Evaluation of Extracts Produced by Some Tropical Fungi as Potential Cholinesterase Inhibitors

Summary Extracts of culture filtrates of fungal taxa belonging to Chaetomium sp., Guignardia mangiferae, Pestalotiopsis guepinii, Phomopsis sp., Physalospora sp., Xylaria sp., and sterile mycelium isolated as endophytes from leaves of members of the plant families Anacardiaceae, Apocynaceae, Leguminosae and Palmae; and seven different species of Penicillium isolated from insects were tested for their in vitro acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibitory activity using Ellman’s colorimetric method adapted for thin layer chromatography. The results showed high rate of cholinesterase inhibitory activity, with the best result being obtained with Penicillium extracts, indicating that they might represent a valuable source for pharmaceutical applications and are worthy of further study.     

Sexual and social behavior of adult saddle-back tamarins (Saguinus fuscicollis), castrated as neonates

The behavior of four adult males castrated as neonates was compared with that of three neonatally sham-castrated males and two untreated males serving as controls. Two males castrated as sexually experienced adults served as additional controls. All males lived in permanent male-female pairs. The interactions of neonatally castrated, sham-castrated, and intact control males with strange conspecifics were tested in a situation analogous to territorial defense. In addition, the interactions of all males with their female pair mates were tested. Neonatally castrated males showed less injurious aggression against strangers and interacted with them less frequently than control males. There were significant differences between both groups of males in some of the agonistic behaviors recorded, but not in all. In pair tests, neonatally castrated males failed to show any coordinated male copulatory behavior but control males did. Males castrated as adults showed rates of mounting and thrusting similar to those of intact controls. Neonatally castrated males tended to interact with their females on a lower level than neonatally sham-castrated and untreated control males. How ever, most differences between both groups in the levels of pair mate interactions were not statistically significant. It is concluded that castration during neonatal life prevents the display of male copulatory behavior in adulthood and selectively affects the display of a number of other behaviors in adulthood. It is not possible at this point to decide whether these results are due to hormonal deficiencies on an organizational or activational level or both. However, data from studies currently in progress suggest that organizational processes might be responsible for part of the effect.     

Anaerobic microbial counts of different potential anaerobic inocula

Sludges of various origin were analysed on the basis of microbial counts of anaerobes in order to investigate potential and alternative sources for inoculating anaerobic digestors. Sludges from neutralization and oil and fat separation tanks of a sugar cane factory, and from a stabilisation pond of a purified terephthalic-acid-producing plant were found to be adequate potential inocula. Activated sludges were found to have both hydrogenotrophic and acetoclastic methanogens and obligate hydrogen-producing acetogens (OHPA). In all sludges tested, hydrogenotrophic methanogens were the dominant strict anaerobes. The ratio between most probable number counts of acetoclastic methanogens and OPHA seems to be different between dispersed sludges and granular sludges. Correspondence to: J. P. Guyot     

Chemophenetics as a Tool for Distinguishing Morphotypes of Annona emarginata (Schltdl.) H. Rainer

Annona emarginata (Annonaceae) has two morphological variations (A. emarginata ‘mirim’ and A. emarginata ‘terra-fria’). The species has agricultural value and produces specialized metabolites of pharmacological interest. The objective of this work was to analyze whether chemical and morphological differences contribute to differentiate A. emarginata ‘terra-fria’ from A. emarginata ‘mirim’, as chemophenetic variations of A. emarginata. The analysis of chemical compounds was based on the quantification, profile of root alkaloids and on the leaves volatile profile, together with morphometric analyses of the leaf blade. The samples were collected in three phenological stages (flowering, fruiting, and vegetative) at two places in São Paulo, Brazil. Differences in the composition of the alkaloid profile and leaf volatiles (in both places and in the three phenological stages) allowed us to separate the two morphotypes by multivariate statistical analysis. These differences agreed with the leaf blade morphology and flower color. This first chemophenetic report of A. emarginata demonstrates that, in addition to morphological variations, the specialized metabolism of roots and leaves can be phytochemical characters, which suggest the existence of at least morphochemotypes of A. emarginata.     

