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In cells undergoing apoptosis, mitochondrial outer-membrane permeabilization (MOMP) is followed by caspase activation promoted by released cytochrome c. Although caspases mediate the apoptotic phenotype, caspase inhibition is generally not sufficient for survival following MOMP; instead cells undergo a >caspase-independent cell death> (CICD). Thus, MOMP may represent a point of commitment to cell death. Here, we identify glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a critical regulator of CICD. GAPDH-expressing cells preserved their clonogenic potential following MOMP, provided that caspase activation was blocked. GAPDH-mediated protection of cells from CICD involved an elevation in glycolysis and a nuclear function that correlated with and was replaced by an increase in Atg12 expression. Consistent with this, protection from CICD reflected an increase in and a dependence upon autophagy, associated with a transient decrease in mitochondrial mass. Therefore, GAPDH mediates an elevation in glycolysis and enhanced autophagy that cooperate to protect cells from CICD. © 2007 Elsevier Inc. All rights reserved., This work was supported by NIH grant AI40646 (D.R.G.) and Association pour la Recherche Contre le Cancer (3642) and Fondation pour la Recherche Médicale (J.-E.R.) and Plan Nacional I+D grant (SAF2005-03923) (A.C.). A.C. received a fellowship from the Secretaria de Estado de Universidades e Investigacion.

Variables associated with physical activity show circadian rhythms in resting subjects; these rhythms have both exogenous (due to the individual's lifestyle and environment) and endogenous (due to the "body clock") components. During exercise, many of the rhythms persist, even though some show decreasing amplitude with increasing severity of exercise. Whilst the value of physical fitness is not disputed (for elite athletes, for individuals who just want to be physically fit, or for patients undertaking physical rehabilitation regimens), there are certain times of the day when special care is needed. These times are soon after waking--when there is the possibility of an increased risk of cardiovascular morbidity and damage to the spine--and late in the day--when there is an increased risk of respiratory difficulties. Since physical exercise is inextricably linked with thermoregulation, there are special considerations to bear in mind when exercise takes place in cold or hot environments. Further, due to the effects of the body clock, exercise and activity during night work and after time-zone transitions presents problems peculiar to these circumstances. In addition, the menstrual cycle affects physical performance, and these circatrigintan rhythms interact with the circadian ones. Bearing in mind these factors, advice that is based upon knowledge of circadian and circatrigintan rhythms can be given to all those contemplating physical activity. Chronobiologically, there is advantage in undertaking physical activity programmes towards the middle of the waking day and not at times when a sleep or nap has just been taken.

Cytotoxic lymphocytes (CL) induce death of their targets by granule exocytosis. During this process, enzymes contained within cytotoxic granules (granzymes) are delivered to the target cell where the enzymes trigger the cell death by cleaving specific substrates. Granzyme B is the only granzyme that has been shown to induce cell death by apoptosis, but the exact pathway by which this is achieved has been the subject of hot debate. Furthermore, several other death-inducing granzymes have been identified; therefore, the exact contribution of granzyme B to CL-induced death is unclear. In this study, we discuss our recent findings on granzyme B-induced cell death and discuss the potential relevance of this pathway to CL-induced death of viral-infected and transformed cells.

A functional immune system is dependent on the generation and selection of a lymphocyte repertoire that is sufficiently diverse to respond to innumerable foreign antigens yet be adequately self-tolerant to avoid the development of autoimmunity. Programmed cell death by a process known as apoptosis is responsible for negative selection of nonreactive leukocyte precursors and autoreactive thymocytes, killing of infected and transformed cells by cytotoxic lymphocytes and deletion of superfluous activated lymphocytes by activation-induced cell death (AICD) and peripheral deletion at the termination of an immune response. Mitochondrial respiration is required to meet the energy requirements of activated and proliferating peripheral lymphocytes. Several mitochondrial proteins have been implicated as regulators of apoptosis in the immune system that are required for prevention of autoimmunity. Recent discoveries have shed light on mitochondrial functions as they relate to cell death, including caspase-dependent and -independent apoptosis, mitochondrial death substrates and events that disable mitochondrial functions during apoptosis. These discoveries, taken with reports that the specific manner by which a cell dies greatly impacts on the nature of subsequent immune responses, highlight an exciting era of research on mitochondrial function and its role in apoptosis and the effects on immune responses.

PROLI/NO is an agent of structure XN(O)==NONa (X = L-prolyl) whose 2-s half-life for nitric oxide (NO) release at physiological pH makes it an excellent prodrug for localizing NO's therapeutic effects at the site of application, but a difficult one to formulate and certify as pure. Despite its extraordinary thermal and hydrolytic instability, however, PROLI/NO could be formulated as an injectable drug by dissolving it in cold 0.1 M sodium hydroxide containing 5% D-mannitol, then quickly ultrafiltering and lyophilizing it in evacuated septum vials. No evidence for decomposition was seen in the contents of these evacuated vials when stored at -20 degrees C over a 140-day observation period, as judged by quantifying NO release in simulated infusate solutions (10 mM carbonate/bicarbonate, pH 10.5). The only hydrolysis products detected were NO, nitrite ion, proline, and N-nitrosoproline, all products of normal human physiological processes.

