A short gamma-ray burst apparently associated with an elliptical galaxy at redshift z = 0.225.

Gamma-ray bursts (GRBs) come in two classes: long (> 2 s), soft-spectrum bursts and short, hard events. Most progress has been made on understanding the long GRBs, which are typically observed at high redshift (z approximately 1) and found in subluminous star-forming host galaxies. They are likely to be produced in core-collapse explosions of massive stars. In contrast, no short GRB had been accurately (< 10'') and rapidly (minutes) located. Here we report the detection of the X-ray afterglow from--and the localization of--the short burst GRB 050509B. Its position on the sky is near a luminous, non-star-forming elliptical galaxy at a redshift of 0.225, which is the location one would expect if the origin of this GRB is through the merger of neutron-star or black-hole binaries. The X-ray afterglow was weak and faded below the detection limit within a few hours; no optical afterglow was detected to stringent limits, explaining the past difficulty in localizing short GRBs.

Inhibition of mono-ADP-ribosyltransferase activity during the execution phase of apoptosis prevents apoptotic body formation.

The objective of this study was to understand factors responsible for apoptotic body formation and release during apoptosis. We have found that inhibition of mono-ADP ribosylation after ultraviolet (UV) light induction of apoptosis in HL-60 cells does not block caspase-3 activation, gelsolin cleavage, or endonucleolytic DNA fragmentation. However, the cytoskeletal features of apoptosis leading to apoptotic body formation and release were inhibited by meta-iodobenzylguanidine (MIBG) and novobiocin, potent inhibitors of arginine-specific mono-ADP-ribosyltransferases (mono-ADPRTs). Suppression of mono-ADP ribosylation as late as 120 min following UV irradiation blocked the depolymerization of actin and release of apoptotic bodies. This suggested that the cytoskeletal changes of apoptosis may be decoupled from the caspase cascade and that there may be a biochemical event either distal to or independent of caspase-3 that regulates apoptotic body formation. To test the hypothesis that ADP ribosylation of actin may occur with the induction of apoptosis, an in vivo assay of mono-ADPRT activity using an antibody against ADP-ribosylarginine was used. An approximately 64% increase in the ADP ribosylation of actin was observed at 2 h following exposure to UV light. When MIBG or novobiocin was present, the ADP ribosylation of actin was only 14-18% above the levels observed in control nonirradiated cells. The current study is the first to demonstrate a relationship between ADP-ribosylation of actin and the formation of apoptotic bodies.

Caspase-3 inhibition partially protects oxidant production in apoptotic human neutrophils.

UNLABELLED: Apoptotic PMN lose functional activity, which emphasizes the tissue injury limiting potential of PMN apoptosis. Caspase-3 activation is the first step in the execution phase of apoptosis. We hypothesized that PMN functional activity, as evidenced by oxidant production, can be restored in apoptotic PMN by inhibition of caspase-3. METHODS: To accelerate PMN apoptosis, PMN were UV-irradiated for 15 min as previously described. PMN were pretreated with the caspase-3 inhibitor DEVD-fmk (100 microM) for 30 min prior to UV. PMN apoptosis was quantitated by flow cytometry with CD16 staining. Oxidant production in response to 10 microM PMA was quantitated fluorometrically using the method of Hyslop and Sklar. Caspase-3 activity was quantitated fluorometrically using a commercially available assay. RESULTS: UV-treated PMN demonstrated a 3-fold increase in caspase-3 activity. This was associated with a significant increase in apoptotic PMN and a 10-fold decrease in oxidant production compared to control PMN. DEVD-fmk blocked increases in caspase-3 activity and significantly reduced PMN apoptosis. Oxidant production was increased 5-fold compared to UV-treated PMN but was still significantly less than control PMN. CONCLUSIONS: In UV-accelerated PMN apoptosis, inhibition of caspase-3 activity partially protects oxidant production in apoptotic PMN. This suggests that signaling events in the initiation phase of PMN apoptosis, which are proximal to caspase-3 activation, may in part be responsible for loss of oxidant production in apoptotic PMN independent of caspase-3 activity.

