Caracterização físico-química de amostras de mel de abelha africanizada dos municípios de Santa Helena e Terra Roxa (PR) / Physicochemical parameters of honey from samples from africanized honeybees in Santa Helena and terra roxa counties (PR)

O presente estudo teve como objetivo a caracterização físico-química de amostras de mel de Apis mellifera coletadas nos municípios de Santa Helena e Terra Roxa, localizados na região oeste do Estado do Paraná, Brasil. Foram coletadas 40 amostras de mel, sendo 20 do município de Santa Helena e 20 de Terra Roxa (PR), coletadas diretamente com os apicultores, as quais foram submetidas a análises físico-químicas de umidade, acidez, pH, cinzas, condutividade elétrica e cor, a fim de verificar se as mesmas apresentavam-se em conformidade com a legislação nacional vigente (Instrução Normativa nº 11), além de conhecer o perfil físico-químico do mel das diferentes localidades, na safra 2008/2009. Os dados encontrados foram submetidos à análise de variância e teste F a 5% de significância. Os resultados apresentaram diferença significativa (P<0,05) apenas entre os valores de acidez, tendo o mel oriundo de Terra Roxa apresentado valor mais alto (33,45±7,7meq.kg-1) que o de Santa Helena (24,53±6,3meq.kg-1). Em sua maioria, as amostras analisadas encontraram-se dentro das especificações determinadas pela legislação para as características físico-químicas, com exceção do parâmetro de umidade, que, apesar de não ter apresentado diferença significativa entre os valores encontrados para os dois municípios, estavam acima do limite estabelecido pela legislação (20 por cento) em 8 amostras do município de Santa Helena e em 7 de Terra Roxa, totalizando 37,5 por cento) das amostras. Essa característica pode ter deixado o produto mais susceptível à fermentação e pode ter sido ocasionada pela colheita imatura do mel...

This study aimed to physicochemically characterize the honey samples of Apis mellifera collected in Santa Helena and Terra Roxa counties, located in the western region of Paraná State, Brazil. Forty honey samples were collected, 20 in Santa Helena and 20 in Terra Roxa (PR) counties, directly from beekeepers, and were subjected to physico-chemical analyzes of moisture, acidity, pH, ashes, electrical conductivity and color, to verify whether they were in accordance with the existing national legislation (Instruction No. 11), in addition to knowing the physicochemical profile of different sites during the 2008/2009 season. The data were submitted to analysis of variance and F test at 5 percent significance level. The results showed significant differences (P<0.05) between values of acidity, and the honey samples from Terra Roxa presented a higher value (33.45±7.7 meq.kg-1) than Santa Helena (24 = 53±6.3 meq.kg-1). Most of the analyzed samples were within the specifications prescribed in the legislation for the physico-chemical parameters except for moisture, which despite having presented a significant difference between values obtained from two counties, were above the limits established by the legislation (20 percent) in eight samples in Santa Helena and seven in Terra Roxa counties, totaling 37.5 percent. This characteristic may have made the product more susceptible to fermentation, and may have been caused by harvesting immature honey...

Isolation and characterization of strains of Flavobacterium columnare from Brazil.

Flavobacterium columnare is an important pathogen of freshwater fish, implicated in skin and gill disease, often causing high mortality. An outbreak of skin disease in fingerling and adult Nile tilapia, Oreochromis niloticus (L.), cultivated in a recirculation system, was investigated. Four strains were isolated and characterized by biochemical reactions, enzyme production, fatty acid profile and analysis of the 16S-23S rDNA intergenic spacer region. All strains were identified as F. columnare. Experimental infection assays with one of these strains (BZ-5-02) were conducted and pathogenicity (by intramuscular route) was demonstrated in Nile tilapia and channel catfish, Ictalurus punctatus (Rafinesque). This is the first report of characterization of Brazilian strains of F. columnare.

Charge-to-alanine mutagenesis of the adeno-associated virus type 2 Rep78/68 proteins yields temperature-sensitive and magnesium-dependent variants.

The adeno-associated virus type 2 (AAV) replication (Rep) proteins Rep78 and 68 (Rep78/68) exhibit a number of biochemical activities required for AAV replication, including specific binding to a 22-bp region of the terminal repeat, site-specific endonuclease activity, and helicase activity. Individual and clusters of charged amino acids were converted to alanines in an effort to generate a collection of conditionally defective Rep78/68 proteins. Rep78 variants were expressed in human 293 cells and analyzed for their ability to mediate replication of recombinant AAV vectors at various temperatures. The biochemical activities of Rep variants were further characterized in vitro by using Rep68 His-tagged proteins purified from bacteria. The results of these analyses identified a temperature-sensitive (ts) Rep protein (D40,42,44A-78) that exhibited a delayed replication phenotype at 32 degrees C, which exceeded wild-type activity by 48 h. Replication activity was reduced by more than threefold at 37 degrees C and was undetectable at 39 degrees C. Stability of the Rep78 protein paralleled replication levels at each temperature, further supporting a ts phenotype. Replication differences resulted in a 3-log-unit difference in virus yields between the permissive and nonpermissive temperatures (2.2 x 10(6) and 3 x 10(3), respectively), demonstrating that this is a relatively tight mutant. In addition to the ts Rep mutant, we identified a nonconditional mutant with a reduced ability to support viral replication in vivo. Additional characterization of this mutant demonstrated an Mg(2+)-dependent phenotype that was specific to Rep endonuclease activity and did not affect helicase activity. The two mutants described here are unique, in that Rep ts mutants have not previously been described and the D412A Rep mutant represents the first mutant in which the helicase and endonuclease functions can be distinguished biochemically. Further understanding of these mutants should facilitate our understanding of AAV replication and integration, as well as provide novel strategies for production of viral vectors.

