Oxidative stress can trigger neuronal cell death and has been implicated in several chronic neurological diseases and in acute neurological injury. Oxidative toxicity can be induced by glutamate treatment in cells that lack ionotrophic glutamate receptors, such as the immortalized HT22 hippocampal cell line and immature primary cortical neurons. Previously, we found that neuroprotective effects of geldanamycin, a benzoquinone ansamycin, in HT22 cells were associated with a down-regulation of c-Raf-1, an upstream activator of the extracellular signal-regulated protein kinases (ERKs). ERK activation, although often attributed strictly to neuronal cell survival and proliferation, can also be associated with neuronal cell death that occurs in response to specific insults. In this report we show that delayed and persistent activation of ERKs is associated with glutamate-induced oxidative toxicity in HT22 cells and immature primary cortical neuron cultures. Furthermore, we find that U0126, a specific inhibitor of the ERK-activating kinase, MEK-1/2, protects both HT22 cells and immature primary cortical neuron cultures from glutamate toxicity. Glutamate-induced ERK activation requires the production of specific arachidonic acid metabolites and appears to be downstream of a burst of reactive oxygen species (ROS) accumulation characteristic of oxidative stress in HT22 cells. However, inhibition of ERK activation reduces glutamateinduced intracellular Ca 2؉ accumulation. We hypothesize that the precise kinetics and duration of ERK activation may determine whether downstream targets are mobilized to enhance neuronal cell survival or ensure cellular demise.

P ainful sensations induced by capsaicin, the pungent substance in hot peppers, are caused by stimulation of vanilloid receptor 1 (VR1), an ion channel protein expressed by nociceptive primary afferent neurons. VR1 also participates in the detection of at least two additional noxious stimuli, acid (pH Ͻ 6) and heat (Ͼ43°C). The urinary bladder is rich with capsaicinsensitive afferent fibers that detect bladder distension or the presence of irritant chemicals and in turn trigger reflex bladder activity. Here, we demonstrate that VR1 is expressed not only by afferent nerves that form close contacts with bladder epithelial (urothelial) cells but also by the urothelial cells themselves. We further show that exogenously applied vanilloids increase intracellular Ca 2ϩ and evoke NO release in urothelial cells and that these responses require VR1. These and other data suggest that urothelial cells work in concert with underlying afferent nerves to detect the presence of irritant stimuli.

Poly(ADP-ribosylation), primarily via poly(ADP-ribose) polymerase-1 (PARP-1), is a pluripotent cellular process important for maintenance of genomic integrity and RNA transcription in cells. However, during conditions of oxidative stress and energy depletion, poly-(ADP-ribosylation) paradoxically contributes to mitochondrial failure and cell death. Although it has been presumed that poly(ADP-ribosylation) within the nucleus mediates this pathologic process, PARP-1 and other poly(ADP-ribosyltransferases) are also localized within mitochondria. To this end, the presence of PARP-1 and poly(ADP-ribosylation) were verified within mitochondrial fractions from primary cortical neurons and fibroblasts. Inhibition of poly(ADP-ribosylation) within the mitochondrial compartment preserved transmembrane potential (⌬⌿ m ), NAD ؉ content, and cellular respiration, prevented release of apoptosis-inducing factor, and reduced neuronal cell death triggered by oxidative stress. Treatment with liposomal NAD؉ also preserved ⌬⌿ m and cellular respiration during oxidative stress. Furthermore, inhibition of poly(ADP-ribosylation) prevented intranuclear localization of apoptosis-inducing factor and protected neurons from excitotoxic injury; and PARP-1 null fibroblasts were protected from oxidative stress-induced cell death. Collectively these data suggest that poly(ADP-ribosylation) compartmentalized to the mitochondria can be converted from a homeostatic process to a mechanism of cell death when oxidative stress is accompanied by energy depletion. These data implicate intra-mitochondrial poly(ADP-ribosylation) as an important therapeutic target for central nervous system and other diseases associated with oxidative stress and energy failure.Poly(ADP-ribose) polymerase-1 (PARP-1 1 ; EC 2.4.2.30), the most abundant poly(ADP-ribosyltransferase) in mammalian cells, plays an essential role in excitotoxic neuronal death both in vitro and in vivo (1-4). The presumptive mechanism for this neurotoxic effect involves, sequentially, increases in [Ca 2ϩ ] i via glutamate receptors, activation of nitric-oxide synthase, generation of the free radical peroxynitrite (ONOO Ϫ ), activation of PARP-1 in response to genomic DNA damage, consumption of NAD ϩ during the formation of poly(ADP-ribose) polymers, and death via energy failure (5). However, the capacity for PARP-1 activation within the nucleus to deplete total cellular energy stores, particularly compartmentalized within mitochondria, remains to be established (4, 6). Because in addition to being abundant in cell nuclei, PARP-1 and other ADP-ribosyltransferases are also prevalent in mitochondria (7-9), where similar to nuclear PARP-1, they facilitate DNA repair in response to oxidative damage (10, 11), we hypothesized that inhibition of mitochondrial poly(ADP-ribosylation) may play a pivotal role in neuronal cell survival under conditions of oxidative stress and excitotoxicity.Here we show that inhibition of mitochondrial poly(ADPribosylation) preserves mitochondrial transmembrane potential (⌬⌿ m...