Nuclear dependence of sulfur mustard-mediated cell death.

Although sulfur mustard (SM) has been reported to be a DNA alkylating agent, it is not clear how much of the cytotoxicity of this agent is secondary to DNA damage. To test the hypothesis that the presence of a nucleus is required for the toxicity of sulfur mustard, enucleated endothelial cytoplasts were treated with SM. Using a combination of biochemical and microscopic assays, we demonstrate that some aspects of SM-induced cell death may be dependent on the presence of a nucleus, while others may not be. For example, it was found that cytoskeletal changes, such as loss of stress fibers and rounding, proceed in response to sulfur mustard treatment even in the absence of a nucleus. However, significant further increases in caspase activity and the associated phosphatidylserine translocation were not observed in cytoplasts treated with 500 microM SM for 6 h (following a 20-h recovery at the end of cytoplast preparation). In contrast, cytoplasts treated with chelerythrine, an agent previously reported to induce rapid apoptosis, demonstrated increases in caspase activity in cytoplasts comparable to that observed in the nucleated cells. This indicates that sulfur mustard-induced alkylation of nuclear DNA may be an important stimulus for activation of caspases in nucleated cells. Interestingly, the baseline caspase activity in cytoplasts was greater than in nucleated cells. Analysis of the time course of caspase activation in untreated adherent cytoplasts indicated that the activity increases initially and then stabilizes by 8 h to a low level that was comparable to the level observed at 26 h in untreated cytoplasts. This indicates that cytoplasts are able to tolerate stable low levels of caspase activity and not proceed immediately into the execution phase of apoptosis. The cytoplast model may be quite useful in the toxicological assessment of agents that are thought to exert their toxicity through DNA damage.

Predictive value of proton magnetic resonance spectroscopy in pediatric closed head injury.

We studied 26 infants (1-18 months old) and 27 children (18 months or older) with acute nonaccidental (n = 21) or other forms (n = 32) of traumatic brain injury using clinical rating scales, a 15-point MRI scoring system, and occipital gray matter short-echo proton MRS. We compared the differences between the acutely determined variables (metabolite ratios and the presence of lactate) and 6- to 12-month outcomes. The metabolite ratios were abnormal (lower NAA/Cre or NAA/Cho; higher Cho/Cre) in patients with a poor outcome. Lactate was evident in 91% of infants and 80% of children with poor outcomes; none of the patients with a good outcome had lactate. At best, the clinical variables alone predicted the outcome in 77% of infants and 86% of children, and lactate alone predicted the outcome in 96% of infants and 96% of children. No further improvement in outcome prediction was observed when the lactate variable was combined with MRI ratios or clinical variables. The findings of spectral sampling in areas of brain not directly injured reflected the effects of global metabolic changes. Proton MRS provides objective data early after traumatic brain injury that can improve the ability to predict long-term neurologic outcome.

Chelerythrine chloride induces rapid polymorphonuclear leukocyte apoptosis through activation of caspase-3.

Polymorphonuclear leukocytes (PMN) play a primary role in the initiation and propagation of inflammatory responses. PMN apoptosis is a major mechanism associated with the resolution of inflammatory reactions. Understanding mechanisms associated with PMN apoptosis will be of critical value in the development of novel pharmacological treatment strategies for local and/or systemic inflammatory disorders. The present study demonstrates that chelerythrine chloride induces human PMN to undergo rapid and synchronous progression into the apoptotic process via a PKC-independent mechanism. The appearance of the morphological features of apoptosis in chelerythrine-treated PMN is preceded by a significant upregulation in caspase-3 activity. GM-CSF (a cytokine that protects PMN in several models of PMN apoptosis) does not protect PMN from chelerythrine chloride-induced apoptosis.

Proton MR spectroscopy in children with acute brain injury: comparison of short and long echo time acquisitions.

