Proteolytic mechanisms of cell injury following glucose -oxygen deprivation in primary neuronal cultures

Researchers have historically emphasized the contribution of caspase-3 to apoptotic but not necrotic cell death, while calpain has been implicated primarily in necrosis and, to a lesser extent, in apoptosis. Activation of these proteases occurs in vivo following various CNS insults including ischemia. In addition, both necrotic and apoptotic cell death phenotypes are detected following ischemia. However, the contributions of calpain and caspase-3 to apoptotic and necrotic cell death phenotypes following CNS insults are relatively unexplored. To date, no study has examined the concurrent activation of calpain and caspase-3 in necrotic and apoptotic cell death phenotypes following any CNS insult. The present study employed oxygen-glucose deprivation (OGD) to determine the relative contributions of caspase-3 and calpain to apoptotic and necrotic cell death following OGD. Experiments characterized a model of OGD by evaluating cell viability and characterizing the cell death phenotypes following OGD in primary septo-hippocampal co-cultures. Furthermore, cell markers (NeuN and MAP2 or GFAP) assessed the effects of OGD on neuronal and astroglial viability, respectively. In addition, calpain and caspase-3 mediated proteolysis of α-spectrin was examined using Western blot techniques. Activation of these proteases in individual cells phenotypically characterized as apoptotic and necrotic was also evaluated by using antibodies specific for calpain or caspase-3 mediated breakdown products to α-spectrin. Administration of appropriate caspase-3 and calpain inhibitors also examined the effects of protease inhibition on cell death. OGD produced prominent expression of apoptotic cell death phenotypes primarily in neurons, with relatively little damage to astroglia. Although Western blot data suggested greater proteolysis of α-spectrin by calpain than caspase-3, co-activation of both proteases was usually detected in cells exhibiting apoptotic or necrotic cell death phenotypes. While inhibition of calpain and caspase-3 activity decreased LDH release following OGD, it was not clear whether this effect was also associated with a decrease in cell death and the appearance of apoptotic cell death phenotypes. These data demonstrate that both calpain and caspase-3 contribute to the expression of apoptotic cell death phenotypes following OGD, and that calpain could potentially have a larger role in the expression of apoptotic cell death than previously thought. ^

YKL -40, a vascular -associated serum marker for glioblastoma, correlates with chronic activation of AKT

YKL-40 is a secreted glycoprotein that has been reported to be expressed in pathologic conditions of extracellular matrix degradation and angiogenesis, such as rheumatoid arthritis, severe osteoarthritis, primary colorectal cancer, metastatic breast cancer, and recurrent ovarian cancer (Dehn, Hogdall et al. 2003). ^ We have identified YKL-40 as a serum marker for glioblastoma multiforme (GBM) using microarray analysis from samples of GBM. We compared the gene expression profile of 19 gliomas to pooled normal brain tissue using the Incyte 10,000 gene expression array. The most differentially expressed gene in this analysis was YKL-40; it was detected in GBM samples with a range of 3 to 62-fold elevation over normal brain. Western blot analysis of glioma samples for YKL-40 protein levels revealed substantial elevation in approximately 65% of GBMs, and undetectable levels in lower-grade gliomas and normal brain tissue. ELISA analysis on serum samples of glioma patients showed that YKL-40 levels were substantially elevated in many of the GBM patients. Statistical analysis indicated that in patients with glioma, serum YKL-40 levels correlate with tumor grade and potentially tumor burden in GBM. ^ Furthermore, we found that YKL-40 expression by in-situ hybridization on a brain tumor tissue array was limited to GBM's and gliosarcomas (GSA), and that YKL-40 expression was specific to the GBM component of GSA. Additional in-situ hybridization analysis, found it to be regionally associated with tumor vasculature as well as activated AKT expression in both human and mouse GBM's. Correlation of elevated YKL-40 with phospho-AKT was confirmed by Western blot analysis on a series of glioblastoma tumors, and inhibition of PI3 Kinase signaling by addition of LY294002 also decreased secretion of YKL-40 over a 7-day period in U87 glioblastoma cell tine. Lastly, YKL-40 expression was induced in response to serum starvation and altered by interaction with specific extracellular matrix (ECM) modules. In summary, we have identified the first accurate serum marker for high-grade gliomas. Furthermore, our findings indicate that YKL-40 is a highly expressed vascular-related glycoprotein in human GBM tissue and that it is affected by the AKT signaling pathway and interaction with components of brain ECM proteins. ^

