Assessment of the current performance of grazing infrastructure across Irish dairy farms.

The increased average Irish dairy herd size in a post-quota environment has put heightened pressure on grazing infrastructure. In a rotational grazing system, grazing infrastructure consists of the paddock system, which delineates the grazing areas into appropriately sized grazing parcels, and the roadway network, which connects these paddocks to the milking parlor. Where herd size has increased without corresponding adaptations to the infrastructure, farm management and roadway network performance has been affected. The links between suboptimal grazing infrastructure and roadway network efficiency are poorly understood and not widely documented. The aims of this study were to (1) analyze the effect of herd expansion and paddock size on pasture allocations per paddock, (2) identify the factors that affect the total distance walked per year, and (3) create a metric to compare the efficiency of roadway networks across farms of varying grazing platforms. A sample population of 135 Irish dairy farms with a median herd size of 150 cows was used for this analysis. Herds were split into the following 5 categories: <100 cows, 100 to 149 cows, 150 to 199 cows, 200 to 249 cows, and ≥250 cows. Herds with ≥250 cows had a greater number of paddocks per farm and rotated around the grazing paddocks more frequently, with 46% of paddocks only suitable for 12 h allocations relative to herd size, compared with just 10% to 27% of paddocks for herds with <100 cows to herds with 200-249 cows. When predicting the total distance walked per year on each study farm, the mean distance from a paddock to the milking parlor was the strongest indicator (R2 = 0.8247). Other metrics, such as herd size, have failed to account for the location of the milking parlor relative to the grazing platform. The creation of the relative mean distance from a paddock to milking parlor (RMDMP) metric allowed the calculation of a farm's roadway network efficiency for moving the herd between paddocks and the milking parlor. The analyzed farms increased their efficiency in terms of RMDMP (0.34-40.74%) as they increased herd size post quota. However, the position of new additional paddocks relative to the milking parlor substantially affected their RMDMP.

Hofstadter's butterfly and the fractal quantum Hall effect in moiré superlattices.

Electrons moving through a spatially periodic lattice potential develop a quantized energy spectrum consisting of discrete Bloch bands. In two dimensions, electrons moving through a magnetic field also develop a quantized energy spectrum, consisting of highly degenerate Landau energy levels. When subject to both a magnetic field and a periodic electrostatic potential, two-dimensional systems of electrons exhibit a self-similar recursive energy spectrum. Known as Hofstadter's butterfly, this complex spectrum results from an interplay between the characteristic lengths associated with the two quantizing fields, and is one of the first quantum fractals discovered in physics. In the decades since its prediction, experimental attempts to study this effect have been limited by difficulties in reconciling the two length scales. Typical atomic lattices (with periodicities of less than one nanometre) require unfeasibly large magnetic fields to reach the commensurability condition, and in artificially engineered structures (with periodicities greater than about 100 nanometres) the corresponding fields are too small to overcome disorder completely. Here we demonstrate that moiré superlattices arising in bilayer graphene coupled to hexagonal boron nitride provide a periodic modulation with ideal length scales of the order of ten nanometres, enabling unprecedented experimental access to the fractal spectrum. We confirm that quantum Hall features associated with the fractal gaps are described by two integer topological quantum numbers, and report evidence of their recursive structure. Observation of a Hofstadter spectrum in bilayer graphene means that it is possible to investigate emergent behaviour within a fractal energy landscape in a system with tunable internal degrees of freedom.

Dietary glycemic index modulates the behavioral and biochemical abnormalities associated with autism spectrum disorder.

Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder of unknown etiology, but very likely resulting from both genetic and environmental factors. There is good evidence for immune system dysregulation in individuals with ASD. However, the contribution of insults such as dietary factors that can also activate the immune system have not been explored in the context of ASD. In this paper, we show that the dietary glycemic index has a significant impact on the ASD phenotype. By using BTBR mice, an inbred strain that displays behavioral traits that reflect the diagnostic symptoms of human ASD, we found that the diet modulates plasma metabolites, neuroinflammation and brain markers of neurogenesis in a manner that is highly reflective of ASD in humans. Overall, the manuscript supports the idea that ASD results from gene-environment interactions and that in the presence of a genetic predisposition to ASD, diet can make a large difference in the expression of the condition.

Global quantitative analysis of phosphorylation underlying phencyclidine signaling and sensorimotor gating in the prefrontal cortex.