The aim of this study was to evaluate comparatively the information given by proton magnetic resonance spectroscopy (MRS) with short echo time (TE 20 msec) stimulated echo acquisition mode and long TE (270 msec) point-resolved spectroscopy in predicting long-term outcome in children suffering from acute brain injury. At 1.5 T, we performed single-voxel proton MRS with both methods in occipital gray matter of 70 children. A linear discriminant analysis used to predict outcomes based on MRS variables was compared with actual neurologic outcome assigned at least 6 months after injury by a pediatric neurologist. Using peak area metabolite ratios and lactate presence, the short and long TE methods were equally predictive in children over 1 month of age. In neonates less than 1 month of age, the long TE method produced a higher percentage of correct outcome predictions (91%) than the short TE method (79%). The long TE method detected lactate more often in all age groups.

N-acetylcysteine and endothelial cell injury by sulfur mustard.

Understanding the underlying mechanisms of cell injury and death induced by the chemical warfare vesicant sulfur mustard (HD) will be extremely helpful in the development of effective countermeasures to this weapon of terror. We have found recently that HD induces both apoptosis and necrosis in endothelial cells (Toxicol. Appl. Pharmacol. 1996; 141: 568-583). Pretreatment of the endothelial cells for 20 h with the redox-active agent N-acetyl-L-cysteine (NAC) selectively prevented apoptotic death induced by HD. In this study, we tested the hypotheses that pretreatment with NAC acts through two different pathways to minimize endothelial injury by HD: NAC pretreatment acts via a glutathione (GSH)-dependent pathway; and NAC pretreatment acts to suppress HD-induced activation of the nuclear transcription factor NFkappaB. We used a fluorescence microscopic assay of apoptotic nuclear features to assess viability and electrophoretic mobility shift assays (EMSAs) to assess the activity of NFkappaB following exposure to HD. The cells were treated with 0-10 mM GSH for 1 h prior to and during exposure to 0 or 500 microM HD for 5-6 h. Cells were also treated with 50 mM NAC or 200 microM buthionine sulfoximine (BSO), an inhibitor of GSH synthesis, alone or in combination overnight prior to exposure to 0 or 500 microM HD for 5-6 h. Externally applied GSH up to a concentration of 5 mM had no toxic effect on the cells. Mild toxicity was associated with 10 mM GSH alone. There was a dose-related enhancement of viability when 2.5 and 5 mM GSH were present during the HD exposure. Pretreatment with BSO alone had no discernible toxicity. However, pretreatment with this inhibitor of GSH synthesis potentiated the toxicity of HD. Pretreatment with 50 mM NAC, as previously reported, provided substantial protection. Combining pretreatment with both BSO and NAC eliminated the protective effect of NAC pretreatment alone on HD injury. These observations are highly suggestive that NAC enhances endothelial survival via GSH-dependent effects and confirms and extends the work of others with different models that externally supplied GSH alone may be a fairly effective countermeasure against HD injury of endothelium. We next examined the hypothesis that HD may activate the nuclear transcription factor NFkappaB by performing EMSAs with nuclear extracts of endothelial cells following exposure to 0, 250 or 500 microM HD. This demonstrated an up to 2.5-fold increase (scanning densitometry) in activation of NFkappaB binding to its consensus sequence induced by 500 microM HD after 5 h of HD exposure. Paradoxically, treatment of the endothelial cells alone with 50 mM NAC activated NFkappaB, although HD-induced activation of NFkappaB was partially suppressed by NAC at 5 h. Factor NFkappaB is an important transcription factor for a number of cytokine genes (e.g. tumor necrosis factor, TNF), which can be activated following stress in endothelial cells. Taken together, these observations suggest that the protective effects of NAC may be mediated by enhanced GSH synthesis. The increased GSH may act to scavenge HD and also prevent oxidative activation of NFkappaB. Under some conditions, NAC may act as an oxidizing agent and thus increase NFkappaB activity. The NFkappaB-dependent gene expression may be important in inducing endothelial cell death as well as in generating a local inflammatory reaction associated with the release of endothelial-derived cytokines.

