The key target of this study was the tau protein kinase II system (TPK II) involving the catalytic subunit cdk5 and the regulatory component p35. TPK II is one of the tau phosphorylating systems in neuronal cells, thus regulating its functions in the cytoskeletal dynamics and the extension of neuronal processes. This research led to demonstration that the treatment of rat hippocampal cells in culture with fibrillary L L-amyloid (AL L) results in a significant increase of the cdk5 enzymatic activity. Interestingly, the data also showed that the neurotoxic effect of 1^20 W WM AL L on primary cultures markedly diminished with co-incubation of hippocampal cells with the amyloid fibers plus the cdk5 inhibitor butyrolactone I. This inhibitor protected brain cells against AL L-induced cell death in a concentration dependent fashion. Moreover, death was also prevented by a cdk5 antisense probe, but not by an oligonucleotide with a random sequence. The cdk5 antisense also reduced neuronal expression of cdk5 compared with the random oligonucleotide. The studies indicate that cdk5 plays a major role in the molecular path leading to the neurodegenerative process triggered by the amyloid fibers in primary cultures of rat hippocampal neurons. These findings are of interest in the context of the pathogenesis of Alzheimer's disease.z 1999 Federation of European Biochemical Societies.

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We have used astrocyte-conditioned medium (ACM) to promote the transdifferentiation of bovine chromaffin cells and study modifications in the exocytotic process when these cells acquire a neuronal phenotype. In the ACM-promoted neuronal phenotype, secretory vesicles and intracellular Ca 2+ rise were preferentially distributed in the neurite terminals. Using amperometry, we observed that the exocytotic events also occurred mainly in the neurite terminals, wherein the individual exocytotic events had smaller quantal size than in undifferentiated cells. Additionally, duration of pre-spike current was significantly shorter, suggesting that ACM also modifies the fusion pore stability. After long exposure (7-9 days) to ACM, the kinetics of catecholamine release from individual vesicles was markedly accelerated. The morphometric analysis of vesicle diameters suggests that the rapid exocytotic events observed in neurites of ACM-treated cells correspond to the exocytosis of large dense-core vesicles (LDCV). On the other hand, experiments performed in EGTA-loaded cells suggest that ACM treatment promotes a better coupling between voltage-gated calcium channels (VGCC) and LDCV. Thus, our findings reveal that ACM promotes a neuronal phenotype in chromaffin cells, wherein the exocytotic kinetics is accelerated. Such rapid exocytosis mode could be caused at least in part by a better coupling between secretory vesicles and VGCC.

ABBREVIATIONS: Aβ, β-amyloid peptide; AD, Alzheimer's disease; CNh, neuronal cell line derived from the cerebral cortex of a normal mouse; CTb, neuronal cell line derived from the cerebral cortex of a trisomy 16 mouse; FBS, fetal bovine serum; PBS, phosphate buffered saline; 2,4-DNP, 2,4-dinitrophenol; 3-NP, 3nitrophenol; 4-AA, 4-anisidine; NGF, nerve growth factor; [Ca 2+ ] i , intracellular Ca 2+ levels; PMSF, phenylmethylsulfonylfluoride.

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In the budding yeast Saccharomyces cerevisiae, the FUN-LOV (FUNgal Light Oxygen and Voltage) optogenetic switch enables high levels of light-activated gene expression in a reversible and tunable fashion. The FUN-LOV components, under identical promoter and terminator sequences, are encoded in two different plasmids, which limits its future applications in wild and industrial yeast strains. In this work, we aim to expand the molecular versatility of the FUN-LOV switch to increase its biotechnological applications. Initially, we generated new variants of this system by replacing the promoter and terminator sequences and by cloning the system in a single plasmid (FUN-LOVSP). In a second step, we included the nourseothricin (Nat) or hygromycin (Hph) antibiotic resistances genes in the new FUN-LOVSP plasmid, generating two new variants (FUN-LOVSP-Nat and FUN-LOVSP-Hph), to allow selection after genome integration. Then, we compared the levels of light-activated expression for each FUN-LOV variants using the luciferase reporter gene in the BY4741 yeast strain. The results indicate that FUN-LOVSP-Nat and FUN-LOVSP-Hph, either episomally or genome integrated, reached higher levels of luciferase expression upon blue-light stimulation compared the original FUN-LOV system. Finally, we demonstrated the functionality of FUN-LOVSP-Hph in the 59A-EC1118 wine yeast strain, showing similar levels of reporter gene induction under blue-light respect to the laboratory strain, and with lower luciferase expression background in darkness condition. Altogether, the new FUN-LOV variants described here are functional in different yeast strains, expanding the biotechnological applications of this optogenetic tool.

The aim of this study was to perform a descriptive study of the morphology, anatomical variations and morphometry of medial talocrural (or deltoid) ligament. We dissected 27 lower limbs obtained from amputations without histories of age, sex or disease. The measurements were made with a caliper, compass and ruler, expressing the results in millimeters. We described the superficial layer morphology of the medial ligament, measuring the size and ligament's thickness. For the deep layer we described and measured the length (l), width (w) and thickness (t). Results: Superficial layer: trapezoid form=66.7% (anterior margin=30.5 mm; posterior margin=27.6 mm; top margin=22.6 mm; bottom margin=50.5 mm), rectangular form=19% (anterior margin=19.3 mm; posterior margin=27.2 mm; top margin=24.4 mm; bottom margin=29.8 mm), triangular form=14.3% (anterior margin=37 mm; posterior margin=37.8 mm; bottom margin=48.3 mm). The average thickness of the superficial layer was 3.6 mm. Deep layer of the medial ligament: l=6.9 mm, w=11 mm, t=5.7 mm; presented rectangular form in 100%. In 76.2% of the specimens, the deep layer was covered completely by the superficial layer; however, in 23.8% the coverage is incomplete, showing the deep layer by posterior angle. The literature is contradictory regarding the anatomy and variations of the medial ligament of the ankle. There are important differences in morphology, attachments, subdivisions and relationships between the two layers of the deltoid ligament. Conclusions: We found significant anatomical variations in the morphology and the relationship between the superficial and deep layers of the deltoid ligament.