Histone H3 is aberrantly phosphorylated in glutamine-repeat diseases

Double-labeling immunohistochemical studies staining with anti-ubiquitin and anti-phosphoserine antibodies and application of an enzymatic dephosphorylation technique reveal neuronal inclusions and affected nuclei to be aberrantly phosphorylated in brain tissues with patients with glutamine-repeat diseases. Regional distribution of the phosphorylated nuclei in neurons correlates with the pathology. To identify the target nuclear protein, transient expression of Huntington's disease exon 1 gene containing an expanded glutamine repeat was generated in a cell culture and nuclear inclusions were isolated with a fluorescence-activated cell sorting system. Immunoblotting studies of the aggregated nuclear proteins using anti-phosphoserine antibody demonstrate the protein of the aberrant phosphorylation as histone H3. The immunoblots of control and diseased brain tissues demonstrate that the phosphorylation of histone H3 is commonly increased in the diseased brains. Aberrant phosphorylation of histone H3 is surmised to be a shared pathological process in glutamine-repeat diseases.     

Divergent roles of GSK3 and CDK5 in APP processing

Glycogen synthase kinase-3 (GSK3) and cyclin-dependent kinase 5 (CDK5) are related serine/threonine kinases that have been well studied for their role in tau hyperphosphorylation, however, little is known about their significance in APP processing. Here we report that GSK3 and CDK5 are involved in APP processing in a divergent manner. Specific inhibition of cellular GSK3 by lithium or GSK3beta antisense elicits a reduction in Abeta. Conversely, negative modulation of cellular CDK5 activity by CDK5 inhibitor, roscovitine, or CDK5 antisense stimulates Abeta production. Neither GSK3 nor CDK5 inhibition by these means significantly affected cellular APP levels or APP maturation. Moreover, oral administration of lithium significantly reduces Abeta production whereas direct ICV administration of roscovitine augmented Abeta production in the brains of PDAPP (APP(V717F)) mice. Our data support a function for both GSK3 and CDK5 in APP processing, further implicating these two kinases in the pathogenesis of Alzheimer's disease.     

1-Methyl-4-phenylpyridinium-induced down-regulation of dopamine transporter function correlates with a reduction in dopamine transporter cell surface expression

The mechanisms whereby 1-methyl-4-phenylpyridinium (MPP(+)) mediates cell death and Parkinsonism are still unclear. We have shown that dopamine transporter (DAT) is required for MPP(+)-mediated cytotoxicity in HEK-293 cells stably transfected with human DAT. Furthermore, MPP(+) produced a concentration- and time-dependent reduction in the uptake of [3H]dopamine. We observed a significant decrease in [3H]WIN 35428 binding in the intact cells with MPP(+). The saturation analysis of the [3H]WIN 35428 binding obtained from total membrane fractions revealed a decrease in the transporter density (B(max)) with an increase in the dissociation equilibrium constant (K(d)) after MPP(+) treatment. Furthermore, biotinylation assays confirmed that MPP(+) reduced both plasma membrane and intracellular DAT immunoreactivity. Taken together, these findings suggest that the reduction in cell surface DAT protein expression in response to MPP(+) may be a contributory factor in the down-regulation of DAT function while enhanced lysosomal degradation of DAT may signal events leading to cellular toxicity.     

Contribution of presenilin/gamma-secretase to calsenilin-mediated apoptosis

Mutant presenilins cause early-onset of familial Alzheimer's disease and render cells vulnerable to apoptosis. Calsenilin/DREAM/KChIP3 is a multifunctional calcium-binding protein that interacts with presenilin and mediates calcium-mediated apoptosis. In the present study, we report that the calsenilin-mediated apoptosis is regulated by presenilin. The expression of calsenilin was highly up-regulated in neuronal cells undergoing Abeta42-triggered cell death. The incidence of calsenilin-mediated apoptosis was diminished in presenilin-1(-/-) mouse embryonic fibroblast cells or neuronal cells stably expressing a loss-of-function presenilin-1 mutant. On the contrary, an array of familial Alzheimer's disease-associated presenilin mutants (gain-of-function) increased calsenilin-induced cell death. Moreover, gamma-secretase inhibitors, including compound E and DAPT, decreased the calsenilin-induced cell death. These results suggest that the pro-apoptotic activity of calsenilin coordinates with presenilin/gamma-secretase activity to play a crucial role in the neuronal death of Alzheimer's disease.     

Cloning and expression analysis of a Parkinson's disease gene, uch-L1, and its promoter in zebrafish

Three genes, alpha-synuclein, parkin, and ubiquitin C-terminal hydrolase L1 (UCH-L1), have been associated with inherited forms of Parkinson's disease (PD), although their in vivo functions have remained largely unknown. To develop an animal model for the molecular study of PD, we cloned zebrafish uch-L1 cDNA and its gene promoter. Sequence analysis revealed that the zebrafish Uch-L1 is highly homologous (79%) to the human UCH-L1, which is a member of the deubiquitinating enzymes. By whole-mount in situ hybridization, we examined the spatiotemporal expression of uch-L1 mRNA in developing zebrafish embryos. The uch-L1 mRNAs are detected in neuronal cells at the first day of embryo development. The expression domain of uch-L1 overlaps with that of tyrosine hydroxylase, a molecular marker for dopaminergic neurons, in the ventral diencephalon, an equivalent structure to the substantia nigra where PD progresses in human. To further analyze the tissue-specific regulation of uch-L1 gene expression, we also tested its gene promoter activity and showed a preferential neuronal expression in transient transgenic zebrafish embryos. These results suggest that uch-L1 may have an important role in the development of neuronal cells in early embryos as well as in the degeneration and disease of neuronal cells in late adult brain.     