Activation of poly [ADP-Ribose] polymerase in endothelial cells and keratinocytes: role in an in vitro model of sulfur mustard-mediated vesication.

Although endothelial cells and keratinocytes appear to be the primary cellular targets of sulfur mustard (SM), the role of the nuclear enzyme poly (ADP-ribose) polymerase (PARP) in SM-induced vesication has not been clearly defined. PARP is thought to play a crucial role in DNA repair mechanisms following exposure to alkylating agents like SM. Using a combination of fluorescence microscopy and biochemical assays, we tested the hypothesis that SM causes activation of PARP in endothelial cells and keratinocytes with subsequent loss of nicotinamide adenine dinucleotide (NAD) and depletion of adenosine triphosphate (ATP) levels. To determine if PARP activation accounts for SM-induced vesication, keratinocyte adherence and permeability of endothelial monolayers were measured as in vitro correlates of vesication. As early as 2 to 3 h after exposure to SM concentrations as low as 250 microM, dramatic changes were induced in keratinocyte morphology and microfilament architecture. Exposure to 500 microM SM induced a fourfold increase in PARP activity in endothelial cells, and a two- to threefold increase in keratinocytes. SM induced a dose-related loss of NAD+ in both endothelial cells and keratinocytes. ATP levels fell to approximately 50% of control levels in response to SM concentrations >/=500 microM. SM concentrations >/=250 microM significantly reduced keratinocyte adherence as early as 3 h after exposure. Endothelial monolayer permeability increased substantially with concentrations of SM >250 microM. These observations support the hypothesis that the pathogenic events necessary for SM-induced vesication (i.e., capillary leak and loss of keratinocyte adherence) at higher vesicating doses of SM (>/=500 microM) may depend on NAD loss with PARP activation and subsequent ATP-dependent effects on microfilament architecture. Vesication developing as a result of exposure to lower concentrations of SM presumably occurs by mechanisms that do not depend on loss of cellular ATP (e.g., apoptosis and direct SM-mediated damage to integrins and the basement membrane).

Granulocyte-macrophage colony-stimulating factor rescues human polymorphonuclear leukocytes from ultraviolet irradiation-accelerated apoptosis.

INTRODUCTION: Bacterial lipopolysaccharide (LPS) and granulocyte-macrophage colony-stimulating factor (GM-CSF) delay PMN apoptosis during in vitro culture. The present study was undertaken to determine if LPS and GM-CSF can rescue UV-irradiated PMN from undergoing apoptosis and to determine the role of extracellular signal-regulated kinase (ERK) in this process. MATERIALS AND METHODS: PMN were preincubated with LPS (20 ng/ml) and GM-CSF (100 units/ml) for 60 min before being UV-irradiated for 15 min. Additional PMN were UV-irradiated for 15 min and then treated with LPS and GM-CSF. To determine the role of ERK in protection or rescue of PMN from apoptosis, PMN were preincubated with PD098059 for 30 min. Morphologic features of apoptosis were determined 4 h after UV irradiation. DNA laddering was confirmed by agarose gel electrophoresis. RESULTS: LPS and GM-CSF pretreatment significantly protected PMN from UV-accelerated apoptosis, although GM-CSF was more effective than LPS. Only GM-CSF rescued PMN that had already been exposed to UV irradiation from undergoing apoptosis. Time response experiments demonstrated that GM-CSF rescued a significant percentage of PMN when added up to 90 min after UV irradiation. Inhibition of ERK with PD098059 abrogated the protective effect of LPS and GM-CSF and blocked rescue of PMN from apoptosis by GM-CSF. CONCLUSIONS: LPS and GM-CSF protect PMN, whereas only GM-CSF can rescue PMN from UV-accelerated apoptosis. The ERK-signaling pathway plays an important role in the protection and/or rescue of PMN from UV-accelerated apoptosis, but appears to be a proximal event in this process. This study provides further insight into factors that regulate PMN apoptosis and provides a novel approach for investigating signal transduction pathways involved in PMN apoptosis.

