High glucose and diabetes increase the release of [3H]-D-aspartate in retinal cell cultures and in rat retinas

Several evidences suggest that glutamate may be involved in retinal neurodegeneration in diabetic retinopathy (DR). For that reason, we investigated whether high glucose or diabetes affect the accumulation and the release of [(3)H]-D-aspartate, which was used as a marker of the glutamate transmitter pool. The accumulation of [(3)H]-D-aspartate did not change in cultured retinal neural cells treated with high glucose (30 mM) for 7 days. However, the release of [(3)H]-D-aspartate, evoked by 50 mM KCl, significantly increased in retinal cells exposed to high glucose. Mannitol, which was used as an osmotic control, did not cause any significant changes in both accumulation and release of [(3)H]-D-aspartate. In the retinas, 1 week after the onset of diabetes, both the accumulation and release of [(3)H]-D-aspartate were unchanged comparing to the retinas of age-matched controls. However, after 4 weeks of diabetes, the accumulation of [(3)H]-D-aspartate in diabetic retinas decreased and the release of [(3)H]-D-aspartate increased, compared to age-matched control retinas. These results suggest that high glucose and diabetes increase the evoked release of D-aspartate in the retina, which may be correlated with the hypothesis of glutamate-induced retinal neurodegeneration in DR.     

High Yield of Methylophilus methylotrophus Cytochrome c″ by Coexpression with Cytochrome c Maturation Gene Cluster from Escherichia coli

Heterologous expression of c-type cytochromes in the periplasm of Escherichia coli often results in low soluble product yield, apoprotein formation, or protein degradation. We have expressed cytochrome c″ from Methylophilus methylotrophus in E. coli by coexpression of the gene encoding the cytochrome (cycA) with the host-specific cytochrome c maturation elements, within the ccmA-H gene cluster. Aerobic cultures produced up to 10 mg holoprotein per liter after induction with IPTG. In the absence of the maturation factors E. coli failed to produce a stable haem protein. Cytochrome c″ isolated from the natural host was compared with the recombinant protein. No structural differences were detected using SDS–PAGE, UV-Visible spectroscopy, differential scanning calorimetry, and ¹H-NMR spectroscopy. The success in expressing the mature cytochrome c″ in E. coli allows the engineering of the cycA gene by site-directed mutagenesis thereby providing an ideal method for producing mutant protein for studying the structure/function relationship.     

Enhanced DNA transfer into fish bone cells using polyethylenimine

The use of in vitro cell culture systems to assess gene function largely depends on the successful transfer of DNA into target cells. Well developed in mammals, transfection methods are still to be optimized for non-mammalian cell culture systems, like fish. Here we describe a rapid, cost-efficient, and successful method to transfer DNA into a fish bone-derived cell line using polyethylenimine (PEI) as the DNA carrier. Using this method, DNA transfer was remarkably enhanced in comparison with commercially available reagents, as demonstrated by the increased activity of both luciferase and green fluorescent protein observed in the transfected cells. Its efficiency in transferring DNA intoa wide range of cell types, including non-mammalian and hard-to-transfect cells, in addition to a low cost, show that PEI is a reagent of choice for nonviral vector transfection.     

Kinetic and metabolic analysis of mouse embryonic stem cell expansion under serum-free conditions

There is a need for a deeper understanding of the biochemical events affecting embryonic stem (ES) cell culture by analyzing the expansion of mouse ES cells in terms of both cell growth and metabolic kinetics. The influence of the initial cell density on cell expansion was assessed. Concomitantly, the biochemical profile of the culture was evaluated, which allowed measuring the consumption of important substrates, such as glucose and glutamine, and the production of metabolic byproducts, like lactate. The results suggest a more efficient cell metabolism in serum-free conditions and a preferential use of glutaminolysis as an energy source during cell expansion at low seeding densities. This work contributes to the development of fully-controlled bioprocesses to produce relevant numbers of ES cells for cell therapies and high-throughput drug screening.     

Local dendritic activity sets release probability at hippocampal synapses

The arrival of an action potential at a synapse triggers neurotransmitter release with a limited probability, p(r). Although p(r) is a fundamental parameter in defining synaptic efficacy, it is not uniform across all synapses, and the mechanisms by which a given synapse sets its basal release probability are unknown. By measuring p(r) at single presynaptic terminals in connected pairs of hippocampal neurons, we show that neighboring synapses on the same dendritic branch have very similar release probabilities, and p(r) is negatively correlated with the number of synapses on the branch. Increasing dendritic depolarization elicits a homeostatic decrease in p(r), and equalizing activity in the dendrite significantly reduces its variability. Our results indicate that local dendritic activity is the major determinant of basal release probability, and we suggest that this feedback regulation might be required to maintain synapses in their operational range.     

Review: When is an antioxidant not an antioxidant? A review of novel actions and reactions of vitamin C

Vitamin C (or ascorbic acid) is regarded as the most important water-soluble antioxidant in human plasma and mammalian cells which have mechanisms to recycle and accumulate it against a concentration gradient, suggesting that the vitamin might also have important intracellular functions. In this review we summarize evidence from human trials that have attempted an association between vitamin C supplementation and an effect on biomarkers of oxidative DNA damage. Most studies reviewed herein showed either a vitamin C-mediated reduction in oxidative DNA damage or a null effect, whereas only a few studies showed an increase in specific base lesions. We also address the possible beneficial effects of vitamin C supplementation for the prevention of cancer and cardiovascular disease. Finally, we discuss the contribution of cell culture studies to our understanding of the mode of action of vitamin C and we review recent evidence that vitamin C is able to modulate gene expression and cellular function, with a particular interest in cell differentiation.