Enzyme activity and liver tissue PCB concentrations of chicks of northern fulmars (F. glacialis) and black-legged kittiwakes (R. tridactyla) from Kongsfjorden

Arctic seabirds are exposed to a wide range of halogenated organic contaminants (HOCs). Exposure occurs mainly through food intake, and many pollutants accumulate in lipid-rich tissues. Little is known about how HOCs are biotransformed in arctic seabirds. In this study, we characterized biotransformation enzymes in chicks of northern fulmars (Fulmarus glacialis) and black-legged kittiwakes (Rissa tridactyla) from Kongsfjorden (Svalbard, Norway). Phase I and II enzymes were analyzed at the transcriptional, translational and activity levels. For gene expression patterns, quantitative polymerase chain reactions (qPCR), using gene-sequence primers, were performed. Protein levels were analyzed using immunochemical assays of western blot with commercially available antibodies. Liver samples were analyzed for phase I and II enzyme activities using a variety of substrates including ethoxyresorufin (cytochrome (CYP)1A1/1A2), pentoxyresorufin (CYP2B), methoxyresorufin (CYP1A), benzyloxyresorufin (CYP3A), testosterone (CYP3A/CYP2B), 1-chloro-2,4-nitrobenzene (CDNB) (glutathione S-transferase (GST)) and 4-nitrophenol (uridine diphosphate glucuronyltransferase (UDPGT)). In addition, the hydroxylated (OH-) polychlorinated biphenyls (PCBs) were analyzed in the blood, liver and brain tissue, whereas the methylsulfone (MeSO2-) PCBs were analyzed in liver tissue. Results indicated the presence of phase I (CYP1A4/CYP1A5, CYP2B, and CYP3A) and phase II (GST and UDPGT) enzymes at the activity, protein and/or mRNA level in both species. Northern fulmar chicks had higher enzyme activity than black-legged kittiwake chicks. This in combination with the higher XOH-PCB to parent PCB ratios suggests that northern fulmar chicks have a different biotransformation capacity than black-legged kittiwake chicks.

A sea urchin Na+K+2Cl- cotransporter is involved in the maintenance of calcification-relevant cytoplasmic cords in Strongylocentrotus droebachiensis larvae

The cellular mechanisms of calcification in sea urchin larvae are still not well understood. Primary mesenchyme cells within the larval body cavity form a syncytium to secrete CaCO3 spicules from intracellular amorphous CaCO3 (ACC) stores. We studied the role of Na+K+2Cl- cotransporter (NKCC) in intracellular ACC accumulation and larval spicule formation of Strongylocentrotus droebachiensis. First, we incubated growing larvae with three different loop diuretics (azosemide, bumetanide, and furosemide) and established concentration-response curves. All loop diuretics were able to inhibit calcification already at concentrations that specifically inhibit NKCC. Calcification was most effectively inhibited by azosemide (IC50 = 6.5 µM), while larval mortality and swimming ability were not negatively impacted by the treatment. The inhibition by bumetanide (IC50 = 26.4 µM) and furosemide (IC50 = 315.4 µM) resembled the pharmacological fingerprint of the mammalian NKCC1 isoform. We further examined the effect of azosemide on the maintenance of cytoplasmic cords and on the occurrence of calcification vesicles using fluorescent dyes (calcein, FM1-43). Fifty micromolars of azosemide inhibited the maintenance of cytoplasmic cords and resulted in increased calcein fluorescence within calcification vesicles. The expression of NKCC in S. droebachiensis was verified by PCR and Western blot with a specific NKCC antibody. In summary, the pharmacological profile of loop diuretics and their specific effects on calcification in sea urchin larvae suggest that they act by inhibition of NKCC via repression of cytoplasmic cord formation and maintenance.