Prepulse inhibition (PPI) is an example of sensorimotor gating and deficits in PPI have been demonstrated in schizophrenia patients. Phencyclidine (PCP) suppression of PPI in animals has been studied to elucidate the pathological elements of schizophrenia. However, the molecular mechanisms underlying PCP treatment or PPI in the brain are still poorly understood. In this study, quantitative phosphoproteomic analysis was performed on the prefrontal cortex from rats that were subjected to PPI after being systemically injected with PCP or saline. PCP downregulated phosphorylation events were significantly enriched in proteins associated with long-term potentiation (LTP). Importantly, this data set identifies functionally novel phosphorylation sites on known LTP-associated signaling molecules. In addition, mutagenesis of a significantly altered phosphorylation site on xCT (SLC7A11), the light chain of system xc-, the cystine/glutamate antiporter, suggests that PCP also regulates the activity of this protein. Finally, new insights were also derived on PPI signaling independent of PCP treatment. This is the first quantitative phosphorylation proteomic analysis providing new molecular insights into sensorimotor gating.

THE EFFECTS OF CONE-BEAM COMPUTED TOMOGRAPHY IMAGING GUIDANCE ON PATIENT RADIATION EXPOSURES IN TRANS-ARTERIAL CHEMOEMBOLISATION FOR HEPATOCELLULAR CARCINOMA.

This study investigated the effects of cone-beam computed tomography (CBCT) guidance in trans-arterial chemoembolisation (TACE) procedures on the number of digital subtraction angiography (DSA) runs acquired and total patient radiation exposure in patients with hepatocellular carcinoma (HCC). A retrospective, analytical cross-sectional, single institution, study was conducted. Dose data were compared across the control (DSA guidance alone) and study (DSA and CBCT guidance) groups. A total of 122 procedures were included within the study. There was a significant reduction in the number of DSA runs (3 vs 5, p < 0.001) and DSA air kerma-area product (PKA) (3077.3 vs 4276.6 µGym2, p = 0.042) for the study group when compared to the control group. Total procedural PKA and total procedural reference air kerma (Ka,r) were shown to be 50 and 73% higher, respectively, for the study group when compared to the control group. CBCT imaging guidance does reduce the number of DSA runs and DSA PKA required to complete the TACE procedure for patients diagnosed with HCC; however, a substantial increase in total procedural PKA is to be expected and it is thus important that this increased dose is carefully considered and justified.

Tissue-dependent regulation of protein tyrosine kinase activity during embryonic development.

Protein tyrosine kinase activity was assayed in a variety of chicken tissues during embryonic development and in the adult. In some tissues protein tyrosine kinase activity decreased during embryonic development; however, in other tissues it remained high throughout development, it contrast to the level of protein tyrosine phosphorylation, which decreased during development. The highest levels of tyrosine kinase activity were detected in 17-d embryonic brain although only low levels of protein tyrosine phosphorylation were observed in this tissue. Several alternatives were examined in an effort to determine the mechanism responsible for the low levels of tyrosine phosphorylated proteins in most older embryonic and adult chicken tissues despite the presence of highly active tyrosine kinases. The results show that the regulation of protein tyrosine phosphorylation during embryonic development is complex and varies from tissue to tissue. Furthermore, the results suggest that protein tyrosine phosphatases play an important role in regulating the level of phosphotyrosine in proteins of many older embryonic and adult tissues.

Nuclear Translocation of fibroblast growth factor (FGF) receptors in response to FGF-2.

Members of the FGF family of growth factors localize to the nuclei in a variety of different cell types. To determine whether FGF receptors are also present within nuclei and if this localization is regulated by FGFs, nuclei were prepared from quiescent and FGF-2-treated Swiss 3T3 fibroblasts and examined for the presence of FGF receptors by immunoblotting with an antibody produced against the extracellular domain of FGF receptor-1 (FGFR-1). Little or no FGFR-1 is detected in nuclei prepared from quiescent cells. When cells are treated with FGF-2, however, there is a time- and dose-dependent increase in the association of FGFR-1 immunoreactivity with the nucleus. In contrast, treatment with either EGF or 10% serum does not increase the association of FGFR-1 with the nucleus. When cell surface proteins are labeled with biotin, a biotinylated FGFR-1 is detected in the nuclear fraction prepared from FGF-2-treated, but not untreated, cells indicating that the nuclear-associated FGFR-1 immunoreactivity derives from the cell surface. The presence of FGFR-1 in the nuclei of FGF-2-treated cells was confirmed by immunostaining with a panel of different FGFR-1 antibodies, including one directed against the COOH-terminal domain of the protein. Fractionation of nuclei from FGF-2-treated cells indicates that nuclear FGFR-1 is localized to the nuclear matrix, suggesting that the receptor may play a role in regulating gene activity.

