SNUFER is a software for the automatic localization and generation of tables used for the presentation of single nucleotide polymorphisms (SNPs). After input of a fasta file containing the sequences to be analyzed, a multiple sequence alignment is generated using ClustalW ran inside SNUFER. The ClustalW output file is then used to generate a table which displays the SNPs detected in the aligned sequences and their degree of similarity. This table can be exported to Microsoft Word, Microsoft Excel or as a single text file, permitting further editing for publication. The software was written using Delphi 7 for programming and FireBird 2.0 for sequence database management. It is freely available for noncommercial use and can be downloaded from http://www.heranza.com.br/bioinformatica2.htm.

Canine monocytic ehrlichiosis caused by Ehrlichia canis is endemic in many regions of Brazil. Since thrombocytopenia is a common finding in infected dogs, many clinicians tend to use it as an indication for antibiotic treatment. Polymerase chain reaction (PCR) and nested PCR were used to study the presence of E. canis, Anaplasma platys and Babesia spp. in thrombocytopenic and non-thrombocytopenic dogs from Ribeirão Preto, Brazil. Despite the high prevalence of E. canis infection among thrombocytopenic dogs, 46.7% of the thrombocytopenic dogs studied were either infected with Babesia spp. or A. platys or not infected with any of the three pathogens. There was a high incidence (25.4%) of E. canis infection in non-thrombocytopenic dogs. Although infection with E. canis should be considered in thrombocytopenic dogs, the final diagnosis needs to be confirmed by complementary tests such as blood smears and PCR to avoid the unnecessary use of antibiotics.

The development of assays for quantitative analysis of the relative transcript levels of ABC transporter genes by real-time reverse transcription-PCR (RT-PCR) might provide important information about multidrug resistance in filamentous fungi. Here, we evaluate the potential of real-time RT-PCR to quantify the relative transcript levels of ABC transporter Atr genes from Aspergillus nidulans. The AtrA to AtrD genes showed different and higher levels in the presence of structurally unrelated drugs, such as camptothecin, imazalil, itraconazole, hygromycin, and 4-nitroquinoline oxide. We also verified the relative transcript levels of the Atr genes in the A. nidulans imazalil-resistant mutants. These genes displayed a very complex pattern in different ima genetic backgrounds. The imaB mutant has higher basal transcript levels of AtrB and -D than those of the wild-type strain. The levels of these two genes are comparable when the imaB mutant is grown in the presence and absence of imazalil. The imaC,-D, and -H mutants have higher basal levels of AtrA than that of the wild type. The same behavior is observed for the relative transcript levels of AtrB in the imaG mutant background.

Two l-amino acid oxidases (LAAOs) were identified by random sequencing of cDNA libraries from the venom glands of Bothrops moojeni(BmooLAAO) and Bothrops jararacussu(Bjussu LAAO). Phylogenetic analysis involving other SV-LAAOs showed sequence identities within the range 83-87% being closely related to those from Agkistrodon and Trimeresurus. Molecular modeling experiments indicated the FAD-binding, substrate-binding, and helical domains of Bmoo and Bjussu LAAOs. The RMS deviations obtained by the superposition of those domains and that from Calloselasma rhodostoma LAAO crystal structure confirm the high degree of structural similarity between these enzymes. Purified BjussuLAAO-I and BmooLAAO-I exhibited antiprotozoal activities which were demonstrated to be hydrogen-peroxide mediated. This is the first report on the isolation and identification of cDNAs encoding LAAOs from Bothrops venom. The findings here reported contribute to the overall structural elucidation of SV-LAAOs and will advance the understanding on their mode of action.

An l-amino acid oxidase isolated from Bothrops moojeni snake venom (BmooLAAO-I) was purified to a high degree using sequential CM-Sepharose ion-exchange and phenyl-Sepharose chromatography. When analyzed by mass spectrometry, the purified BmooLAAO-I presented a molecular weight of 64,889 and 130,779 under denaturing and nondenaturing conditions, respectively. BmooLAAO-I is a homodimeric acidic glycoprotein with a pI approximately 4.7, and the N-terminal sequence shows close structural similarity to other snake venom LAAOs. This enzyme was inactivated by freezing or low pH, and secondary structural analysis by circular dichroism revealed 48% alpha-helix, 20% beta-sheet, 12% beta-turn, and 20% random coil structures. BmooLAAO-I exhibited bactericidal, antitumoral, trypanocidal, edematogenic, and platelet-aggregating activities. All of these effects were inhibited by catalase, suggesting that these biological effects are mediated by the production of H(2)O(2). BmooLAAO-I induced typical apoptotic DNA fragmentation in HL-60 cells, which was also inhibited by catalase. These results point to the potential use of BmooLAAO-I as a therapeutic agent for treatment of diseases in which induction of H(2)O(2) production can be beneficial.

A repetitive DNA sequence (RPS) from Petunia hybrida had previously been shown to enhance expression variegation in petunia and tobacco and to carry a hot spot for de novo DNA methylation. Here we show that a strong de novo hypermethylation site is located within a palindromic segment of the RPS and present indirect evidence, based on sequence homologies to other repeat units within the RPS, for the formation of secondary structures at the methylation site in vivo. We demonstrate that the palindromic RPS element, which is moderately to highly repetitive in petunia, does not predominantly localise to constitutive heterochromatin. To test whether the RPS is subject to de novo methylation due to its repetitive nature or to intrinsic signals within the RPS, we integrated the RPS into the genome of Arabidopsis thaliana, a plant lacking homology to the RPS. Our data indicate that the palindromic element also acts as a de novo hypermethylation site in the non-repetitive genomic background of Arabidopsis, strongly suggesting a signal function of the palindromic RPS element.