ATP converts necrosis to apoptosis in oxidant-injured endothelial cells.

Cell death due to necrosis results in acute inflammation, while death by apoptosis generally does not. The effect of adenosine triphosphate (ATP) on the pattern of cell death induced by oxidants was examined in bovine endothelial cells. ATP levels were altered by hydrogen peroxide (H2O2), glutamine (Gln), and metabolic inhibition (MI), to determine if necrosis can be shifted to apoptosis during oxidant injury. The form of cell death was determined by fluorescence microscopic techniques and the pattern of DNA degradation on agarose gels. ATP levels were measured using the luciferase-luciferin assay. Apoptosis occurred with 100 microM H2O2 without an alteration in ATP levels. ATP was significantly lowered with 5 mM H2O2, and necrosis occurred. MI, in combination with 100 microM H2O2, decreased ATP and resulted in necrosis. MI alone, however, did not cause cell death. Gln partially restored ATP levels in cells injured with 5 mM H2O2 and resulted in a significant increase in apoptosis. DNA laddering on agarose gels confirmed the apoptotic changes seen by fluorescence microscopy. In summary, a threshold level of ATP 25% of basal levels is required for apoptosis to proceed after oxidant stress, otherwise necrosis occurs. Agents like glutamine that enhance ATP levels in oxidant-stressed cells may be potent means of shifting cell death during inflammation to the noninflammatory form of death--apoptosis.

Lipopolysaccharide protects polymorphonuclear leukocytes from apoptosis via tyrosine phosphorylation-dependent signal transduction pathways.

BACKGROUND: The present study was undertaken to determine if tyrosine phosphorylation signal transduction pathways, which are known to be activated in polymorphonuclear leukocytes (PMN) by lipopolysaccharide (LPS), play a role in priming of PMN oxidative burst and protection of PMN from apoptosis by LPS, and to determine if an interface between these two signaling pathways exists. METHODS: PMN were combined with or without 10-fold serial dilutions (0.1 ng-1 microgram/ml) of LPS and incubated at 37 degrees C/5% CO2. After 24 h PMN apoptosis was assessed using fluorescence microscopy and DNA agarose gel electrophoresis. Additional PMN were pretreated with the tyrosine kinase inhibitors genistein and herbamycin A before addition of LPS. Tyrosine phosphorylation was detected by immunoblotting. Oxidant production was quantitated by following the oxidation of a chromophore to its fluorescent product. RESULTS: LPS delayed the onset of apoptosis and prolonged the survival of PMN in a dose-dependent fashion. Both tyrosine kinase inhibitors blocked the protective effect of LPS on PMN apoptosis; however, only genistein blocked the priming effect of LPS on PMN oxidative burst. CONCLUSIONS: Tyrosine phosphorylation signal transduction pathways are central to protection of PMN from apoptosis by LPS. Although tyrosine phosphorylation pathways also play a role in priming of the oxidative burst in PMN, our data suggest that there is not an interface between these important signaling pathways.

Autocrine/paracrine modulation of polymorphonuclear leukocyte survival after exposure to Candida albicans.

Polymorphonuclear leukocytes (PMN) play a central role in the host response to injury and infection. These terminally differentiated phagocytes have a limited life span, after which they undergo spontaneous apoptosis. PMN life span can be significantly prolonged by several naturally occurring cytokines, and PMN are now known to be capable of cytokine production in response to various antigenic stimuli. These facts suggest the possibility that PMN possess an autocrine/paracrine mechanism for the control of their own survival. The present study was undertaken to test this hypothesis. Supernatants from PMN that had been incubated with Candida albicans for 18 h significantly decreased the number of fresh PMN demonstrating features of apoptosis and increased the percentage of viable PMN during in vitro culture. This was demonstrated by monitoring morphologic features of apoptosis with fluorescence microscopy and DNA endonuclease activity with agarose gel electrophoresis. Significant levels of tumor necrosis factor (TNF) were detectable in the supernatants of PMN that had been stimulated with C. albicans, as determined using a TNF-sensitive cell line. Neutralization of TNF biologic activity with a specific monoclonal antibody partially abrogated the supernatant-mediated prolongation of PMN survival. The present study demonstrates that PMN possess a mechanism for the modulation of their own survival, which in part may be through the production of TNF.