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Experimental asphaltene precipitation data on several liveoil/solvent mixtures at reservoir conditions were measured to study the effects of temperature, pressure, and composition on precipitate formation and the relationships between critical properties, PVT phase behavior, and precipitate formation. Data generated by the model can be used to identify operating conditions conducive to precipitate formation.

Chromosomal instability is a key step in the generation of the cancer cell karyotype. An indicator of unstable chromosomes is the presence of chromatin bridges during anaphase. We examined in detail the fate of anaphase bridges in cultured oral squamous cell carcinoma cells in real-time. Surprisingly, chromosomes in bridges typically resolve by breaking into multiple fragments. Often these fragments give rise to micronuclei (MN) at the end of mitosis. The formation of MN is shown to have important consequences for the cell. We found that MN have incomplete nuclear pore complex (NPC) formation and nuclear import defects and the chromatin within has greatly reduced transcriptional activity. Thus, a major consequence of the presence of anaphase bridges is the regular sequestration of chromatin into genetically inert MN. This represents another source of ongoing genetic instability in cancer cells.

Transforming growth factor (TGF)-β inhibits T cell proliferation and differentiation. TGF-β has been shown to inhibit the expression of transcription factors such as GATA-3 and T-bet that play important roles in T cell differentiation. Here we show that TGF-β inhibits T cell differentiation at a more proximal step. An early event during T cell activation is increased intracellular calcium levels. Calcium influx in activated T cells and the subsequent activation of transcription factors such as NFATc, events essential for T cell differentiation, are modulated by the Tec kinases that are downstream of the T cell receptor and CD28. We show that in stimulated CD4+ T cells, TGF-β inhibits phosphorylation and activation of the Tec kinase Itk, increase in intracellular Ca2+ levels, NFATc translocation, and activation of the mitogen-activated protein kinase ERK that together regulate T cell differentiation. Our studies suggest that by inhibiting Itk, and consequently Ca2+ influx, TGF-β limits T cell differentiation along both the Th1 and Th2 lineages.

Calpain activity is required for de-adhesion of the cell body and rear to enable productive locomotion of adherent cells during wound repair and tumor invasion. Growth factors activate m-calpain (calpain 2, CAPN2) via ERK/mitogen-activated protein kinases, but only when these kinases are localized to the plasma membrane. We thus hypothesized that m-calpain is activated by epidermal growth factor (EGF) only when it is juxtaposed to the plasma membrane secondary to specific docking. Osmotic disruption of NR6 fibroblasts expressing the EGF receptor demonstrated m-calpain being complexed with the substratum-adherent membrane with this increasing in an EGF-dependent manner. m-Calpain colocalized with phosphoinositide biphosphate (PIP 2 ) with exogenous phospholipase C removal of phosphoinositides, specifically, PI(4,5)P 2 but not PI(4)P 1 or PIP 3 , releasing the bound m-calpain. Downregulation of phosphoinositide production by 1-butanol resulted in diminished PIP 2 in the plasma membrane and eliminated EGF-induced calpain activation. This PIP 2 -binding capacity resided in domain III of calpain, which presents a putative C2-like domain. This active conformation of this domain appears to be partially masked in the holoenzyme as both activation of m-calpain by phosphorylation at serine 50 and expression of constitutively active phosphorylation mimic glutamic acid-increased m-calpain binding to the membrane, consistent with blockade of this cascade diminishing membrane association. Importantly, we found that m-calpain was enriched toward the rear of locomoting cells, which was more pronounced in the plasma membrane footprints; EGF further enhanced this enrichment, in line with earlier reports of loss of PIP 2 in lamellipodia of motile cells. These data support a model of m-calpain binding to PIP 2 concurrent with and likely to enable ERK activation and provides a mechanism by which cell de-adhesion is directed to the cell body and tail as phospholipase C-␥ hydrolyzes PIP 2 in the protruding lamellipodia.Cell motility is a complex process involving a sequence of events consisting of extension of a lamellipodium, formation of new adhesions at the leading edge, contraction of the cell body, and detachment of the rear of the cell (35, 47). These separate events must work in a coordinated effort to provide persistent cell movement in one direction. Rear detachment has been shown to be a rate-limiting step during both haptokinetic (26, 45) and growth factor-induced chemokinetic (22, 32, 56) motility. This subcellular asymmetry of processes occurs even in the absence of an externally imposed gradient of cues (35, 62), suggesting an intracellular segregation of biochemical controls. While progress has been made in deciphering the signaling gradients during ameboid movement in yeast (28, 37), the situation in mammalian fibroblasts and epithelial cells is less clear (47).Extrinsic signals, including growth factors and the extracellular matrix, initiate intracellular signal cascades leading to biophysical changes in the cell (60,...