Loss of Bid confers clonogenic survival to granzyme B-treated cells, however the exact role of Bid-induced mitochondrial damage--upstream or downstream of caspases--remains controversial. Here we show that direct cleavage of Bid by granzyme B, but not caspases, was required for granzyme B-induced apoptosis. Release of cytochrome c and SMAC, but not AIF or endonuclease G, occurred in the absence of caspase activity and correlated with the onset of apoptosis and loss of clonogenic potential. Loss of mitochondrial trans-membrane potential (DeltaPsim) was also caspase independent, however if caspase activity was blocked the mitochondria regenerated their DeltaPsim. Loss of DeltaPsim was not required for rapid granzyme B-induced apoptosis and regeneration of DeltaPsim following cytochrome c release did not confer clonogenic survival. This functional dissociation of cytochrome c and SMAC release from loss of DeltaPsim demonstrates the essential contribution of Bid upstream of caspase activation during granzyme B-induced apoptosis.

Here, we explore the chemistry of the previously undocumented E form of diazeniumdiolates having the structure R(1)R(2)NN(O)=NOR(3). Reported crystallographic studies have uniformly revealed the Z configuration, and our attempts to observe a Z --E conversion through thermal equilibration or photochemical means have, until now, consistently failed to reveal a significant amount of a second conformer. As a typical example, the NMR spectrum of trimethyl derivative Me(2)NN(O)=NOMe revealed no evidence for a second configuration. Electronic structure calculations attribute this finding to a prohibitively high interconversion barrier of approximately 40 kcal/mol. A similar result was obtained when we considered the case of R(1) = Me = R(3) and R(2) = H at the same levels of theory. However, when MeHNN(O)=NOMe was ionized by dissociating the N-H bond, the barrier was calculated to be lower by approximately 20 kcal/mol, with the E form of the anion being favored over Z. This circumstance suggested that an E isomer might be isolable if a Z anion were formed and given sufficient time to assume the E configuration, then quenched by reaction with an electrophile to trap and neutralize the E form and restore the putatively high interconversion barrier. Consistent with this prediction, basifying iPrHNN(O)=NOCH(2)CH(2)Br rapidly led to a six-membered heterocycle that was crystallographically characterized as containing the -N(O)=NO- functional group in the E configuration. The results suggest an approach for generating pairs of Z and E diazeniumdiolates for systematic comparison of the rates at which the individual isomers release bioactive NO and of other physicochemical determinants of their biomedical utility.

A functional immune system is dependent on the generation and selection of a lymphocyte repertoire that is sufficiently diverse to respond to innumerable foreign antigens yet be adequately self-tolerant to avoid the development of autoimmunity. Programmed cell death by a process known as apoptosis is responsible for negative selection of nonreactive leukocyte precursors and autoreactive thymocytes, killing of infected and transformed cells by cytotoxic lymphocytes and deletion of superfluous activated lymphocytes by activation-induced cell death (AICD) and peripheral deletion at the termination of an immune response. Mitochondrial respiration is required to meet the energy requirements of activated and proliferating peripheral lymphocytes. Several mitochondrial proteins have been implicated as regulators of apoptosis in the immune system that are required for prevention of autoimmunity. Recent discoveries have shed light on mitochondrial functions as they relate to cell death, including caspase-dependent and -independent apoptosis, mitochondrial death substrates and events that disable mitochondrial functions during apoptosis. These discoveries, taken with reports that the specific manner by which a cell dies greatly impacts on the nature of subsequent immune responses, highlight an exciting era of research on mitochondrial function and its role in apoptosis and the effects on immune responses.

Granzyme B, a protease released from cytotoxic lymphocytes, has been proposed to induce target cell death by cleaving and activating the pro-apoptotic Bcl-2 family member Bid. It has also been proposed that granzyme B can induce target cell death by activating caspases directly, by cleaving caspase substrates, and/or by cleaving several non-caspase substrates. The relative importance of Bid in granzyme B-induced cell death has therefore remained unclear. Here we report that cells isolated from various tissues of Bid-deficient mice were resistant to granzyme B-induced cell death. Consistent with the proposed role of Bid in regulating mitochondrial outer membrane permeabilization, cytochrome c remained in the mitochondria of Bid-deficient cells treated with granzyme B. Unlike wild type cells, Bid-deficient cells survived and were then able to proliferate normally, demonstrating the critical role for Bid in mediating granzyme B-induced apoptosis.

