Validação do método de deteção de eritropoietina recombinante humana em urina humana por focalização isoelétrica

A eritropoietina (EPO) é uma substância que estimula a produção de eritrócitos, aumentando a oxigenação muscular, sendo segregada de forma natural pelo organismo e excretada na urina em baixas concentrações. Devido às suas propriedades e características, a EPO foi rapidamente introduzida no mundo do desporto, como substância ilícita, proporcionando vantagens no rendimento desportivo. No início de 2000 foi desenvolvido um método de deteção direta de EPO Recombinante (rHuEPO) em urina humana por Lasne, baseado na focalização isoelétrica (IEF) em gel de poliacrilamida, seguido de duplo blote, tendo este sido publicado e validado. Em 2002, a Agência Mundial Antidopagem (AMA) implementou este mesmo método, sendo atualmente um dos métodos oficiais utilizado pelos laboratórios acreditados pela AMA. Desta forma, o ponto de partida para a realização deste trabalho consistiu na necessidade de implementar e validar o método de referência de IEF para a deteção de rHuEPO em urina humana. O trabalho foi realizado no Laboratório de Análises e Dopagem (LAD) do Instituto do Desporto de Portugal (IDP), atual Instituto Português do Desporto e Juventude (IPDJ). O principal objetivo deste trabalho consistiu no estudo/investigação de diferentes parâmetros de validação (especificidade/seletividade; capacidade de identificação; limite de deteção; exatidão e repetibilidade), de acordo com o protocolado no Procedimento Geral interno do Laboratório de Análises de Dopagem de Lisboa (LAD). O referido método de triagem e confirmação revelou possuir características de desempenho conformes com os requisitos aplicáveis, pelo que é considerado validado e apto.

Concomitant rotavirus serotypes 1 and 4 infections in a diarrhoeic child from Belém, Brazil

Concomitant serotypes 1 and 4 infections were detected in a 15-month old female child with community-acquired diarrhoea which lasted 7 days and coursed with moderate dehydration. The evidence for dual rotavirus infection was offered by the following findings: a) enzyme-linked immunosorbent assay (ELISA) positive reactions to both 1 and 4 serotypes; and b) extra-migrating bands at electro-phoresis of RNA in polyacrylamide gel (PAGE). These results suggest that children living under poor sanitation conditions are heavily exposed to rotavirus infections; in addition, the co-circulation of different serotypes in the same setting sustains the current concept that a rotavirus vaccine should be rnultivalent, in order to protect children against the four epidemiologically important rotavirus G serotypes.

Salivary gland proteins of the human malaria vector, Anopheles dirus B (Diptera: Culicidae)

Salivary gland proteins of the human malaria vector, Anopheles dirus B were determined and analyzed. The amount of salivary gland proteins in mosquitoes aged between 3 - 10 days was approximately 1.08 ± 0.04 µg/female and 0.1 ± 0.05 µg/male. The salivary glands of both sexes displayed the same morphological organization as that of other anopheline mosquitoes. In females, apyrase accumulated in the distal regions, whereas alpha-glucosidase was found in the proximal region of the lateral lobes. This differential distribution of the analyzed enzymes reflects specialization of different regions for sugar and blood feeding. SDS-PAGE analysis revealed that at least seven major proteins were found in the female salivary glands, of which each morphological region contained different major proteins. Similar electrophoretic protein profiles were detected comparing unfed and blood-fed mosquitoes, suggesting that there is no specific protein induced by blood. Two-dimensional polyacrylamide gel analysis showed the most abundant salivary gland protein, with a molecular mass of approximately 35 kilodaltons and an isoelectric point of approximately 4.0. These results provide basic information that would lead to further study on the role of salivary proteins of An. dirus B in disease transmission and hematophagy.

