Genetic variability of matrix (M), small hydrophobic (SH) and attachment (G) proteins of human metapneumovirus circulating in children in Beijing from 2006 to 2010 / 病毒学报

Human metapneumovirus (hMPV) is associated with acute respiratory tract infections (ARTI) in all age groups. However, there is limited information of genetic analysis of hMPV circulating in Beijing. To learn the characteristics of structural protein genes of human metapneumovirus circulating in children in Beijing, sequence analysis of matrix (M), small hydrophobic (SH) and attachment (G) proteins of hMPV from 2006 to 2010 was performed. Phylogenetic analysis of nucleotide sequences of 42 full length M genes, 49 SH gene and 55 G gene revealed that the hMPVs from pediatric patients were divided into sub-genotypes A2, B1 and B. There were highly conserved identities among M gene, with 7 conserved mutations of amino acids between A and B genotypes which were fairly conserved in the same genotype A or B. The amino acid identities of SH were 60.7% to 64.4% between different genotypes, 93.3% - 100% among same sub-genotype and 84.7% - 88.7% between different sub-genotypes. Use of alternative transcription-termination codon, nucleotide deletion and insertion resulted in variable length of nucleotide and deduced amino acid of G protein. Amino acid identities within same genotype ranged from 81.5% - 100%, whereas sequence identities between two genotypes ranged from 34.0% - 38.6% at the amino acid level. A new cluster of G genes in sub-genotype B2 appeared due to the same mutations and insertion of two amino acids in G protein encoding genes amplified from specimens collected from 2008 to 2010. Prediction of antigen sites of SH and G protein indicated that the variation of antigen sites between different sub-genotypes existed.

Mapping the molecular characteristics of Brazilian human T-cell lymphotropic virus type 1 Env (gp46) and Pol amino acid sequences for vaccine design

This study was carried out to evaluate the molecular pattern of all available Brazilian human T-cell lymphotropic virus type 1 Env (n = 15) and Pol (n = 43) nucleotide sequences via epitope prediction, physico-chemical analysis, and protein potential sites identification, giving support to the Brazilian AIDS vaccine program. In 12 previously described peptides of the Env sequences we found 12 epitopes, while in 4 peptides of the Pol sequences we found 4 epitopes. The total variation on the amino acid composition was 9 and 17 percent for human leukocyte antigen (HLA) class I and class II Env epitopes, respectively. After analyzing the Pol sequences, results revealed a total amino acid variation of 0.75 percent for HLA-I and HLA-II epitopes. In 5 of the 12 Env epitopes the physico-chemical analysis demonstrated that the mutations magnified the antigenicity profile. The potential protein domain analysis of Env sequences showed the loss of a CK-2 phosphorylation site caused by D197N mutation in one epitope, and a N-glycosylation site caused by S246Y and V247I mutations in another epitope. Besides, the analysis of selection pressure have found 8 positive selected sites (w = 9.59) using the codon-based substitution models and maximum-likelihood methods. These studies underscore the importance of this Env region for the virus fitness, for the host immune response and, therefore, for the development of vaccine candidates.

Clonagem e expressão da glicoproteína transmembrana do vírus linfotrópico de células T humanas em sistema procarioto / Cloning and expression of the transmembranic glycoprotein from human T cell lymphotropic virus in a prokaryotic system

O HTLV-1 é o vírus causador da leucemia/linfoma de célula T no adulto e de uma desordem neurológica conhecida por mielopatia associada ao HTLV ou paraparesia espástica tropical. Um dos modos de transmissão é pelo sangue contaminado e seus subprodutos e, devido ao risco de infecções associadas ao HTLV sua pesquisa na triagem de doadores de sangue foi introduzida no Brasil a partir de 1993. Os kits diagnósticos utilizados nos bancos de sangue nacionais são na sua maioria comprados de empresas estrangeiras. O Brasil não detém a tecnologia para produção deste material e há a necessidade de produção de sistemas de diagnóstico com tecnologia nacional. Neste trabalho, mostramos a expressão da gp21/HTLV-1 em Escherichia coli e sua reatividade frente a anticorpos monoclonais e de pacientes infectados. Expressar tais proteínas é o primeiro passo para obtenção de conjuntos diagnósticos com tecnologia brasileira.

HTLV-1 is the virus that causes T cell lymphoma/leukemia in adults and a neurological disorder known as HTLV-associated myelopathy or tropical spastic paraparesis. One of the transmission means is through contaminated blood and its byproducts. Because of the risk of HTLV-associated infections, screening for HTLV was introduced for Brazilian blood donors in 1993. Most of the diagnostic kits used in the national blood banks are bought from foreign companies. Brazil does not have the technology to produce this material and there is a need to produce diagnostic systems with national technology. In this study, we show the expression of gp21/HTLV-1 in Escherichia coli and its reactivity towards monoclonal antibodies and the antibodies of infected patients. Expressing these proteins is the first step towards obtaining diagnostic kits with Brazilian biotechnology.

Ets oncogene family.