Tyrosine phosphorylated proteins in different tissues during chick embryo development.

A high affinity polyclonal antibody specific for phosphotyrosyl residues has been used in immunoblotting experiments to survey developing embryonic chicken tissues for the presence and characteristics of tyrosine phosphorylated proteins. Proteins phosphorylated on tyrosine were found to be present in all the embryonic tissues examined, including heart, thigh, gizzard, intestine, lung, liver, kidney, brain, and lens, from 7 to 21 d of development in ovo, but were greatly reduced or absent in the same tissues taken from adult chickens. A limited number of major tyrosine phosphorylated proteins were seen in all the tissues examined and they ranged in molecular mass from 35 to 220 kD. Most of the tissues contained proteins phosphorylated on tyrosine with apparent molecular masses of 120, 70, 60, and 35 kD, suggesting that the substrates of tyrosine protein kinases in different tissues may be related proteins. One-dimensional peptide mapping of the 120- and 70-kD protein bands indicated a close structural relationship among the phosphotyrosine-containing proteins of 120 kD, and similarly among those of 70 kD, from the different tissues.

Regulation of antioxidant metabolism by translation initiation factor 2alpha.

Oxidative stress and highly specific decreases in glutathione (GSH) are associated with nerve cell death in Parkinson's disease. Using an experimental nerve cell model for oxidative stress and an expression cloning strategy, a gene involved in oxidative stress-induced programmed cell death was identified which both mediates the cell death program and regulates GSH levels. Two stress-resistant clones were isolated which contain antisense gene fragments of the translation initiation factor (eIF)2alpha and express a low amount of eIF2alpha. Sensitivity is restored when the clones are transfected with full-length eIF2alpha; transfection of wild-type cells with the truncated eIF2alpha gene confers resistance. The phosphorylation of eIF2alpha also results in resistance to oxidative stress. In wild-type cells, oxidative stress results in rapid GSH depletion, a large increase in peroxide levels, and an influx of Ca(2+). In contrast, the resistant clones maintain high GSH levels and show no elevation in peroxides or Ca(2+) when stressed, and the GSH synthetic enzyme gamma-glutamyl cysteine synthetase (gammaGCS) is elevated. The change in gammaGCS is regulated by a translational mechanism. Therefore, eIF2alpha is a critical regulatory factor in the response of nerve cells to oxidative stress and in the control of the major intracellular antioxidant, GSH, and may play a central role in the many neurodegenerative diseases associated with oxidative stress.

Importin beta-mediated nuclear import of fibroblast growth factor receptor: role in cell proliferation.

Although growth factor receptors are generally thought to carry out their role in signal transduction at the cell surface, many of these transmembrane proteins translocate to the nucleus after ligand stimulation. Here, we show that the nuclear translocation of fibroblast growth factor receptor (FGFR)1 occurs via a mechanism distinct from classical nuclear import but dependent on importin beta, a component of multiple nuclear import pathways. Furthermore, we show that nuclear FGFR1 induces c-Jun and is involved in the regulation of cell proliferation. These data are the first description of a nuclear import pathway for transmembrane growth factor receptors and elucidate a novel signal transduction pathway from the cell surface to the nucleus.

Immunocytochemical studies of cardiac myofibrillogenesis in early chick embryos. I. Presence of immunofluorescent titin spots in premyofibril stages.