Partial characterization of proteolytic activity in Giardia duodenalis purified proteins

This report describes a preliminary characterization of proteolytic activity of proteins isolated from lysate of Giardia trophozoites of an axenic Brazilian strain. Fractions obtained by high-performance liquid chromatography (FPLC) were tested in SDS-polyacrylamide gel for the protein profiles, and the proteases activity was analyzed using gelatin impregnated SDS-PAGE. The proteases characterization was based on inhibition assays employing synthetic inhibitors for cysteine (E-64, IAA), serine (PMSF, TPCK, TLCK, and elastatinal), metalo (EDTA) and aspartic (pepstatin) proteases. Among thirty eluted fractions, polypeptide bands were observed in eight of them, however, proteolytic activity was detected in four ones (F23, F24, F25 and F26). Protein profiles of these fractions showed a banding pattern composed by few bands distributed in the migration region of 45 to < 18 kDa. The zymograms revealed proteolytic activity in all the four fractions assayed, mainly distributed in the migration region of 62 to 35 kDa. Among the profiles, the main pronounced zones of proteolysis were distinguished at 62, 55, 53, 50, 46 and 40 kDa. In inhibition assays, the protease activities were significantly inhibited by cysteine (E-64) and serine proteases (TPCK, TLCK and elastatinal) inhibitors. Gels incubated with other cysteine and serine protease inhibitors, IAA and PMSF, respectively, showed a decrease in the intensity of hydrolysis zones. Indeed, in the assays with the inhibitors EDTA for metalloproteases and pepstatin for aspartic proteases, none inhibition was detected against the substrate. These observations are relevants, especially if we consider that to define the real role of the proteases in host-parasite interaction, the purification of these enzymes for detailed studies may be warranted.

Standardization of an ELISA test using a recombinant nucleoprotein from the Junin virus as the antigen and serological screening for arenavirus among the population of Nova Xavantina, State of Mato Grosso

INTRODUCTION: Arenavirus hemorrhagic fever is a severe emerging disease. METHODS: Considering that the levels of antibodies against arenavirus in the Brazilian population are completely unknown, we have standardized an ELISA test for detecting IgG antibodies using a recombinant nucleoprotein from the Junin virus as the antigen. This protein was obtained by inserting the gene of the Junin virus nucleoprotein into the genome of Autographa californica nucleopolyhedrovirus, using the Bac-to-Bac baculovirus expression system. This recombinant baculovirus was used to infect S. frugiperda cells (SF9). RESULTS: The infection resulted in synthesis of high concentrations of recombinant protein. This protein was detected on 12.5% polyacrylamide gel and by means of Western blot. Using the standardized ELISA test, 343 samples from the population of Nova Xavantina were analyzed. We observed that 1.4% of the serum samples (five samples) presented antibody titers against arenavirus. CONCLUSIONS: These results show the population studied may present exposure to arenavirus infection.

The twist, writhe and overall shape of supercoiled DNA change during counterion-induced transition from a loosely to a tightly interwound superhelix. Possible implications for DNA structure in vivo.

A cryo-electron microscopy study of supercoiled DNA molecules freely suspended in cryo-vitrified buffer was combined with Monte Carlo simulations and gel electrophoretic analysis to investigate the role of intersegmental electrostatic repulsion in determining the shape of supercoiled DNA molecules. It is demonstrated here that a decrease of DNA-DNA repulsion by increasing concentrations of counterions causes a higher fraction of the linking number deficit to be partitioned into writhe. When counterions reach concentrations likely to be present under in vivo conditions, naturally supercoiled plasmids adopt a tightly interwound conformation. In these tightly supercoiled DNA molecules the opposing segments of interwound superhelix seem to directly contact each other. This form of supercoiling, where two DNA helices interact laterally, may represent an important functional state of DNA. In the particular case of supercoiled minicircles (178 bp) the delta Lk = -2 topoisomers undergo a sharp structural transition from almost planar circles in low salt buffers to strongly writhed "figure-eight" conformations in buffers containing neutralizing concentrations of counterions. Possible implications of this observed structural transition in DNA are discussed.

RecA-mediated annealing of single-stranded DNA and its relation to the mechanism of homologous recombination.