During cytotoxic lymphocyte (CL) mediated killing of target cells, granzyme B is released from the CL into the immune synapse. Recent studies have found that ELISPOT-detection of granzyme B correlated well with conventional assays for CL mediated killing. In this way, the released granzyme B can be used to mark the spot where a target cell was murdered. We discuss the benefits and potential limitations of using this assay to measure CL mediated killing of target cells.

A rapid, sensitive, and specific high-performance liquid chromatography-electrospray ionization-multistage mass spectrometry (MS) method for measuring endogenous ketolic estrogen metabolites in human urine has been developed. The method requires a single hydrolysis/extraction/derivatization step and only 2.5 mL of urine, yet is able to simultaneously quantify estrone and its 2-methoxy and 2-, 4-, and 16alpha-hydroxy derivatives, 16-ketoestradiol, and 2-hydroxyestrone-3-methyl ether metabolites. The combination of a simple hydrazone derivatization step with multistage MS greatly enhances the sensitivity and specificity of the analysis of endogenous estrogen within human urine. Standard curves are linear over a 100-fold concentration range with linear regression correlation coefficients typically greater than 0.99. The lower limit of quantitation for each ketolic estrogen is 0.2 ng/2.5-mL urine sample (10 pg on column), with an accuracy of 93-103% and an overall precision, including the hydrolysis, extraction, and derivatization steps, of 1-13% relative standard derivation (RSD) for samples prepared concurrently and 8-16% RSD for samples prepared in separate batches. This method also allows for the identification of 2-hydroxyestrone-3-methyl ether in urine obtained from both pre- and postmenopausal women. This potentially protective estrogen metabolite has been previously reported only in the urine of pregnant women. Since individual patterns of estrogen metabolism may influence the risk of breast cancer, accurate and specific measurement of estrogen metabolites in biological matrixes will facilitate future research on breast cancer prevention, screening, and treatment.

Translocation of proteins from the mitochondrial intermembrane space to the cytoplasm is a critical event during apoptosis. There are several methods for assaying this event cited in the literature. In this chapter, we highlight separation of cytosolic and mitochondrial fractions of cultured cells using digitonin as the method for measuring cytochrome c release that, in our hands has been the simplest and most reproducible.

Translocation of proteins from the mitochondrial intermembrane space to the cytoplasm is a critical event during apoptosis. There are several methods for assaying this event cited in the literature. In this chapter, we highlight separation of cytosolic and mitochondrial fractions of cultured cells using digitonin as the method for measuring cytochrome c release that, in our hands has been the simplest and most reproducible.

Most mammalian cells are constantly threatened by viral infection and oncogenic transformation. To maintain healthy function of organs and tissues it is critical that afflicted cells are efficiently detected and removed. Cytotoxic lymphocytes (CL) are chiefly responsible for efficiently seeking out and eliminating damaged or infected cells. It is known that CLs must specifically recognize and bind to their targets, but the molecular events that occur within the target cell that lead to its death are still poorly understood. The two main processes initiated by CLs to induce target cell death are mediated by ligation of surface receptors or release of toxic proteins from secretory granules (granule exocytosis) of the CL. Here we review some of the key findings that have defined our knowledge of the granule exocytosis-mediated pathways to CL-mediated killing and discuss recent insights that challenge conventional views in the important area of CL effector function.

Antarctic exploration and research have led to some significant although localized impacts on the environment. Human impacts occur around current or past scientific research stations, typically located on ice-free areas that are predominantly soils. Fuel spills, the most common occurrence, have the potential to cause the greatest environmental impact in the Antarctic through accumulation of aliphatic and aromatic compounds. Effective management of hydrocarbon spills is dependent on understanding how they impact soil properties such as moisture, hydrophobicity, soil temperature, and microbial activity. Numbers of hydrocarbon-degrading bacteria, typically Rhodococcus, Sphingomonas, and Pseudomonas species for example, may become elevated in contaminated soils, but overall microbial diversity declines. Alternative management practices to the current approach of "dig it up and ship it out" are required but must be based on sound information. This review summarizes current understanding of the extent and effects of hydrocarbon spillage on Antarctic soils; the observed physical, chemical, and biological responses of such soils; and current gaps in knowledge.

We measured small mammal response to several different group selection silvicultural systems that varied by opening size (0.03, 0.13, and 1.0 ha) but maintained a consistent 30% area removal. The southern redbacked vole (Clethrionomys gapperi), followed by the common shrew (Sorex cinereus) and dusky shrew (S. monticolus), were the most abundant species pre- and post-harvest. There was no evidence that the minimum number alive estimates for red-backed voles differed significantly (α = 0.05) among treatments pre-harvest (ρ = 0.67) or post-harvest (1993, ρ = 0.98; 1994, ρ = 0.84). However, red-backed voles used harvested openings less than the surrounding forest within each treatment. Common shrews showed some preference for the unlogged controls and the treatment units containing 1.0-ha openings. Dusky shrews showed no treatment preference. Overall, we conclude that the group selection silvicultural systems did not substantially change the relatively rich, abundant small mammal community present before harvesting.