Our initial attempts to immunolabel intact myocardial walls of 4-12 somite stage chick embryos were hindered by the presence of the cardiac jelly that covers the inner myocardial wall surface and prevents the access of antibodies to that surface. We overcame this difficulty by treating the specimens with hyaluronidase, which made the cardiac jelly permeable to the antibodies. An additional nonionic detergent treatment made the two or more cell layers of the myocardial wall accessible to the antibodies from both surfaces of the wall. Specimens treated in this manner were fluorescently labeled with antibodies to titin, myosin, or actin or with NBD-phallacidin for F-actin and examined as whole mount preparations or cut into semithin sections after resin embedding. These preparations and sections revealed that titin, a putative scaffolding protein of sarcomeres, is present in a punctate state and also in a diffuse form throughout the cytoplasm of cardiac myocytes in the premyofibril stages (4-7 somite stages) as well as in the early stages of myofibril formation. We interpreted the punctate and diffuse states to represent an aggregated state of several titin molecules and a dispersed state of individual titin molecules, respectively. In the 4-7 somite cardiac primodia, myosin and actin show only a uniform labeling throughout the cytoplasm of the myocytes. These observations are in contrast to a previous report that titin and myosin are tightly linked during in vitro skeletal myofibrillogenesis (Hill, C. S., S. Duran, Z. Ling, K. Weber, and H. Holtzer, 1986, J. Cell Biol., 103:2185-2196). In the 8-11 somite stage hearts, the number of individual titin spots rapidly reduces, while the number of myofibrils with periodically aligned titin spots increases, which strongly suggests that the titin spots are incorporated into the newly arising myofibrils. Titin spots were seen as doublets only after titin spots were incorporated into the first myofibrils. However, the fact that the distance between the components of the narrowest doublet was close to the resolution limit of the light microscope left open the possibility that undiscernible doublets of submicroscopic separations might exist in the premyofibril stages. The myosin labeling revealed the sarcomeric periodicity in an earlier stage of myofibril development than the F-actin labeling. In addition, we made two morphogenic observations.(ABSTRACT TRUNCATED AT 400 WORDS)

Immunocytochemical studies of cardiac myofibrillogenesis in early chick embryos. II. Generation of alpha-actinin dots within titin spots at the time of the first myofibril formation.

In whole mount preparations of the 9 somite stage chick embryonic hearts that were immunofluorescently double labeled for titin and alpha-actinin, presumptive myofibrils were recognized as rows of several periodically aligned titin spots. Within these titin spots, smaller alpha-actinin dots were observed. These periodical arrangements of titin spots and alpha-actinin dots were not found in the 7 somite stage hearts. In wide myofibrils in the 10 somite stage hearts, the alpha-actinin dots and titin spots simultaneously became 'lines.' To study the ultrastructural features of the titin-positive regions in the 6-9 somite stage hearts, the thoracic portions of the embryos were immunofluorescently labeled for titin and embedded in resin. Ultrathin sections were mounted on electron microscopic grids and examined in immunofluorescence optics. The titin-positive regions thus identified were then examined in the electron microscope. No readily discernable specific ultrastructural features were found in titin-positive regions of the 6 somite stage cardiac primodia. Examination of the sections of the 9 somite stage hearts, on the other hand, revealed the occasional presence of small dense bodies, Z bodies, in the titin-positive regions. These observations strongly suggest that these Z bodies are the ultrastructural counterparts of the alpha-actinin dots seen by immunofluorescence optics and that they are formed nearly at the time of the formation of the first myofibrils. In some of the nascent myofibrils the Z bodies were found to be considerably narrower than the myofibrils, implying that the Z bodies are required not for the assembly of myofibrils per se but for their stabilization. Immunofluorescent labeling for titin and alpha-actinin revealed that the length of the shortest sarcomeres in the first myofibrils is approximately 1.5 micron, approximately the width of the A bands of mature myofibrils. The possibility that the A bands might define the initial length of nascent sarcomeres was indicated.

Heat shock induces protein tyrosine phosphorylation in cultured cells.

We examined the effect of heat shock on protein tyrosine phosphorylation in cultured animal cells using antiphosphotyrosine antibodies in immunoblotting and immunofluorescence microscopy experiments. Heat shock significantly elevated the level of phosphotyrosine in proteins in most of the cultured cells examined, including fibroblasts, epithelial cells, nerve cells, and muscle cells, but not in Rous sarcoma virus-transformed fibroblasts. The increase in protein tyrosine phosphorylation induced by heat shock occurred in proteins with a wide range of molecular masses and was dependent on the temperature and duration of the heat shock.

Requirement for cGMP in nerve cell death caused by glutathione depletion.