We demonstrate that RecA protein can mediate annealing of complementary DNA strands in vitro by at least two different mechanisms. The first annealing mechanism predominates under conditions where RecA protein causes coaggregation of single-stranded DNA (ssDNA) molecules and where RecA-free ssDNA stretches are present on both reaction partners. Under these conditions annealing can take place between locally concentrated protein-free complementary sequences. Other DNA aggregating agents like histone H1 or ethanol stimulate annealing by the same mechanism. The second mechanism of RecA-mediated annealing of complementary DNA strands is best manifested when preformed saturated RecA-ssDNA complexes interact with protein-free ssDNA. In this case, annealing can occur between the ssDNA strand resident in the complex and the ssDNA strand that interacts with the preformed RecA-ssDNA complex. Here, the action of RecA protein reflects its specific recombination promoting mechanism. This mechanism enables DNA molecules resident in the presynaptic RecA-DNA complexes to be exposed for hydrogen bond formation with DNA molecules contacting the presynaptic RecA-DNA filament.

Protein-binding site at the immunoglobulin mu membrane polyadenylylation signal: possible role in transcription termination.

mRNAs specifying immunoglobulin mu and delta heavy chains are encoded by a single large, complex transcription unit (mu + delta gene). The transcriptional activity of delta gene segments in terminally differentiated, IgM-secreting B lymphocytes is 10-20 times lower than in earlier B-lineage cells expressing delta mRNA. We find that transcription of the mu + delta gene in IgM-secreting murine myeloma cells terminates within a region of 500-1000 nucleotides immediately following the mu membrane (mu m) polyadenylylation site. Transcription decreases only minimally through this region in murine cell lines representative of earlier stages in B-cell development. A DNA fragment containing the mu m polyadenylylation signal gives protein-DNA complexes with different mobilities in gel retardation assays with nuclear extracts from myeloma cells than with nuclear extracts from earlier B-lineage cells. However, using a recently developed "footprinting" procedure in which protein-DNA complexes resolved in gel retardation assays are subjected to nucleolytic cleavage while still in the polyacrylamide gel, we find that the DNA sequences protected by factors from the two cell types are indistinguishable. The factor-binding site on the DNA is located 5' of the mu m polyadenylylation signal AATAAA and includes the 15-nucleotide-long A + T-rich palindrome CTGTAAACAAATGTC. This type of palindromic binding site exhibits orientation-dependent activity consistent with the reported properties of polymerase II termination signals. This binding site is followed by two sets of directly repeated DNA sequences with different helical conformation as revealed by their reactivity with the chemical nuclease 1,10-phenanthroline-copper. The close proximity of these features to the signals for mu m mRNA processing may reflect a linkage of the processes of developmentally regulated mu m polyadenylylation and transcription termination.

Genetic markers between Biomphalaria glabrata snails susceptible and resistant to Schistosoma mansoni infection

The analysis of the genetic variability related to susceptibility to Schistosoma mansoni infection in the vector of the genus Biomphalaria is important in terms of a better understanding of the epidemiology of schistosomiasis itself, the possible pathological implications of this interaction in vertebrate hosts, and the formulation of new strategies and approaches for disease control. In the present study, the genetic variability of B. glabrata strains found to be resistant or susceptible to S. mansoni infection was investigated using DNA amplification by random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR). The amplification products were analyzed on 8% polyacrylamide gel and stained with silver. We selected 10 primers, since they have previously been useful to detect polymorphism among B. glabrata and/or B. tenagophila. The results showed polymorphisms with 5 primers. Polymorphic bands observed only in the susceptible strain. The RAPD-PCR methodology represents an adequate approach for the analysis of genetic polymorphisms. The understanding of the genetic polymorphisms associated to resistance may contribute to the future identification of genomic sequences related to the resistance/susceptibility of Biomphalaria to the larval forms of S. mansoni and to the development of new strategies for the control of schistosomiasis.

Use of the comet test to assess DNA damage in children with ataxia-telangiectasia and their relatives.