A Golgi PKD Activity Reporter Reveals a Crucial Role of PKD in Nocodazole-Induced Golgi Dispersal

The protein kinase D (PKD) family comprises multifunctional serine/threonine-specific protein kinases with three mammalian isoforms: PKD1, PKD2 and PKD3. A prominent PKD function is the regulation of basolateral-targeted transport carrier fission from the trans -Golgi network (TGN). To visualize site-specific PKD activation at this organelle, we designed a molecular reporter consisting of a PKD-specific substrate sequence fused to enhanced green fluorescent protein (EGFP), specifically targeted to the TGN via the p230 GRIP domain. Quantitative analyses using a phosphospecific antibody and ratiometric fluorescence imaging revealed that Golgi-specific phosphorylation of the reporter was strictly dependent on stimulation of endogenous PKD or transient expression of active PKD constructs. Conversely, PKD-specific pharmacological inhibitors and siRNA-mediated PKD knockdown suppressed reporter phosphorylation. Using this reporter we investigated a potential role for PKD in the regulation of Golgi complex morphology. Interestingly, nocodazole-induced Golgi complex break-up and dispersal was associated with local PKD activation as measured by reporter phosphorylation and this was efficiently blocked by expression of a dominant-negative PKD mutant or PKD depletion. Our data thus identify a novel link between PKD activity and the microtubule cytoskeleton, whereby Golgi complex integrity is regulated.     

Protein and Genome Evolution in Mammalian Cells for Biotechnology Applications

Mutation and selection are the essential steps of evolution. Researchers have long used in vitro mutagenesis, expression, and selection techniques in laboratory bacteria and yeast cultures to evolve proteins with new properties, termed directed evolution. Unfortunately, the nature of mammalian cells makes applying these mutagenesis and whole-organism evolution techniques to mammalian protein expression systems laborious and time consuming. Mammalian evolution systems would be useful to test unique mammalian cell proteins and protein characteristics, such as complex glycosylation. Protein evolution in mammalian cells would allow for generation of novel diagnostic tools and designer polypeptides that can only be tested in a mammalian expression system. Recent advances have shown that mammalian cells of the immune system can be utilized to evolve transgenes during their natural mutagenesis processes, thus creating proteins with unique properties, such as fluorescence. On a more global level, researchers have shown that mutation systems that affect the entire genome of a mammalian cell can give rise to cells with unique phenotypes suitable for commercial processes. This review examines the advances in mammalian cell and protein evolution and the application of this work toward advances in commercial mammalian cell biotechnology.     

Evaluation of the Presence of Mutagenic Dyes in Sediments from Cristais River

Azo dyes are largely used by coloring textiles and can contaminate the aquatic environment, including the sediment, through their release through effluent discharges. In this work the presence of mutagenic azo dyes was evaluated using Thin Layer Chromatography in sediment samples of the Cristais River upstream and downstream of an azo dye processing plant discharge area. Mutagenicity of the sediment samples was also analyzed using the Salmonella/microsome assay with the strain YG1041 in the presence and absence of S9. Extracts of benthic organisms collected in the same area were analyzed for the presence of dyes. The dyes CI Disperse Blue 373 and CI Disperse Orange 37 as well as three unknown fluorescent compounds were detected only in the sediment samples collected downstream of the industrial discharge. Activity was detected with the Salmonella assay in the three samples analyzed but higher values were obtained after the azo dye processing plant when compared to the reference site. This effect could be partially explained by the presence of the mutagenic dyes detected, considering their mutagenic potencies. No dyes were found in the extracts of the organisms. Further studies should be performed to evaluate the fate and effects of these dyes in the sediment and in the aquatic community and their potential to be transferred to the water column.     

Characterization of the molecular defect in a null allele of the opaque-2 locus of maize

The molecular defect in an opaque-2 (o2) mutant, previously characterized as a null allele, has been identified as containing an insertion of the transposable element of the Bergamo (Bg) family. Restriction mapping and partial sequence analysis of the Bg in the o2 null allele indicates that this element is distinct from the previously described Bg as well as the defective Bg (rbg) of the o2m(r) allele. It is, however, inserted at the same site in O2 as the rbg of 62m(r) and can transpose when Bg is present. This study shows that, depending on genetic background, this allele may not behave as a stable null which could dramatically influence the conclusions drawn from experiments based on this particular mutant.