This qualitative study was designed to record the perception by Newcastle children of the influences on their choice of drinks and their knowledge of the dental health problems caused by acidic drinks.Four focus groups, each involving 8 Newcastle schoolchildren (4 boys and 4 girls) formed the basis of the study. Two age groups, 13-14-year-olds and 8-9-year-olds, and two socio-economic groups were investigated, using state schools in Newcastle upon Tyne. A moderator guided the children to discuss their choice of drink and its dental effects amongst themselves.In total, 32 children participated in the focus groups and the results suggested that 8-9-year-olds preferred still, fruit-flavoured drinks whilst 13-14-year-olds preferred carbonated drinks. Taste was the most important influence on drink choice in all age groups. Parents and friends were more influential in younger children, whilst cost, availability and thirst were more important to older children. Younger children did not believe advertisements whilst older children thought they might work if seen enough times. Dental knowledge was confused in all age groups and only the 13-14-year-old-high socio-economic groups knew that acidic drinks were bad for the teeth. Different methods for addressing the problem of erosion were suggested by different age groups. There was very little difference between the socio-economic groups in the areas discussed.The children's knowledge of dental diseases and the effect of drinks on the teeth were confused. The factors that influence drink choice appear to change with age, rather than socio-economic status.

Histone deacetylase inhibitors (HDACIs) are a new class of chemotherapeutic drugs able to induce tumor cell apoptosis and/or cell cycle arrest; however, the molecular mechanisms underpinning their anticancer effects are poorly understood. Herein, we assessed the apoptotic pathways activated by three HDACIs, suberoylanilide hydroxamic acid, oxamflatin, and depsipeptide. We determined that all three drugs induced the accumulation of cells with a 4n DNA content and apoptosis mediated by the intrinsic apoptotic pathway. HDACI-induced mitochondrial membrane damage and apoptosis were inhibited by overexpression of Bcl-2, but not by the polycaspase inhibitor N-tert-butoxy-carbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk). Moreover, induction of a G(1)-S checkpoint through overexpression of p16(INK4A) or suppression of de novo protein synthesis also inhibited HDACI-induced cell death. Proteolytic cleavage of caspase-2, which is poorly inhibited by zVAD-fmk, was concomitant with HDACI-induced death; however, full processing of caspase-2 to the p19 active form was blocked by Bcl-2. Whereas all three drugs induce the activation of the proapoptotic Bcl-2 protein Bid upstream of mitochondrial membrane disruption, Bid cleavage in response to depsipeptide was significantly attenuated by zVAD-fmk. Suberoylanilide hydroxamic acid and oxamflatin could kill both P-glycoprotein (P-gp)(+) MDR cells and their P-gp(-) counterparts, whereas depsipeptide was shown to be a substrate for P-gp and was less effective in killing P-gp(+) cells. These data provide insight into the functional profile of three HDACIs and are important for the development of more rational approaches to chemotherapy, where information regarding the genetic profile of the tumor is matched with the functional profile of a given chemotherapeutic drug to promote favorable clinical responses.

The circadian structure (i.e. rest-activity cycle) was evaluated by actometry in a group of 25 advanced NSCLC patients. A better circadian profile was observed in the 6 patients treated with corticosteroïds. However those patients had also a shortened survival.

Cell cycle checkpoints act to protect cells from external stresses and internal errors that would compromise the integrity of the cell. Checkpoints are often defective in cancer cells. Drugs that target checkpoint mechanisms should therefore be selective for tumor cells that are defective for the drug-sensitive checkpoint. Histone deacetylase inhibitors typify this class of agents. They trigger a G2-phase checkpoint response in normal cells but are cytotoxic in tumor cells in which this checkpoint is defective. In this study, we investigated the molecular basis of the tumor-selective cytotoxicity of these drugs and demonstrated that it is due to the disruption of two cell cycle checkpoints. The first is the histone deacetylase inhibitor-sensitive G2-phase checkpoint, which is defective in drug-sensitive cells and permits cells to enter an aberrant mitosis. The second is the drug-dependent bypass of the mitotic spindle checkpoint that normally detects aberrant mitosis and blocks mitotic exit until the defect is rectified. The disruption of both checkpoints results in the premature exit of cells from an abortive mitosis followed by apoptosis. This study of histone deacetylase inhibitors demonstrates that drugs targeting cell cycle checkpoints can provide the selectivity and cytotoxicity desired in effective chemotherapeutic agents.