The electrophoresis of cells in alkaline medium (comet assay) is a valid technique for quantifying DNA damage in patients with ataxia-telangiectasia and their relatives.

Strategy for optimizing DNA amplification in a peripheral blood PCR assay used for diagnosis of human brucellosis.

We studied two of the possible factors which can interfere with specific DNA amplification in a peripheral-blood PCR assay used for the diagnosis of human brucellosis. We found that high concentrations of leukocyte DNA and heme compounds inhibit PCR. These inhibitors can be efficiently suppressed by increasing the number of washings to four or five and decreasing the amount of total DNA to 2 to 4 microg, thereby avoiding false-negative results.

Phylogenetic relationships among four species of the guarani group of Drosophila (Diptera, Drosophilidae) as inferred by molecular and morphological analyses

Traditionally, the Drosophila guarani species group has been divided into two subgroups: the guarani and the guaramunu subgroups. Two, out of the four species included in this research, are members of the guarani subgroup (D. ornatifrons Duda, 1927 and D. subbadia Paterson & Mainland, 1943) and two are included in the guaramunu subgroup (D. maculifrons Duda, 1927 and D. griseolineata Duda, 1927). However, some authors have suggested that D. maculifrons and D. griseolineata are much closer to some species of the Drosophila tripunctata group than to some of the species of the guarani group. To add new data to the matter under dispute, Polyacrylamide Gel Eletrophoresis (PAGE-SDS) was used for the analysis and comparison of protein composition and Random Amplified Polymorphic DNA (RAPD) analysis to find differences in genomic DNA, in addition to the analysis of quantitative morphological characters previously described. Analysis of PAGE-SDS results in a dendrogram that pointed out D. subbadia as being the most distant within the Drosophila guarani group. However, these results were not supported either by RAPD analysis or by the analysis of continuous morphological characters, which supplied the clustering of D. subbadia with D. ornatifrons. Although our data give strong support to the clustering of D. subbadia and D. ornatifrons, none of the dendrograms provided a clade comprising D. maculifrons and D. griseolineata. Thus, this research does not support the traditional subdivision of the D. guarani group into those two subgroups.

Evidence against close linkage of the loci for fraXq of Martin-Bell syndrome and for factor IX.

Linkage between the loci for fraXq of Martin-Bell syndrome and factor IX was studied in nine families exhibiting this syndrome by means of a restriction fragment length polymorphism at the factor IX locus. Computer analysis of the data indicates there to be no evidence for close linkage between the syndrome and the factor IX locus.

Isolation and characterization of carcinoembryonic antigen (CEA) extracted from normal human colon mucosa.

Carcinoembryonic antigen (CEA) was identified in perchloric acid (PCA)_extract from normal colon mucosa by 2 immunological criteria: a line of identity in double diffusion and a parallel inhibition curve in radioimmunoassay (RIA), both with reference colon carcinoma-CEA (CEA-Tu). The average concentration of CEA in normal colon mucosa (CEA-No) was 35 times lower than in primary large bowel carcinomas and 230 times lower than in metastatic colon or rectum carcinomas. CEA-No was purified from PCA extracts of normal colon mucosa by Sephadex G-200 filtration and immunoadsorbent columns. Purified CEA-No had quatitatively the same inhibition activity in RIA as the British Standard CEA coded 73/601. Purified CEA-No was labelled with 125I. The percentage of binding of labelled CEA-No to a specific goat anti-CEA-Tu antiserum was similar to that of CEA-Tu. Labelled CEA-No could be used as radioactive tracer in RIA as well as labelled CEA-Tu. The physico-chemical properties of purified CEA-Tu as demonstrated by Sepharose 6 B filtration, SDS Polyacrylamide gel analysis and cesium chloride density gradient, were found to be almost identical to those of reference CEA-Tu. Preliminary results showed that CEA-No and CEA-Tu contained the same types of carbohydrates in similar proportions. A rabbit antiserum against CEA-No was obtained which demonstrated the same specificity as conventional anti-CEA-Tu antisera.