Ultraviolet irradiation accelerates apoptosis in human polymorphonuclear leukocytes: protection by LPS and GM-CSF.

Polymorphonuclear leukocytes (PMN) play a central role in host response to injury and infection. Understanding factors that regulate PMN survival may therefore have a major influence on the development of novel treatment strategies for controlling life-threatening infections, as well as local and systemic inflammatory responses. Unfortunately, the presently utilized in vitro culture model of PMN apoptosis makes the examination of early biochemical events surrounding PMN apoptosis very difficult. This study demonstrates that a short course of UV irradiation (15 min) can be used to induce rapid progression of PMN through the apoptotic process with 70-90% of PMN displaying features of apoptosis by 4 h after UV exposure. Bacterial lipopolysaccharide and granulocyte-macrophage colony-stimulating factor, which are known to prolong PMN survival during in vitro culture, also protected PMN from UV-accelerated apoptosis. The UV-accelerated model of PMN apoptosis provides another valuable tool for the investigation of early signaling pathways associated with inducing or delaying PMN apoptosis.

1H-magnetic resonance spectroscopy-determined cerebral lactate and poor neurological outcomes in children with central nervous system disease.

By using proton magnetic resonance spectroscopy ((1)H-MRS), cerebral lactate has been shown to be elevated in a wide variety of pediatric and adult neurological diseases. In this study we compared 36 newborns, infants, and children with elevated lactate peaks on (1)H-MRS with 61 patients without an identifiable lactate signal. (1)H-MRS was acquired from the occipital gray and parietal white matter (8 cm3 volume, STEAM sequence with echo time = 20 msec, repetition time = 3.0 seconds) and data were expressed as ratios of different metabolite peak areas (N-acetylaspartate [NA]/creatine [Cr], NA/choline [Ch], and Ch/Cr) and the presence of a characteristic lactate doublet peak at 1.3 ppm. Outcomes (Pediatric Cerebral Performance Category Scale score; PCPCS) were assigned 6 to 12 months after injury. Patients with lactate peaks were more likely to have suffered a cardiac arrest, were more often hyperglycemic, and had lower Glasgow Coma Scale scores on admission. They were also more likely to have abnormal metabolite ratios when compared with age-matched controls or with patients without detectable lactate. Of prognostic importance, patients with increased lactate were more likely to be severely disabled (39% vs 10%), survive in a persistent vegetative state (13% vs 2%), or have died (39% vs 7%). In contrast, patients with similar conditions without increased lactate were more likely to have had a good outcome (23% vs 3%) or recovered to a mild (38% vs 6%) or moderate disability (20% vs 0%). Our data suggest that (1)H-MRS is useful in the prediction of long-term outcomes in children with neurological disorders. Patients with elevated cerebral lactate are more likely to die acutely or are at greater risk for serious long-term disability.

Proton MR spectroscopy after acute central nervous system injury: outcome prediction in neonates, infants, and children.