The mechanism of p53-dependent apoptosis is still only partly defined. Using early-passage embryonic fibroblasts (MEF) from wild-type (wt), p53(-/-) and bax(-/-) mice, we observe a p53-dependent translocation of Bax to the mitochondria and a release of mitochondrial Cytochrome c during stress-induced apoptosis. These events proceed independent of zVAD-inhibitable caspase activation, are not prevented by dominant negative FADD (DN-FADD), but are negatively regulated by Mdm-2. Bcl-x(L) expression prevents the release of mitochondrial Cytochrome c and apoptosis, but not Bax translocation. At a single-cell level, enforced expression of p53 is sufficient to induce Bax translocation and Cytochrome c release. Real-time RT-PCR analysis reveals a significant induction of RNA expression of Noxa and Bax in p53(+/+), but not in p53(-/-) MEF. Noxa protein expression becomes detectable prior to Bax translocation, and downregulation of endogenous Noxa by RNA interference protects wt MEF against p53-dependent apoptosis. Hence, in oncogene-expressing MEF p53 induces apoptosis by BH3 protein-dependent caspase activation.

We have previously shown that nitric oxide (NO) inhibits growth and induces differentiation and apoptosis in acute myeloid leukemia cells, with the HL-60 human myeloid leukemia line being particularly sensitive to NO-mediated cytolysis. With the goal of identifying a prodrug that can target NO to the leukemia cells without inducing NO-mediated systemic hypotension, we have screened a series of O(2)-aryl diazeniumdiolates designed to be stable at physiological pH but to release NO upon reaction with glutathione. O(2)-(2,4-Dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate (JS-K) proved to be the most active antiproliferative agent among those tested in HL-60 cells, with an IC(50) of 0.2-0.5 microM. After 5 days of exposure to 0.5 micro M JS-K, HL-60 cells had differentiated and acquired some of the phenotypic features of normal monocytes. One- to 2-day treatment with JS-K at concentrations of 0.5-1 microM resulted in apoptosis induction in a concentration- and caspase-dependent manner. JS-K also inhibited the growth of solid tumor cell lines but to a lesser extent than HL-60 cells. JS-K was administered i.v. to nonobese diabetic-severe combined immune deficient mice at doses of up to 4 micromol/kg without inducing significant hypotension. The growth of s.c. implanted HL-60 cells was reduced by approximately 50% when the mice received i.v. injections three times/week with 4 micromol/kg boluses of JS-K. Histological examination of tumor explants from JS-K-treated animals revealed extensive necrosis. Similar results were seen with s.c. human prostate cancer (PPC-1) xenografts. Our data indicate that JS-K is a promising lead compound for the possible development of a novel class of antineoplastic agents.

As the number of Australians who attended the fifth European Workshop on Cell Death (EWCD) was sufficient to field a soccer team capable of taking on the rest of the World, it seemed fitting that we should host an Australian version. ‘Death on the Reef', the First Australian WCD was therefore held in August 2011 on a secluded island location off the coast of Queensland to discuss the latest research in this field. The meeting attracted predominantly young scientists who presented short talks on their current research, and highlighted that not only does Australia, ‘abound in nature's gifts', as proclaimed by the national anthem, but also abounds in high quality cell death research. Although it was not possible to cover all of the talks here because of editorial restrictions, the quality of these talks made a good argument for a biennial meeting to complement the EWCD.

Patients with chronic fatigue syndrome (CFS) show evidence of circadian rhythm disturbances. We aimed to determine whether CFS symptoms were alleviated by melatonin and bright-light phototherapy, which have been shown to improve circadian rhythm disorders and fatigue in jet-lag and shift workers.Thirty patients with unexplained fatigue for6 months were initially assessed using placebo and then received melatonin (5 mg in the evening) and phototherapy (2500 Lux for 1 h in the morning), each for 12 weeks in random order separated by a washout period. Principal symptoms of CFS were measured by visual analogue scales, the Shortform (SF-36) Health Survey, Mental Fatigue Inventory and Hospital Anxiety and Depression Scale. We also determined the circadian rhythm of body temperature, timing of the onset of melatonin secretion, and the relationship between these.Neither intervention showed any significant effect on any of the principal symptoms or on general measures of physical or mental health. Compared with placebo, neither body temperature rhythm nor onset of melatonin secretion was significantly altered by either treatment, except for a slight advance of temperature phase (0.8 h; P = 0.04) with phototherapy.Melatonin and bright-light phototherapy appear ineffective in CFS. Both treatments are being prescribed for CFS sufferers by medical and alternative practitioners. Their unregulated use should be prohibited unless, or until, clear benefits are convincingly demonstrated.

