological variability was detected among isolates in regard to colony morphology and to conidia coloration. The molecular characterization was based on the RAPD markers, telomeric fingerprinting and ITS sequencing. A total of 78 RAPD primers were used and 8 presented differences in band patterns with 54 percent of the amplified polymorphic fragments. The monomorphic fragments of 600 bp (P. expansum) and 594 bp (P. griseoroseum) were amplified. The only internal transcribed spacer region variation detected between the two species was the additional six initial nucleotides. The analysis by telomeric fingerprinting showed polymorphism between both species and the chromosome minimal numbers estimated were three. The polymorphism observed in the organization of the subtelomeric region in the genome of two Penicillium species within the high homogeneous Penicillium subgenus is for the first time reported and perhaps can be employed in future phylogenetic studies.

Penicillium expansum e P. griseoroseum foram caracterizados morfológica e molecularmente. Variações na morfologia das colônias e coloração dos conídeos foram observadas entre os isolados. A caracterização molecular foi baseada em marcadores RAPD, sequenciamento da região do espaçador interno transcrito do DNA ribossomal e "fingerprinting" telomérico. Foi usado um total de 78 primers RAPD, sendo que 8 apresentaram 54 por cento de fragmentos de DNA polimórficos. Os produtos da amplificação da região ITS de P. expansum e P. griseoroseum foram de 600 e 594 pb, respectivamente. Não foi detectada nenhuma variação na seqüência de nucleotídeos dessa região, comparando-se P. expansum e P. griseoroseum, exceto em relação aos seis nucleotídeos iniciais adicionais. Observou-se a ocorrência de polimorfismo na organização da região subtelomérica no genoma destes fungos e estimou-se um número mínimo de três cromossomos para estas espécies. Este é o primeiro trabalho que descreve a existência de polimorfismo na organização da região subtelomérica do genoma de espécies de fungos pertencentes ao gênero Penicillium, altamente homogêneo, indicando uma possível utilização da abordagem empregada neste estudo para pesquisas filogenéticas futuras.

Molecular discrimination between individual metacercariae of Paragonimus heterotremus and P. westermani occurring in Thailand.

To accurately discriminate between individual metacercariae of Paragonimus heterotremus and P. westermani occurring in Thailand, polymerase chain reaction (PCR)-based molecular methods were established and subjected to an evaluation. We first amplified and sequenced the second internal transcribed spacer (ITS2) region of the nuclear ribosomal DNA of the two species. Based on their nucleotide differences, P. heterotremus and P. westermani were unequivocally discriminated from each other. These nucleotide differences were further utilized to select the ApaL1 endonuclease site for PCR-restriction fragment length polymorphism (PCR-RFLP) analyses and to design species-specific primers for multiplex PCR reactions. Both PCR-RFLP and multiplex PCR methods allowed a more rapid and labor-effective species discrimination. Furthermore, the multiplex PCR method enabled the most efficient discrimination because species identification involved a single round of PCR in a single tube. In Thailand, P. heterotremus is the only species affecting humans. Thus, the methods established in the present study can be used as reliable tools to identify the lung fluke metacercariae that cause human disease.

El mapa de la variabilidad genética del humano, los polimorfismos de un solo nucleótido (SNPïs) y algunas de sus aplicaciones en medicina (editorial) / The human genetic variability map, single nucleotide polymorphisms (SNPïs) and some of their applicarions to medicine (editoial)