PURPOSE: To evaluate the usefulness of proton magnetic resonance (MR) spectroscopy in predicting 6-12-month neurologic outcome in children after central nervous system injuries. MATERIALS AND METHODS: Localized single-voxel, 20-msec-echo-time MR spectra (including N-acetylaspartate [NAA], choline [Ch], creatine and phosphocreatine [Cr]) were obtained in the occipital gray matter in 82 patients and 24 control patients. Patient age groups were defined as neonates (< or = 1 month [n = 23]), infants (1-18 months [n = 31]), and children (> or = 18 months [n = 28]). Metabolite ratios and the presence of lactate were determined. Linear discriminant analysis-with admission clinical data, proton MR spectroscopy findings, and MR imaging score (three-point scale based on severity of structural neuroimaging changes)-was performed to help predict outcome in each patient. Findings were then compared with the actual 6-12-month outcome assigned by a pediatric neurologist. RESULTS: Outcome on the basis of proton MR spectroscopy findings combined with clinical data and MR imaging score was predicted correctly in 91% of neonates and in 100% of infants and children. Outcome on the basis of clinical data and MR imaging score alone was 83% in neonates, 84% in infants, and 93% in children. The presence of lactate was significantly higher in patients with poor outcome than in patients with good-moderate outcomes in all three age groups (neonates, 38% vs 5%; infants, 87% vs 5%; children, 64% vs 10% [chi 2 test, P < .02]). In children with poor outcomes, NAA/Cr ratios were significantly lower in infants (P = .006) and children (P < .001), and NAA/Ch ratios were significantly lower in infants (P = .001) and neonates (P = .05). CONCLUSION: Findings at proton MR spectroscopy helped predict long-term neurologic outcomes in children after central nervous system injury.

Prognostic value of 1H-MRS in perinatal CNS insults.

The authors studied 37 term neonates (38-42 gestational weeks) at 1-11 days after central nervous system insult to determine whether proton magnetic resonance spectroscopy (1H-MRS) of the occipital gray/parietal white matter was useful in predicting outcomes. Etiologies included asphyxia, 18; sepsis/meningitis, 8; metabolic disorders, 5; stroke, 4; and trauma, 2. 1H-MRS data (1.5T; 8 cm3 vol, stimulated echo acquisition mode sequence, TE = 20 ms, TR = 3000 ms) were expressed as metabolite peak area ratios (NAA/Cr, NAA/Cho, Cho/Cr) and the presence or absence of lactate. Outcomes were assessed at 6 to 12 months post-insult using the Pediatric Cerebral Performance Scale and were dichotomized as follows: good/moderate outcome (good, mild or moderate disability) or poor outcome (severe disability, persistent vegetative state, death). Neonates with poor outcomes had significantly lower NAA/Cho and significantly higher Cho/Cr ratios in the occipital region, as compared with patients with good/moderate outcomes. No neonates with good/moderate outcomes had metabolite ratios that exceeded 2 standard deviations from the mean. In addition, the absence of lactate on 1H-MRS correlated with a good/moderate outcome. The study also showed that 1H-MRS metabolite ratio data, added to either the Sarnat or EEG scores, enhanced the correlation between these prognostic factors and outcomes. 1H-MRS provides additional objective data early after a wide variety of perinatal neurologic insults to enhance outcome prediction.

Sulfur mustard induces apoptosis and necrosis in endothelial cells.

Sulfur Mustard (SM) is a vesicant or blistering chemical warfare agent, for which there still is no effective therapy. Endothelial cells are one of the major cellular targets for SM. The mechanism of endothelial cell death during SM injury is poorly understood. We studied the effect of exposure of endothelial cells to 0-1000 microM SM over the time course of 2-24 hr to determine the role of apoptotic and necrotic patterns of cell death in endothelial injury induced by SM. SM concentrations < or = 250 microM induced exclusively apoptosis which was observed after 5 hr in 30% of endothelial cells. Exposure to SM concentrations > or = 500 microM caused apoptosis and necrosis to the same extent in 60-85% of all cells after 5 to 6 hr. Necrosis was accompanied by a significant (approximately 50%) depletion of intracellular ATP, while in apoptotic cells ATP remained at the level similar to healthy cells. Interestingly, disruption of the long actin filament stress fibers and rounding of cells preceded other features of apoptosis--DNA fragmentation, membrane budding, and apoptotic body formation. In apoptotic cells, microfilaments formed constricted perinuclear bands, which were not observed in necrotic cells. Pretreatment with 50 mM N-acetyl-L-cysteine (NAC), a sulfhydryl donor and antioxidant, nearly eliminated the apoptotic features of cell death but did not prevent necrosis in response to SM. NAC pretreatment alone induced reorganization of actin filaments into an enhanced network of long stress fibers instead of a dominant cortical band of actin. NAC pretreatment prevented loss of cell adherence and cell rounding following exposure to 250 microM SM. The effect of NAC on cytoskeletal organization and its ability to eliminate SM-induced apoptosis suggests that actin filament organization may be an important element in cellular susceptibility to apoptotic stimuli.