A sensitive, precise and accurate stable isotope dilution high-performance liquid chromatography–electrospray ionization mass spectrometry method has been developed for measuring endogenous 2- and 4-hydroxyestrones, the main catechol estrogens in human urine. Compared to the published methods using gas chromatography–mass spectrometry, this approach simplifies sample preparation and increases the throughput of analysis. The unique part of our method is the use of a simple and rapid derivatization step that forms a hydrazone at the C-17 carbonyl group of catechol estrogens. This derivatization step has greatly enhanced method sensitivity as well as HPLC separability of 2- and 4-hydroxyestrones. Standard curves were linear over a 100-fold calibration range with correlation coefficients for the linear regression curves typically greater than 0.996. The lower limit of quantitation for each catechol estrogen is 1 ng per 10-ml urine sample, with an accuracy of 97–99% and overall precision, including the hydrolysis, extraction and derivatization steps, of 1–3% for samples prepared concurrently and 2–11% for samples prepared in several batches. This method is adequate for measuring the low endogenous levels of catechol estrogens in urine from postmenopausal women.

The induction of cell death by cytotoxic T-lymphocytes (CTL) or natural killer (NK) cells is one of the main ways by which higher organisms protect themselves from rogue cells, including those infected by a virus, or posing a risk of cancer. Considering the rapidity of viral replication and spread to uninfected cells, CTL and NK are extremely efficient killers. This is at least partly due to the variety of pathways that these cytolytic lymphocytes (CL) can use to ensure the death of a cell. Primarily, CL utilize two independently initiated pathways involving either ligation of death receptors or perforin mediated trafficking of granzyme B to the target cell cytosol to activate a family of death proteases (caspases) in the target cell. The caspases then orchestrate the orderly dismantling of that cell by cleavage of a set of critical substrates. If caspases are inactivated, due either to mutations in proteins that signal their activation or direct inhibition by a viral gene product, CL can utilize a caspase-independent pathway to ensure the death of the target cell. Here we will discuss the mechanisms by which these stellar killers achieve their goal.

Status epilepticus (SE) is a common neurologic emergency with a high mortality. Immediate recognition and rapid treatment are essential. After initial stabilization of airway and circulation, the patient should be treated as soon as possible with an intravenous (IV) benzodiazepine, followed immediately by IV fosphenytoin. If SE persists, general anesthesia should be initiated, with intubation and cardiac monitoring. Electroencephalogram must also be monitored to ensure suppression of all seizures. Etiology of SE should be assessed through history, examination, blood tests, and brain imaging.

Identification of pro-apoptotic activities for a variety of proteins normally resident in the mitochondrial inter-membrane space has substantiated the role of mitochondria as integral to the apoptotic process. Cytochrome c is involved in apoptosome formation and caspase activation, SMAC/Diablo deregulates the inhibitor of apoptosis proteins, apoptosis-inducing factor may play a role in chromatin condensation and release of other proteins such as adenylate kinase may adversely affect cellular metabolism and contribute to the death of a cell if the downstream apoptotic pathway is blocked. It is still unclear how these proteins are released from the mitochondria. Recent advances in our knowledge of mitochondrial outer-membrane permeabilization and the consequences of this event on mitochondria will be discussed.

Prostaglandin E2 (PGE2) has been suggested as an indicator of irreversible pulpitis in permanent teeth [1]. There is scant information on the role of chemical mediators in primary molar pulp inflammation. The aim of this preliminary study was to investigate the levels of PGE2 in blood harvested from root pulp stumps following coronal pulp amputation in vital primary molar teeth with carious exposures.Seventy-nine cariously exposed primary molars underwent treatment by one of two vital pulp therapy techniques. Blood was harvested from 38 teeth and volume and concentration of PGE2 ([PGE2]) determined [2]. Treatment outcome was assessed from both clinical and radiographic evidence.PGE2 was detected in all samples, with a wide concentration range (1-2641 ng/mL). The distribution was skewed, requiring log transformation. The difference in the mean (log) [PGE2] for radiological success (3.12, SD 1.60 and failure (4.62, SD 1.80) was significant, t = 2.05, P = 0.047. The difference in the mean (log) [PGE2] for clinical success (3.24, SD 1.65) and failure (5.44, SD 1.43 was near-significant, t = 1.84, P = 0.074.[PGE2] correlated positively with radiological outcome following vital pulp therapy.

High-performance liquid (HPLC) and thin-layer (TLC) chromatographic methods for the detection and quantification of diazeniumdiolates are described. The HPLC determinations were made using a Rocket Platinum NH2 column (7 x 53 mm, 100-A pore size, 3-microm particle size), under isocratic conditions with mixtures of acetonitrile, methanol, and water containing 0.1% diethylamine (v/v), at a flow rate of 2.5 ml/min, a column temperature of 22 degrees C, and UV detection at 250 nm. The TLC determinations were similarly made using Merck NH2 F254S precoated glass plates (approximately 2 x 5 cm, 5-microm particle size, 0.2-mm layer thickness) with mixtures of acetonitrile, methanol, and water containing 0.1% diethylamine (v/v). Preexisting traces of carcinogenic nitrosamines were detected in some samples of diazeniumdiolates. The methods provide a more efficient means of characterizing the purity of diazeniumdiolate samples prepared for use in biomedical applications than older procedures which rely on differential absorbance measurements at 250 and 350 nm, respectively.