First member of Ets gene family was discovered a decade ago by studying avian erythroblastosis virus, E26. This virus encodes a tripartite protein gag-myb-ets with a molecular weight of 135 kDa. Subsequently, a series of cellular Ets genes were isolated (Ets-1, Ets-2, Erg, Elk-1, Sap-1, PEA-3, PU.1, Fli-1, Pok/Yan, Etv-1 etc.). These genes share sequence homology to E26 Ets gene (v-ets or viral ets). Ets genes are highly conserved in phylogenetically divergent species from Drosophila to man. Mammalian Ets genes are located on different chromosomes. Ets gene products are transcriptionally active sequence-specific DNA binding proteins and are differentially regulated. Ets genes are involved in certain chromosomal translocations leading to the formation of chimeric fusion proteins that are associated with certain leukemias and soft tissue cancers. Ets genes also have a role in T-cell development and molecular and genetic analysis of Down Syndrome patients have implicated the human Ets-2 and Erg genes in the disease. Down Syndrome afflicted patients have immunologic and thymic disorders as well as a greater risk for leukemic disease. Thus, Ets genes having homology to viral oncogenes, may be instrumental in regulating cellular growth and differentiation, as well as organismal development. Alteration of these genes and their products may cause deregulation of normal cell growth and differentiation and result in a disease.

Conformational dynamism in d-(GAATTCCGTTATT) containing the complementary Myb responsive element d-CCGTTA: NMR and MD investigations.

Conformational features of the DNA segment d-(GAATTCCGTTATT) containing the complementary Myb responsive element CCGTTA has been studied by NMR and molecular dynamics calculations with a view to see the role of 3D structure in specific DNA recognition. From the low field imino proton NMR spectra, the DNA sequence is seen to exist as a duplex with pyrimidine mismatches in the centre. The 2D NMR spectra however show that teh molecule exhibits substantial dynamism even at 1 degree C. Several extra cross peaks, more than the expected number, are seen in particular regions in all the spectra. These observations indicate that the duplex undergoes slow transitions between base-paired and unbase-paired states due to mismatches in the centre. Hence, to characterise those transitions a restrained verlet dynamics has been performed for 50ps using X-PLOR force field. Structural intermediates at regular intervals have been analysed, and we see that the dynamism in the molecule results in substantial fluctuations in the different torsion angles. The mismatch sites are seen to exhibit the highest degree of fluctuations, with the bases stacking in and looping out of the duplex. The sugar geometry is seen to be fairly steady around the S domain for most of the residues.

Cáncer gástrico: características clinico-patológicas y expresión del oncogen c-erbB-2 en 122 casos / Gastric cancer: clinico-pathological characteristics and expression of c-erbB-2 oncogen in 122 cases

Se presenta una serie de 119 pacientes com 122 carcinomas gástricos tratados con gastrectomía y con seguimento mayor de 5 años. Eran 80 (67,2 por ciento) hombres y 39 (32,8 por ciento) mujeres con una edad promedio general de 66,4 años. En 101 (84,9 por ciento) se hizo diagnóstico endoscópico de cáncer. El dolor fue el síntoma más frecuente (55,5 por ciento). Setenta y dos (59 por ciento) estaban localizados en el antro, 26 (21 por ciento) en cardias y 24 (19,7 por ciento) en el cuerpo. El 43,4 por ciento medía menos de 5 cm y el 56,6 por ciento, 5 o más cm. Ochenta y nueve (73 por ciento) eran de tipo intestinal, 15 (12,3 por ciento) difuso, y 18 (14,8 por ciento mixto. Ochenta (65,6 por ciento) eran de grado histológico bajo y 42 (34,4 por ciento) alto. Diez (9,2 por ciento) fueron tempranos y 112 (91,8 por ciento) avanzados. Se observó amplificación del oncogen c-erbB-2 en 13 (10,6 por ciento). La amplificación del c-erbB-2 mostró una asociación estadísticamente significativa con los tumores menores de 5 cm (p = 0,05) y con los de grado histológico bajo. La sobrevida a 5 años se correlacionó con tumores menores de 5 cm, con invasión de la pared sin sobrepasar la muscular propia y con ganglios libres de metástasis. No se encontró asociación entre la amplificación del c-erbB-2 y la sobrevida

Detection of px gene product of bovine leukemia virus in infected cells.

Bovine leukemia virus (BLV), like its closest relatives human T-cell leukemia virus-I and II, contain a 'px' gene, between the 'env' gene and the 3' long terminal repeat in its genome. A monoclonal antibody prepared against a synthetic oligopeptide whose sequence was deduced from highly conserved region of 'px' gene of BLV, was used to detect the presence of 'px' gene product in chronically BLV infected synchronised cells. By immunoperoxidase staining the 'px' gene product was detected maximum after 6-9 hr after synchronization in the nucleus of the cells which demonstrated the close interaction of it with viral DNA which is integrated with host cell genome.

Binding of phytohemagglutinin to bovine B lymphocytes and its role in stimulation of expression of bovine leukemia virus genome.

Phytohemagglutinin (PHA) is known to increase the synthesis of bovine leukemia virus (BLV) particles and viral antigens in short-term culture of BLV-infected neoplastic and non-neoplastic lymphocytes. This stimulation of BLV expression has been shown to be due to enhanced transcription of the viral genome by a PHA-induced protein. We have investigated the binding of 125I-labelled PHA to BLV-infected bovine B lymphocytes and subsequent events that may lead to the stimulation of BLV-p25 synthesis. We found that PHA binding to the infected cells were rapid, but only a small fraction of the bound PHA is translocated to nucleus. However, bound PHA dissociated rapidly from the cell membrane, but not from the nucleus when PHA is removed from the culture medium. Furthermore, continuous presence of PHA was not essential for optimal stimulatory activity, instead a minimum incubation with PHA for 6 hr. followed by culturing the infected cells in its absence, was sufficient to exhibit maximal stimulatory activity. Our results raise the possibility that interaction of PHA with the plasma membrane probably triggers synthesis of a protein which in turn enhances the transcription of BLV genome in vitro.