Actin polymerization and depolymerization during apoptosis in HL-60 cells.

Little is known about the biochemical "machinery" responsible for the morphological features of apoptosis, although the cytoskeleton is presumed to be involved. Using flow cytometry, polyacrylamide gel electrophoresis, and fluorescence microscopy, we show that apoptosis induced by ultraviolet (UV) irradiation or 80 micrograms/ml etoposide correlates with early transient polymerization and later depolymerization of filamentous (F)-actin and dramatic changes in visible microfilament organization. Depolymerization of F-actin began before the formation of apoptotic bodies and was ultimately composed of decreases in both the detergent-insoluble (40%) and detergent-soluble (50%) pools of F-actin. Dihydrocytochalasin B (H2CB), which blocked apoptotic body formation, depolymerized F-actin in the detergent-insoluble pool only. Visually, H2CB treatment disrupted microfilament organization, resulting in short, brightly stained microfilaments dispersed throughout the cytoplasm. In contrast, apoptotic cells contained a network of fine microfilaments with bright staining concentrated at the site of apoptotic body formation. Together, these results suggest that reorganization of the microfilament network is necessary for the formation of apoptotic bodies and that depolymerization of F-actin may also be a necessary component of the process of apoptosis.

Proton magnetic resonance spectroscopy in the evaluation of children with congenital heart disease and acute central nervous system injury.

We studied nine infants and children, aged 1 week to 42 months, with severe acute central nervous system injuries associated with cardiac disease or corrective operations by means of single-voxel proton magnetic resonance spectroscopy to determine whether this technique would be useful in predicting neurologic outcome. Proton magnetic resonance spectroscopic data were acquired from the occipital gray and parietal white matter (8 cm3 volume, stimulated echo-acquisition mode sequence with echo time of 20 msec and repetition time of 3.0 seconds) a median of 9 days after operation (range 3 to 42 days). Data were expressed as ratios of areas under metabolite peaks, including N-acetyl compounds, choline-containing compounds, creatine and phosphocreatine, and lactate. Four patients had cerebral insults before operation, one had both a preoperative and a perioperative insult, three had perioperative insults, and one had a prolonged cardiac arrest 2 days after operation. Outcomes (Glasgow Outcome Scale scores) were assigned at discharge and 6 to 12 months after injury. Six patients were in a vegetative state or had severe impairment at discharge, and two still had severe impairment at 6- to 12-month follow-up. Proton magnetic resonance spectroscopy showed lactate in these two patients, along with markedly reduced ratios of N-acetyl compounds to creatine compounds. The other four patients with severe impairment recovered to a level of mild disability at follow-up. Proton magnetic resonance spectroscopy showed no lactate in these four patients; however, one patient showed moderately reduced ratio of N-acetyl compounds to creatine compounds. The three patients who had mild or moderate impairment at discharge showed no lactate and mild or no changes in metabolite ratios; follow-up revealed normal or mild outcomes. Overall, we found that the presence of lactate and markedly reduced ratios of N-acetyl compounds to creatine compounds were predictive of severe outcomes at discharge and long-term follow-up, whereas no lactate and mild or no changes in ratios suggested potential for recovery with at least a mild disability. Continuing investigations are in progress to determine the optimal selection of candidates and timing of proton magnetic resonance spectroscopic studies.