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.     

Die Feinstruktur des Augenfleckes beiAcetabularia-Gameten und sein Verhalten nach der Gametenfusion

Summary Light and electron microscope studies on the fine structure of the stigma ofAcetabularia gametes and on its behaviour after gamete-fusion are described.The stigma, located within the single gamete-chloroplast, is composed of two layers of nearly hexagonal units, separated by double membranes.After gamete-fusion the young zygote cell contains two eye-spots, according to its two chloroplasts derived from each gamete cell. In later stages of zygote development the eyespots appear fragmented. There are indications that stigma fragments become distributed to newly formed chloroplasts.In zygote cells neither fusion of the gamete-chloroplasts nor of the eye-spots were observed.