H. B. Machado

Different parkinsonism models produce a time-dependent induction of COX-2 in the substantia nigra of rats.

The present study investigated the effects on general activity, COX-2 and TH protein expression of intranigral neurotoxins LPS, MPTP or 6-OHDA infusion in rats. Results indicate that LPS produced an increase in locomotion frequency (3 and 7 days after surgery) and a strong up-regulation of COX-2 protein 16 and 24 h after surgery, as observed in the substantia nigra (SN). The MPTP model generated impairment in locomotion frequency 24 h after surgery. Besides, MPTP caused a marked up-regulation in COX-2 protein observed in the SN 16 h after surgery. Moreover, the 6-OHDA model produced severe motor impairment indicated by the decrease in locomotion (24 h) and rearing (24 h, 3 and 7 days) frequencies and also an increase in latency (24 h, 3 and 7 days) and immobility (24 h and 3 days) times. We also demonstrated an up-regulation of COX-2, which occurred in the SN 4-24 h after surgery. TH protein did not appear to be reduced in the striatum in the groups lesioned with the neurotoxins. In contrast, the TH content of SN was significantly reduced in the groups lesioned with the very same neurotoxins. For all the models analyzed, we observed no statistical differences in the expression of COX-2 in the striatum along the time-points. The results of the present study suggest that COX-2 induction patterns differ in function of the neurotoxin tested. Such time-dependent induction has been found to be relatively constant, a fact of great significance considering the importance of the neuroinflammatory process in Parkinsons disease.

Identification and characterization of the two-component NtrY/NtrX regulatory system in Azospirillum brasilense

Two Azospirillum brasilense open reading frames (ORFs) exhibited homology with the two-component NtrY/NtrX regulatory system from Azorhizobium caulinodans. These A. brasilense ORFs, located downstream to the nifR3ntrBC operon, were isolated, sequenced and characterized. The present study suggests that ORF1 and ORF2 correspond to the A. brasilense ntrY and ntrX genes, respectively. The amino acid sequences of A. brasilense NtrY and NtrX proteins showed high similarity to sensor/kinase and regulatory proteins, respectively. Analysis of lacZ transcriptional fusions by the beta-galactosidase assay in Escherichia coli ntrC mutants showed that the NtrY/NtrX proteins failed to activate transcription of the nifA promoter of A. brasilense. The ntrYX operon complemented a nifR3ntrBC deletion mutant of A. brasilense for nitrate-dependent growth, suggesting a possible cross-talk between the NtrY/X and NtrB/C sensor/regulator pairs. Our data support the existence of another two-component regulatory system in A. brasilense, the NtrY/NtrX system, probably involved in the regulation of nitrate assimilation.

Regulation of nif genes expression in Azospirillum brasilense and Herbaspirillum seropedicae

Several ethylenediamine (EDA)-resistant mutants of A. brasilense were isolated and found to depress nitrogenase in the presence of high NH 4 + concentrations. These mutants were defective in NH 4 + uptake and capable of excreting NH4 + in N-free semi-solid medium. A gene library of H. seropedicae strain HZ78 was constructed in cosmid pVK102. A recombinant plasmid pEMS1, capable of complementing a nifA - mutant of A. brasilense, was isolated and is being characterized.

Cloning of the nifA and nifB Genes of Azospirillum Brasilense Strain FP2

A genomic library of Azospirillum brasilense was constructed and used to isolate the nifA gene by complementation of a nifA - mutant of A. brasilense (FP10). A recombinant plasmid, pMAK7, was isolated and found to contain a functional nifA gene and to hybridize with nifA and nifB probes from Herbaspirillum seropedicae.