Los polimorfísmos de un solo nucleótido estan presentes a lo largo del genoma humano con un espacimiento promedio de 1.5 x 10 6 bases (aproximadamente 2 x 106 en todo el genoma). Se cuenta con un mapa de alrededor de 1.4 millones de SNPs a lo largo del genoma, 60,000 de los cuales (alrededor del 3 por ciento) se encuentran dentro de regiones codificantes o exones. La diversidad nucleotídica de los SNP's presenta variaciones importantes a lo largo del genoma. Este mapa de SNP's, ahora de dominio publico, permitirá la identificación de posibles haplotipos relacionados con rasgos bioquímicos o patológicos, y el mapeo de los genes implicados en el desarrollo de distintas enfermedades, principalmente aquéllas de etiología genética compleja. Esto a su vez permitirá al corto plazo, la implementación de estrategias diagnósticas moleculares para estas enfermedades de alta prevalencia en la población.Los polimorfismos de una solo nucleótido (SNP's) como su nombre lo indica, son variaciones de secuencia que involucran la sustitución de un solo nucleótido cuando se comparan dos cromosomas homólogos (Fig 1A). Estos cambios de secuencia se encuentran presentes a lo largo del genoma humano en promedio cada 1000-2000 bases. En la publicación de Nature 49: 928-933, 2001 se describe un mapa de alrededor de 1.4 millones de estos polimorfismos en el genoma humano, su distribución en cada cromosoma así como un estudio preliminar de su heterozigocidad en distintos grupos étnicos (la posibilidad de encontrar variación en la secuencia de un SNP al comparar cromosomas homólogos de individuos de distintas poblaciones). Los SNP's a diferencia de otro tipo de marcadores genéticos como los marcadores microsatélites, tienen una tasa menor de mutación por lo que pueden ser utilizados en estudios de genética de poblaciones para entender fenómenos de migración y evolución de la raza humana.

Oligonucleotide fingerprint analysis of a wild strain & its attenuated variant of Japanese encephalitis virus.

A strain of Japanese encephalitis (JE) virus was passaged serially through primary chick kidney cell cultures (45 times) and primary baby hamster kidney cell cultures (21 times). The resultant virus lost its lethal effect to 3 wk old mice by the ic route and 10 day old mice by the ip route. The oligonucleotide fingerprint analysis of the parent and the passaged strains showed 64 spots in common; 17 spots were present in the parent strain which were absent in the passaged virus, while the latter had acquired 9 spots which were not present in the parent virus.

Desarrollo de un metodo mas simple y rapido para el analisis bidimensional de ologonucleotidos del virus de la fiebre aftosa: minifingerprinting de ARN / Developement of a more rapid and simple procedure for two-dimensional oligonucleotide analysis of foot-and-mouth disease virus: RNA minifingerprinting

Una característica significativa del virus de la fiebre aftosa (VFA) es su alto grado de variabilidad, lo cual constiruye un importante obstáculo para el control de la enfermedad. Actualmente se sispone de varias técnicas bioquímicas como fingerprinting de ARN, ADN recombinante y secuencimiento rápido para estudiar, con significativa precisión, las características genéticas de las cepas virales. La tecnica de fingerprinting resulta muy adecuada para evaluar las relaciones evolutivas entre cepas virales muy relacionadas y en el caso del VFA tambien se ha probado su utilidad para fines de diagnostico. Con la creciente aplicacion de estudios epidemiologicos a nivel molecular que requieren el analisis de un gran numero de muestras, se hace evidente que el empleo de tecnicas mas practicas y rapidas y de menor costo, seria de gran utilidad para la caracterizacion del ARN genomico

Oligonucleotide fingerprint analysis of Japanese encephalitis virus strains of different geographical origin.

RNA fingerprint analysis was carried out with different strains of Japanese encephalitis virus which were isolated from Japan, China, India and Sri Lanka. From the similarity ratios, a similarity matrix was worked out which yielded a dendrogram. Geographical proximity of the place of isolation did not contribute much to the similarity of the fingerprint of the strains, nor did temporal proximity. The Japanese Nakayama strain had greater similarity with the Asansol strain from West Bengal. However, another strain from West Bengal, the Bankura strain, showed marked difference. Similarly the Bhopal and Beijing (China) strains were relatively close to each other while the Japanese JaGAr15460 strain was nearer to the strain from Gorakhpur. Serial mouse passage of the Asansol strain did not change the fingerprint pattern drastically.

Acido desoxirribonucleico (DNA) en pruebas de filiación, paternidad y medicina forense

A new precise method of personal identification with significant implications for civil and criminal paternity cases, as well as for other forensic purposes and genetic studies is presented. DNA multi-locus analysis offers a discrimination of 1 in 30 billions, cosntituting the most precise determination in paternity testing. If we consider that the world populations is around 5 billions people, and that less that 2.5 billions are males, of the wich approximately 1/3 are not adults, then we can see how the possibility of error is extremely low. It makess other paternity with HLA, blood groups, enzimes and proteins ineffective in paternity disputes. All courts and legal personnel should be awere of the scientific implications of this new available test in our media