ACE inhibition actively promotes cell survival by altering gene expression

We tested the effect of ACE inhibition on the survival of bovine retinal (REC) and choroidal (CEC) endothelial cells (EC) in culture. The ACE inhibitor captopril delayed the apoptotic tube collapse of REC on Matrigel for >15 days. Captopril treatment of confluent monolayers (2-8 weeks) followed by slow starvation (2-4 weeks) increased EC viability by approximately 200%. Two-week captopril exposures were sufficient to confer maximal protection. Only vehicle-treated EC demonstrated apoptotic features such as membrane blebbing and DNA laddering. By RT-PCR, the starvation marker p202 was upregulated only in starved cells. In REC, captopril upregulated the pro-survival proteins mortalin-2, uPA, and uPAR while downregulating the anti-growth sprouty-4 and tPA. In CEC, captopril also upregulated tPA and its inhibitor PAI-1. Amiloride (uPA inhibitor) blocked the captopril-induced increase in EC survival, secondary sprouting, and invasion in Matrigel. The pro-survival effects of captopril involve the reprogramming of genes involved in cell survival and immortalization.     

Challenges in structure prediction of oligomeric proteins at the united-residue level: searching the multiple-chain energy landscape with CSA and CFMC

A revised version of the Conformational Space Annealing (CSA) global optimization method is developed, with three separate measures of structural similarity, in order to overcome the inability of a single distance measure to evaluate multiple-chain protein structures adequately. A second search method, Conformational Family Monte Carlo (CFMC), involving genetic-type moves, Monte Carlo-with-minimization perturbations, and explicit clustering of the population into conformational families, is adapted to treat multiple-chain proteins. These two methods are applied to two oligomeric proteins, the retro-GCN4 leucine zipper and the synthetic domain-swapped dimer. CFMC proves superior to CSA in its search for low-energy representatives of its conformational families, but both methods encounter difficulty in finding the native packing arrangements in the absence of native-like symmetry constraints, even when native monomers are present in the population.     

Organic tissues in rotating bioreactors: fluid-mechanical aspects, dynamic growth models, and morphological evolution

This analysis deals with advances in tissue-engineering models and computational methods as well as with novel results on the relative importance of "controlling forces" in the growth of organic constructs. Specifically, attention is focused on the rotary culture system, because this technique has proven to be the most practical solution for providing a suitable culture environment supporting three-dimensional tissue assemblies. From a numerical point of view, the growing biological specimen gives rise to a moving boundary problem. A "volume-of-fraction" method is specifically and carefully developed according to the complex properties and mechanisms of organic tissue growth and, in particular, taking into account the sensitivity of the construct/liquid interface to the effect of the fluid-dynamic shear stress (it induces changes in tissue metabolism and function that elicit a physiological response from the biological cells). The present study uses available data to introduce a set of growth models. The surface conditions are coupled to the transfer of mass and momentum at the specimen/culture-medium interface and lead to the introduction of a group of differential equations for the nutrient concentration around the sample and for the evolution of tissue mass displacement. The models are then used to show how the proposed surface kinetic laws can predict (through sophisticated numerical simulations) many of the known characteristics of biological tissues grown using rotating-wall perfused vessel bioreactors. This procedure provides a validation of the models and associated numerical method and also gives insight into the mechanisms of the phenomena. The interplay between the increasing size of the tissue and the structure of the convective field is investigated. It is shown that this interaction is essential in determining the time evolution of the tissue shape. The size of the growing specimen plays a critical role with regard to the intensity of convection and the related shear stresses. Convective effects, in turn, are found to impact growth rates, tissue size, and morphology, as well as the mechanisms driving growth. The method exhibits novel capabilities to predict and elucidate experimental observations and to identify cause-and-effect relationships.     

A divergent synthesis of [1-14C]-mono-E isomers of fatty acids

A convenient synthesis of [1-14C]-mono-trans fatty acid using olefin inversion as a key-step is described. This methodology allows for a facile synthesis of [1-14C]-labelled mono-trans analogues of oleic, linoleic and linolenic acids. As an example, only eleven steps were necessary to obtain the [1-14C]-mono-E isomers of linolenic acid from its commercial all-Z form. In the first step, Barton's decarboxylation procedure yielded a bromo intermediate. Epoxidation of this compound resulted in the formation of three monoepoxides, which could be separated by HPLC. After identification by 1H NMR and MS, the pure monoepoxides were then subjected to inversion consisting of a stereospecific deoxygenation followed by a beta-elimination step. Finally, the labelling was introduced by substitution of the bromine by a [14C]-cyano group followed by hydrolysis.