High resolution immunogold analysis reveals distinct subcellular compartmentation of protein kinase Cγ and δ in rat Purkinje cells

High resolution immunogold cytochemistry was used to investigate the subcellular distribution of protein kinase Cγ and δ in Purkinje cells of the rat cerebellum. Postembedding incubation with an antibody raised to a peptide sequence near the C-terminus of protein kinase Cγ resulted in strong labelling along the dendrosomatic plasma membrane. A quantitative analysis indicated that this labelling reflected the existence of two pools of protein kinase Cγ; one membrane associated pool and one cytoplasmic pool located within 50 nm of the plasma membrane. The labelling along the plasma membrane showed a pronounced and abrupt increase when moving from the cell body into the axon initial segment. Gold particles signalling protein kinase Cγ were also enriched in putative Purkinje axon terminals in the dentate nucleus. The only organelle showing a consistent immunolabelling for protein kinase Cγ was the Golgi apparatus where the gold particles were restricted to the trans face. Protein kinase Cγ immunoreactivity also occurred in the Purkinje cell spines, with an enrichment in or near the postsynaptic density. Antibodies to protein kinase Cδ produced a very different labelling pattern in the Purkinje cells. Most of the gold particles were associated with rough endoplasmic reticulum, particularly with those cisternae that were located close to the nucleus or in the nuclear indentations. No significant protein kinase C5 immunolabelling was detected at the plasma membrane or in Purkinje cell spines. The present data point to a highly specific compartmentation of the two major protein kinase C isozyme in Purkinje cells and suggest that these isozymes act on different substrates and hence have different regulatory functions within these neurons

The Silk-protein Sericin Induces Rapid Melanization of Cultured Primary Human Retinal Pigment Epithelial Cells by Activating the NF-κB Pathway

Restoration of the retinal pigment epithelial (RPE) cells to prevent further loss of vision in patients with age-related macular degeneration represents a promising novel treatment modality. Development of RPE transplants, however, requires up to 3 months of cell differentiation. We explored whether the silk protein sericin can induce maturation of primary human retinal pigment epithelial (hRPE) cells. Microarray analysis demonstrated that sericin up-regulated RPE-associated transcripts (RPE65 and CRALBP). Upstream analysis identified the NF-κB pathway as one of the top sericin-induced regulators. ELISA confirmed that sericin stimulates the main NF-κB pathway. Increased levels of RPE-associated proteins (RPE65 and the pigment melanin) in the sericin-supplemented cultures were confirmed by western blot, spectrophotometry and transmission electron microscopy. Sericin also increased cell density and reduced cell death following serum starvation in culture. Inclusion of NF-κB agonists and antagonists in the culture medium showed that activation of the NF-κB pathway appears to be necessary, but not sufficient, for sericin-induced RPE pigmentation. We conclude that sericin promotes pigmentation of cultured primary hRPE cells by activating the main NF-κB pathway. Sericin’s potential role in culture protocols for rapid differentiation of hRPE cells derived from embryonic or induced pluripotent stem cells should be investigated

Colorimetric Methods for Determining Fe, V, and Ni Contents in Coke and Anodes

Prebaked carbon anodes are used in the electrolytic production of aluminum. They are made of petroleum coke, butts, recycled anodes, and coal tar pitch. The anode quality, which depends on the raw material quality and the production conditions, has an important impact on the cell performance. Metallic impurities (V, Ni, and Fe) found in cokes and anodes increase the carbon consumption by catalyzing the air and CO2 reactivities. In turn, this increases the production cost, energy consumption, and the emission of greenhouse gases. The current methods for detecting the metallic impurities in carbon are time consuming and require intensive sample preparation, skilled personnel, and costly reagents. In this work, simple, rapid, and effective tools were developed using colorimetric methods