The adeno-associated virus type 2 p40 promoter requires a proximal Sp1 interaction and a p19 CArG-like element to facilitate Rep transactivation.

We have identified the sequence elements that are required for adeno-associated virus type 2 p40 promoter activity. Mutation of specific promoter elements showed that two Sp1 sites at approximately -50 (Sp1-50) and -70 (GGT-70) bp upstream of the start of the p40 messages were necessary for maximal promoter activity. As expected, the TATA site at -30 was also essential. In vitro DNA binding experiments confirmed that the Sp1-50 and GGT-70 sites were bound by Sp1 or Sp1-like proteins. Two other transcription elements, the ATF-80 and AP1-40 sites, may play a role in p40 activity. Mutation of these elements resulted in a modest decrease in p40 transcription, but DNA binding experiments did not clearly demonstrate binding of transcription factors to these sites. In contrast, a major late transcription factor site at -110 was shown to bind the transcription factor, but mutation of this site had no effect on p40 activity. In a previous report, we have shown that transactivation of the p40 promoter by the viral Rep proteins required an upstream Rep binding element (in the terminal repeat or the p5 promoter), an unidentified p19 promoter element, and a p40 promoter element (D. J. Pereira and N. Muzyczka, J. Virol. 71:1747-1756, 1997). Here we demonstrate that the CArG-140 element in the p19 promoter and the Sp1-50 element in the p40 promoter are the specific p19 and p40 elements required for Rep induction of p40. As in the case of the p19 promoter, Sp1 facilitates interaction of Rep with the p40 promoter by interaction of the two proteins. Furthermore, electron microscopy experiments demonstrated that when Rep is bound to an upstream Rep binding element, it can interact with a proximal Sp1 site by protein contacts and create a loop in the intervening DNA. This finding suggests a common mechanism whereby the Rep binding element in the TR or the p5 promoter induces p19 and p40 activity by interaction with their respective Sp1 sites.

The cellular transcription factor SP1 and an unknown cellular protein are required to mediate Rep protein activation of the adeno-associated virus p19 promoter.

Control of adeno-associated virus (AAV) transcription from the three AAV promoters (p5, p19, and p40) requires the adenovirus E1a protein and the AAV nonstructural (Rep) proteins. The Rep proteins have been shown to repress the AAV p5 promoter yet facilitate activation of the p19 and p40 promoters during a productive infection. To elucidate the mechanism of promoter regulation by the AAV Rep proteins, the cellular factors involved in mediating Rep activation of the p19 promoter were characterized. A series of protein-DNA binding experiments using extracts derived from uninfected HeLa cells was performed to identify cellular factors that bind to the p19 promoter. Electrophoretic mobility shift assays, DNase I protection analyses, and UV cross-linking experiments demonstrated specific interactions with the cellular factor SP1 (or an SP1-like protein) at positions -50 and -130 relative to the start of p19 transcription. Additionally, an unknown cellular protein (cellular AAV activating protein [cAAP]) with an approximate molecular mass of 34 kDa was found to interact with a CArG-like element at position -140. Mutational analysis of the p19 promoter suggested that the SP1 site at -50 and the cAAP site at -140 were necessary to mediate Rep activation of p19. Antibody precipitation experiments demonstrated that Rep-SP1 protein complexes can exist in vivo. Although Rep was demonstrated to interact with p19 DNA directly, the affinity of Rep binding was much lower than that seen for the Rep binding elements within the terminal repeat and the p5 promoter. Furthermore, the interaction of purified Rep68 with the p19 promoter in vitro was negligible unless purified SP1 was also added to the reaction. Thus, the ability of Rep to transactivate the p19 promoter is likely to involve SP1-Rep protein contacts that facilitate Rep interaction with p19 DNA.

The adeno-associated virus (AAV) Rep protein acts as both a repressor and an activator to regulate AAV transcription during a productive infection.