Publisher Summary This chapter discusses the study of mitochondria in apoptosis. Accumulating evidence has shown that mitochondria also play an integral role in the death of a cell by apoptosis. Because of the mitochondrial cytochrome c release and its regulation by Bcl-2 family members appears to be a common mitochondrial change during apoptosis, the chapter focuses on the methods used to study cytochrome c release. The chapter also discusses methods to examine other mitochondrial parameters that may relate to the apoptosis, including the identification of proteins released from the mitochondria concomitantly with cytochrome c . Many inducers of apoptosis have shown to induce the release of cytochrome c from the mitochondria of cultured cells. Radiation, topoisomerase inhibitors, protein synthesis inhibitors, and protein kinase inhibitors are all commonly used apoptotic stimuli that trigger cytochrome c release. None of these toxins appears directly to induce the release of cytochrome c ; rather they use signaling pathways that may involve Bax, Bid, or other proapoptotic Bcl-2 family members. Various toxins act directly on mitochondria to induce cytochrome c release. Some of these toxins induce a phenomenon known as permeability transition (PT) in isolated mitochondria.

Cytotoxic T lymphocytes and natural killer cells kill virus-infected or transformed cells through the combined actions of a pore forming protein (perforin) and a family of serine proteases (granzymes). 1 Upon the formation of a stable conjugate between the two cells, the exocytosis of granule toxins from the lymphocyte results in the induction of specific pathways to apoptosis in the target cell. Perforin is both permissive and indispensable for apoptosis, as it enables the granzymes to access their key substrates in the target cell cytoplasm to bring about caspase-dependent and -independent cell death. While considerable progress has been made in our understanding of pro-apoptotic pathways activated by the granzymes, the precise molecular role played by perforin remains poorly understood. Over recent years, we have developed a number of robust expression modalities for mouse and human perforin, in order to characterise its molecular and cellular functions. These have included the capacity to express wild-type and mutated perforin molecules in a cellular context where cytoxicity can be measured, and in baculovirus-based systems where purified protein can be analysed for its cytotoxic activity. By combining these approaches, we have been able to map some of perforin’s functional domains, including a determination of the key residues responsible for calcium binding. 2 In addition, a repository of missense mutations identified in patients with the rare familial immune deficiency syndrome, familial haemophagocytic lymphohistiocytosis (FHL) has proven invaluable in identifying perforin residues that result in either presynaptic protein instability or post-synaptic dysfunction at the level of the target cell membrane. 3 Mutagenesis and modelling have recently allowed us to define the mechanism through which perforin forms transmembrane olgomers. 4 The functional implications of certain common human perforin polymorphisms such as Ala91Val and much rarer mutations will also be discussed, particularly with respect to perforin’s role in human cancer susceptibility. 5

The rest/activity circadian cycle has been used as a reference for chemotherapy administration at specific times to improve tolerability and efficacy. Because cancer processes may be associated with alterations of circadian rhythms, the rest/activity cycle was monitored noninvasively to assess its relationship with tumor response, survival, and quality of life in 200 patients with metastatic colorectal cancer. Patients wore an actigraph, a wristwatch that records the number of accelerations per minute, for 3 days before receiving chronomodulated chemotherapy. The circadian rhythms in activity were estimated by two robust parameters: the autocorrelation coefficient at 24 h (r24), and the dichotomy index (IO) for comparing amounts of activity when in bed and out of bed. Accurate data for inclusion, quality of life, response, and survival were available for 192 patients. Survival at 2 years was 5-fold higher (P = 10(-4)) in patients with marked activity rhythm (IO in upper quartile) than in those with rhythm alteration (IO in lower quartile). These results were supported by the multivariate Cox analysis. Multivariate regression analysis showed that circadian rhythms in activity (IO; P = 3 x 10(-4)) and in WBCs (P = 0.03) as well as performance status (P = 0.02) were jointly prognostic of response. Patients with marked rest/activity rhythms also had better quality of life and reported significantly less fatigue. The individual rest/activity cycle provides a novel independent prognostic factor for cancer patients' survival and tumor response as well as a quantitative indicator for quality of life.