Glutathione depletion occurs in several forms of apoptosis and is associated with Parkinson's disease and HIV toxicity. The neurotransmitter glutamate kills immature cortical neurons and a hippocampal nerve cell line via an oxidative pathway associated with glutathione depletion. It is shown here that soluble guanylyl cyclase (sGC) activity is required for nerve cell death caused by glutathione depletion. Inhibitors of sGC block glutamate toxicity and a cGMP analogue potentiates cell death. Glutamate also induces an elevation of cGMP that occurs late in the cell death pathway. The resultant cGMP modulates the increase in intracellular calcium that precedes cell death because sGC inhibitors prevent calcium elevation and the cGMP analogue potentiates the increase in intracellular calcium. These results suggest that the final pathway of glutamate induced nerve cell death is through a cGMP-modulated calcium channel.

High affinity immunoreactive FGF receptors in the extracellular matrix of vascular endothelial cells--implications for the modulation of FGF-2.

We recently characterized three FGF-binding proteins (FGF-BPs) which are soluble forms of the extracellular domains of the high affinity FGF receptors (Hanneken, A. M., W. Ying, N. Ling, and A. Baird. Proc. Natl. Acad. Sci. USA. 1994. 91:9170-9174). These proteins circulate in blood and have been proposed to modulate the biological activity of the FGF family of proteins. Immunohistochemical studies now demonstrate that these soluble, truncated FGF receptors are also present in the basement membranes of retinal vascular endothelial cells. These immunoreactive proteins can be detected with antibodies raised to the extracellular domain of FGFR-1 but not with antibodies raised to either the juxtamembrane domain or the cytoplasmic domain of FGFR-1. Western blotting of human retinal extracts with the antibody raised to the extracellular domain of FGFR-1 detects specific, low molecular mass proteins at 85 kD and 55 kD, corresponding in size to the FGF-BPs, which are not detected with antibodies against the cytoplasmic domain of the receptor. The interaction of this receptor with the extracellular matrix is not dependent on the presence of FGF-2. Immunoreactive receptors are still detected in vascular basement membranes after the removal of FGF-2 with heparitinase. In addition, the recombinant extracellular domain of FGFR-1 continues to bind to corneal endothelial cell matrix after endogenous FGF-2 has been removed with 2 M NaCl. Acid treatment, which has been shown to disrupt protein interactions with the extracellular matrix, leads to a significant reduction in the presence of the matrix form of the FGF receptor. This loss can be restored with exogenous incubations of the recombinant extracellular domain of FGFR-1. This report is the first demonstration that a truncated form of a high affinity growth factor receptor can be localized to the extracellular matrix. These findings add to the list of binding proteins associated with the extracellular matrix (IGFBP-5) and suggest a potentially new regulatory mechanism for controlling the biological availability of FGF, and other peptide growth factors, in the extracellular matrix.

Distributions of vimentin and desmin in developing chick myotubes in vivo. II. Immunoelectron microscopic study.

The distribution of the intermediate filament proteins vimentin and desmin in developing and mature myotubes in vivo was studied by single and double immunoelectron microscopic labeling of ultrathin frozen sections of iliotibialis muscle in 7-21-d-old chick embryos, and neonatal and 1-d-old postnatal chicks. This work is an extension of our previous immunofluorescence studies of the same system (Tokuyasu, K. T., P. A. Maher and S. J. Singer, 1984, J. Cell Biol., 98:1961-1972). In immature myotubes of 7-11-d embryos, significant labeling for desmin and vimentin was found only in intermediate filaments, and these proteins coexisted in the same individual filaments. Each of the two proteins was present in irregular clusters along the entire length of a filament. No exclusively vimentin- or desmin-containing filaments were observed at this stage. In the early myotubes, the intermediate filaments were essentially all longitudinally oriented, even when they contained three times as much desmin as vimentin. No special relationship was recognized between the dispositions of the filaments and the organization of the myofibrils. Occasionally, several myofibrils were already aligned in lateral registry at this early stage, but labeling for desmin and vimentin was largely absent at the level of the Z bands. Instead, the Z bands appeared to be covered by elements of the sarcoplasmic reticulum. The confinement of intermediate filaments to the level of the Z bands occurred in the myotubes of later embryos after the extensive lateral registry of the Z bands. Thus, intermediate filaments are unlikely to play a primary role in producing the lateral registration of myofibrils during myogenesis, but may be important in determining the polarization of the early myotube and the alignment of its organelles. Throughout the development of myotubes, desmin and vimentin remained in the form of intermediate filaments, although the number of filaments per unit volume of myotube appeared to be reduced as myofibrils increased in number in maturing myotubes. This observation indicated that the transverse orientation of intermediate filaments in mature myotubes does not result from the de novo polymerization of subunits from Z band to Z band, but a continuous shifting of the positions and directions of intact filaments.