Adeno-associated virus (AAV) uses three promoters, p5, p19, and p40, to regulate viral gene expression. The p5 and p19 promoters direct the synthesis of the viral regulatory proteins, Rep78 and -68 and Rep52 and -40, respectively. The p5 Rep proteins bind a linear 22-bp sequence, the Rep binding element (RBE), that is within both the terminal repeat (TR) and the p5 promoter. In the absence of helper virus, all four Rep proteins have been shown to reduce transcription from the viral p5 and p19 promoters. In this report, we focus on the roles of these proteins and the RBEs in controlling transcription during a productive infection, that is, in the presence of adenovirus. We find that in the presence of adenovirus, the p5 RBE represses p5 transcription while the RBE in the TR activates p5. However, both the TR RBE and the p5 RBE transactivate the p19 and p40 promoters. The fact that the p5 RBE-Rep complex can transactivate p19 and p40 while repressing p5 suggests that Rep78/68 is both a repressor and a transactivator. Rep repression of p5 is specific for the p5 RBE, as other p5 promoter elements do not support this activity. We also demonstrate that in the presence of adenovirus, the p19 Rep proteins, which do not bind to the RBE, can eliminate repression of the p5 promoter by Rep78 and Rep68. This may occur by the association of Rep52 with Rep78 or Rep68 to produce a Rep78/68-Rep52 protein complex which can be detected in vivo by immunoprecipitation. Finally, two Rep mutants that were deficient in RBE binding and transactivation but positive for p5 repression were identified. These mutants may define interaction domains involved in making contacts with other proteins that facilitate repression. These observations suggest a mechanism for controlling the p5 and p19 mRNA levels during a productive AAV infection.

Identification of linear DNA sequences that specifically bind the adeno-associated virus Rep protein.

We have used baculovirus-expressed Rep68 that has been purified to homogeneity to reexamine the binding properties of the Rep protein. We find that Rep68 is capable of binding to a linear DNA sequence that is contained within a 25-bp sequence of the A stem of the adeno-associated virus (AAV) terminal repeat proximal to the B and C palindromes. This has been shown conclusively by demonstrating that Rep68 could specifically bind to a synthetic oligonucleotide containing the 25-bp region in the absence of the other sequences within the terminal repeat. Rep78 was also capable of binding the A stem recognition element, as demonstrated by the fact that a DNA affinity column containing the 25-bp sequence can be used to purify Rep78. The ability to recognize the linear DNA sequence within the A stem provides a mechanism by which the Rep protein can be oriented on the terminal repeat so that only the correct strand is cut at the terminal resolution site (trs site) during terminal resolution. In addition, computer analysis suggests that sequences similar to the A stem element are present within the three AAV promoter regions. Electrophoretic mobility shift experiments clearly demonstrate that the p5 promoter contains a Rep binding sequence. DNase protection experiments indicate that the Rep binding sequence within the p5 promoter is located between the YY1 initiator sequence and the TATA binding site. This position immediately suggests a mechanism by which the Rep protein could act as a repressor or a transactivator of p5 transcription by interacting with either YY1 or TBP. In addition, gel shift experiments suggest that the p19 promoter also contains a Rep binding site. The presence of Rep binding sites upstream of both promoters suggests that these sites may be involved in coordinate regulation of AAV transcription. In addition, we have identified a heterologous Rep binding sequence within pBR322 DNA. A comparison of the sequences within the A stem, p5, and pBR322 binding sites suggests that a repeating GAGC motif is at least part of the Rep recognition sequence. In the accompanying report (D. M. McCarty, J. H. Ryan, S. Zolutukhin, X. Zhou, and N. Muzyczka, J. Virol. 68:4998-5006, 1994), we examine the relative affinity of Rep to the A stem site and the complete terminal repeat. Finally, we also have reexamined the ability of Rep68 and Rep78 to cut at the trs site in substrates that do not contain the B and C palindromes or any apparent secondary structure.(ABSTRACT TRUNCATED AT 400 WORDS)

[Cesium-157 in milk produced on farms of the Angra dos Reis Region, RJ]. / Césio-137 em leite produzido em fazendas da Região de Angra dos Reis, RJ.

The first Brazilian Nuclear Power Plant is located in the Angra dos Reis county, about 130 km West of Rio de Janeiro. Among the radionuclides that will be released to the environment by the Nuclear Plant, tritium and radioisotopes of Cs, I, Co and Sr are the ones of greatest potential impact on the local population. During the preliminary phase of the pre-operational environmental radiological program, 137Cs resulting from nuclear explosion fallout was detected in milk samples from only one farm, among the ones included in the monitoring program. This finfing seemed odd, leading to believe on the possibility of a soil anomaly on the region, in which 137Cs would be more available for plant uptake than in normal areas, as it has been observed in some areas in various countries. Trying to explain this issue, the Radioisotopes Laboratory of the Biophysics Institute and the Radioecology Laboratory of FURNAS decided to carry on a series of analyses of 137Cs in milk, pasture and soil collected in the four farms of the program. The results demonstrated the non-existence of a soil anomaly in the region regarding the 137Cs behavior Cesium-137 concentrations in milk varied from 0.06 to 0.93 Bq/l but the differences of the average values in the four farms were not statistically significant. In one farm, occasional high peaks of 137Cs concentrations in milk were observed, which seemed to be related to the cattle management. Apparently during certain periods, the cattle grazes in sectors whose 137Cs concentrations in soil and pasture are higher than in other areas of the same farm or the region, due to the influence of micro-climate and erosion of superficial soil.