RESUMO
Dipeptidyl aminopeptidase IV (DAP IV or DPP IV) from Pseudoxanthomonas mexicana WO24 (PmDAP IV) preferentially cleaves substrate peptides with Pro or Ala at the P1 position [NH2-P2-P1(Pro/Ala)-P1'-P2' ]. For crystallographic studies, the periplasmic form of PmDAP IV was overproduced in Escherichia coli, purified and crystallized in complex with the tripeptide Lys-Pro-Tyr using the hanging-drop vapour-diffusion method. Kinetic parameters of the purified enzyme against a synthetic substrate were also determined. X-ray diffraction data to 1.90â Å resolution were collected from a triclinic crystal form belonging to space group P1, with unit-cell parameters a = 88.66, b = 104.49, c = 112.84â Å, α = 67.42, ß = 68.83, γ = 65.46°. Initial phases were determined by the molecular-replacement method using Stenotrophomonas maltophilia DPP IV (PDB entry 2ecf) as a template and refinement of the structure is in progress.
Assuntos
Dipeptidil Peptidase 4/química , Dipeptidil Peptidase 4/isolamento & purificação , Periplasma/enzimologia , Xanthomonadaceae/enzimologia , Cristalização , Cristalografia por Raios X , Dipeptidil Peptidase 4/metabolismo , Cinética , Conformação Proteica , Xanthomonadaceae/classificaçãoRESUMO
Dipeptidyl aminopeptidase BII from Pseudoxanthomonas mexicana WO24 (DAP BII) is able to cleave a variety of dipeptides from the amino-terminus of substrate peptides. For crystallographic studies, DAP BII was overproduced in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. X-ray diffraction data to 2.3 Å resolution were collected using an orthorhombic crystal form belonging to space group P2(1)2(1)2(1), with unit-cell parameters a = 76.55, b = 130.86, c = 170.87 Å. Structural analysis by the multi-wavelength anomalous diffraction method is in progress.
Assuntos
Cristalografia por Raios X/métodos , Dipeptidil Peptidases e Tripeptidil Peptidases/química , Xanthomonadaceae/enzimologia , Cristalização , Conformação Proteica , Proteínas Recombinantes/químicaRESUMO
Although dogs are considered to be the principal transmitter of rabies in Brazil, dog rabies had never been recorded in South America before European colonization. In order to investigate the evolutionary history of dog rabies virus (RABV) in Brazil, we performed a phylogenetic analysis of carnivore RABV isolates from around the world and estimated the divergence times for dog RABV in Brazil. Our estimate for the time of introduction of dog RABV into Brazil was the late-19th to early-20th century, which was later than the colonization period but corresponded to a period of increased immigration from Europe to Brazil. In addition, dog RABVs appeared to have spread to indigenous animals in Brazil during the latter half of the 20th century, when the development and urbanization of Brazil occurred. These results suggest that the movement of rabid dogs, along with human activities since the 19th century, promoted the introduction and expansion of dog RABV in Brazil.
Assuntos
Doenças do Cão/epidemiologia , Doenças do Cão/virologia , Evolução Molecular , Polimorfismo Genético , Vírus da Raiva/classificação , Vírus da Raiva/genética , Raiva/veterinária , Animais , Brasil/epidemiologia , Análise por Conglomerados , Doenças do Cão/história , Cães , Genótipo , História do Século XIX , História do Século XX , História do Século XXI , Dados de Sequência Molecular , Filogenia , RNA Viral/genética , Raiva/epidemiologia , Raiva/história , Raiva/virologia , Vírus da Raiva/isolamento & purificação , Análise de Sequência de DNARESUMO
The low rates of nonsynonymous evolution observed in natural rabies virus (RABV) isolates are suggested to have arisen in association with the structural and functional constraints operating on the virus protein and the infection strategies employed by RABV within infected hosts to avoid strong selection by the immune response. In order to investigate the relationship between the genetic characteristics of RABV populations within hosts and the virus evolution, the present study examined the genetic heterogeneities of RABV populations within naturally infected dogs and foxes in Brazil, as well as those of bat RABV populations that were passaged once in suckling mice. Sequence analyses of complete RABV glycoprotein (G) genes showed that RABV populations within infected hosts were genetically highly homogeneous whether they were infected naturally or experimentally (nucleotide diversities of 0-0.95x10(-3)). In addition, amino acid mutations were randomly distributed over the entire region of the G protein, and the nonsynonymous/synonymous rate ratios (d(N)/d(S)) for the G protein gene were less than 1. These findings suggest that the low genetic diversities of RABV populations within hosts reflect the stabilizing selection operating on the virus, the infection strategies of the virus, and eventually, the evolutionary patterns of the virus.
Assuntos
Mutação , Vírus da Raiva/genética , Raiva/virologia , Animais , Antígenos Virais/química , Antígenos Virais/genética , Encéfalo/virologia , Brasil , Quirópteros , Bases de Dados de Ácidos Nucleicos , Cães , Raposas , Glicoproteínas/química , Glicoproteínas/genética , Vírus da Raiva/isolamento & purificação , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genéticaRESUMO
The patient is a 24-year-old woman who first came for consultation at age 10 years. Based on clinical phenotype and thin-layer chromatography of urinary oligosaccharides, peripheral leukocytes were sent for beta-galactosidase assay. This testing showed a deficiency in enzyme activity, and gene mutation analysis identified a previously reported mutation p.H281Y (875C > T) and a novel mutation p.W273R (817T > C). Unlike previously reported patients, mutant enzymes in this patient's cultured skin fibroblasts did not respond to treatment with a chaperone compound, N-octyl-4-epi-beta-valienamine.