Zeugmatin: a new high molecular weight protein associated with Z lines in adult and early embryonic striated muscle.

Monoclonal antibodies were generated to a purified preparation of the fascia adherens domains of the intercalated discs of chicken cardiac cell membranes. One of these antibodies, McAb 20, immunofluorescently labeled the Z lines of adult skeletal muscle, the Z lines and intercalated discs of adult cardiac muscle, and the dense bodies and dense plaques of adult gizzard smooth muscle. In addition, McAb 20 was found to label regenerating muscle cells in a cross-striated pattern much like that of Z lines in 24-h muscle cell cultures before the appearance of Z lines was detectable by phase or Nomarski optics and before the concentration of alpha-actinin occurred at the Z lines. Thus, McAb 20 appears to be directed against an antigen involved in early myofibrillar organization. Preliminary biochemical characterization of the antigen recognized by McAb 20 indicates that it is a high molecular weight doublet of over 5 X 10(5) kD that is highly susceptible to proteolysis. By virtue of its presence in Z lines, and its possible role in the end-on attachment of microfilaments to Z lines and membranes, we have named this protein zeugmatin (xi epsilon nu gamma mu alpha identical to yoking).

Distributions of vimentin and desmin in developing chick myotubes in vivo. I. Immunofluorescence study.

Antibodies against chicken erythrocyte vimentin and gizzard desmin were affinity purified and then cross-absorbed with the heterologous antigen. They were used to study the in vivo distributions of these proteins in developing and mature myotubes by immunofluorescence microscopy of 0.5-2-micron frozen sections of iliotibialis muscle in 7-21-day chick embryos, neonatal and 1-d postnatal chicks, and adult chickens. The distributions of vimentin and desmin were coincidental throughout the development of myotubes, but the concentration of vimentin was gradually reduced as the myotubes matured and became largely undetectable at the time of hatching. The process of confining these proteins to the level of Z line from the initial uniform distribution occurred subsequent to the process of bringing myofibrils into lateral registry: in-register lateral association of several myofibrils was occasionally seen as early as in 7-11-d embryos, whereas the cross-striated immunofluorescence pattern of desmin and vimentin was only vaguely discerned in myotubes of 17-d embryos, just 4 d before hatching. In some myotubes of 21-d embryos, myofibrils were in lateral registry as precisely as in adult myofibers but desmin was still widely distributed around Z line in an irregular manner. Nevertheless, in many other myotubes of prenatal or neonatal chicks, desmin became confined to the level of Z line in a manner similar to that seen in adult myofibers, thus essentially completing its redistribution to the confined state of adult myofibers in coincidence with the time of hatching. In extracts from iliotibialis and posterior latissimus dorsi muscles of adult chickens, we detected a hitherto unidentified protein that was very similar to vimentin in molecular weight but did not react with our antivimentin antibody. We discuss the possibility that this protein was confused with vimentin in the past.

The regulation of reactive oxygen species production during programmed cell death.

Reactive oxygen species (ROS) are thought to be involved in many forms of programmed cell death. The role of ROS in cell death caused by oxidative glutamate toxicity was studied in an immortalized mouse hippocampal cell line (HT22). The causal relationship between ROS production and glutathione (GSH) levels, gene expression, caspase activity, and cytosolic Ca2+ concentration was examined. An initial 5-10-fold increase in ROS after glutamate addition is temporally correlated with GSH depletion. This early increase is followed by an explosive burst of ROS production to 200-400-fold above control values. The source of this burst is the mitochondrial electron transport chain, while only 5-10% of the maximum ROS production is caused by GSH depletion. Macromolecular synthesis inhibitors as well as Ac-YVAD-cmk, an interleukin 1beta-converting enzyme protease inhibitor, block the late burst of ROS production and protect HT22 cells from glutamate toxicity when added early in the death program. Inhibition of intracellular Ca2+ cycling and the influx of extracellular Ca2+ also blocks maximum ROS production and protects the cells. The conclusion is that GSH depletion is not sufficient to cause the maximal mitochondrial ROS production, and that there is an early requirement for protease activation, changes in gene expression, and a late requirement for Ca2+ mobilization.