Today it is commonplace for people to fly across several time zones on business or on holiday. Moreover, many people work at night rather than the conventional 9-to-5, not only to provide public services but also to use expensive equipment economically and engage in world-wide commerce. Both groups can experience ill-effects, known respectively as 'jet-lag' and 'shiftworker's malaise'l 4. In the short term, they feel tired and unable to concentrate, yet cannot sleep uninterruptedly during the night; they perform physical and mental tasks less well than usual and with more errors; they have no appetite and they experience indigestion or bowel upsets. As well as being troublesome at a personal level, these effects might have wider implications for example, by hampering international negotiations or reducing dexterity in a technical procedure. In the longer term also there are negative effects. For example, there is evidence for raised frequencies of menstrual disorders in airline hostessess and of gastrointestinal ulcers3 and cardiovascular morbidity in nightworkers6. A full explanation of these effects will doubtless indicate that the causes are complex and multiple, but a key issue is the 'body clock'. Here I outline its function and some of the non-pharmacological ways it can be influenced to alleviate jet-lag or shiftworker's malaise. The accompanying article by Arendt7 focuses on pharmacotherapy.

Whether we view the mitochondria as the headquarters for the leader of a crack suicide squad or as a prison for the leader of a militant coup, the role of the mitochondria in the apoptotic process is now well established. During apoptosis the integrity of the mitochondria is breeched, the mitochondrial transmembrane potential drops, the electron transport chain is disrupted. and proteins from the mitochondrial intermembrane space (MIS) such as cytochrome c are released into the cytosol, although not necessarily in that order. In the cytosol, cytochrome c forms part of a proteinaceous complex that directly activates caspase-9, one of the apical enzymes responsible for the dismantling of the cell. In this way a mitochondrial factor which is normally locked away from the rest of the cell can directly trigger apoptosis. The need to regulate the release of cytochrome c suggests that the mitochondria may be the decision center for whether a cell lives or dies. Various hypotheses have been formulated to explain how proteins of the MIS are released and how this process is regulated. These include the Bcl-2-regulated opening of a permeability transition pore or an increase in mitochondrial transmembrane potential followed by outer membrane rupture. It remains to be clarified which mitochondria specific events are essential for apoptosis and which are merely consequences of apoptosis.

Physical exercise activates blood coagulation and enhances fibrinolytic activity. To investigate whether these activations of blood coagulation and fibrinolysis are balanced post-exercise and during the period of recovery, 11 moderately active young men were examined immediately after a standardised cycle ergometer test and during the 24 h period of recovery. Blood samples were obtained at rest, immediately after exercise, and 2, 6 and 24 h after exercise. All post-exercise values were corrected for any change in plasma volume. Exercise induced a significant increase in factor VIII activity and this occurred with a significant shortening of activated partial thromboplastin time. A concomitant enhancement of tissue plasminogen activity resulted in significant increases in tissue plasminogen activity antigen and total fibrin/fibrinogen degradation products, and a significant decrease in tissue plasminogen activator inhibitor-1 activity. Increases in coagulation and fibrinolytic activity changed in parallel during exercise. However, during recovery, while the increase in factor VIII activity post-exercise persisted 2 and 6 h into recovery, fibrinolytic activity demonstrated a sharp fall. It is concluded that whereas the enhanced fibrinolytic activity during exercise appears to counterbalance the increase in blood coagulability, this haemostatic balance is not maintained during recovery. This perturbed blood haemostasis could constitute an enhanced risk for coronary artery thrombosis and may contribute to exercise-related cardiovascular events.

Status epilepticus (SE) is a common neurological and medical emergency. Despite the significant mortality associated with SE, no human data have been available regarding cardiovascular changes prior to death in patients with this condition. This study was conducted to measure hemodynamic trends in the 24 h prior to death in a series of 24 prospectively evaluated SE patients. Two distinct cardiovascular patterns of mean arterial pressure (MAP) and heart rate (HR) were observed. Ten patients had a gradual decline in MAP and/or HR, and this group was designated as having gradual cardiac decompensation (GCD). The remaining 14 patients showed no significant changes in either MAP or HR up to the time of death. This group of patients was designated as having acute cardiac decompensation (ACD). The changes in MAP and HR over the last 24 h prior to death between the GCD and ACD groups were statistically significant. Ninety percent of the GCD patients had a history of multiple risk factors for arteriosclerotic cardiovascular disease (ASCVD), while only 30% of the ACD group had a history of multiple risk factors for ASCVD. The results provide the first human data of cardiovascular events immediately preceding death in SE patients. We propose that further investigation of the cardiovascular pathophysiology of SE may provide new therapeutic interventions which could decrease the significant mortality associated with SE.

The onset of apoptosis is coupled to the proteolytic activation of a family of cysteine proteases, termed caspases. These proteases cleave their target proteins after an aspartate residue. Following caspase activation during apoptosis, a number of specific proteins have been shown to be cleaved. Here we show that Nedd4, a ubiquitin-protein ligase containing multiple WW domains and a calcium/lipid-binding domain, is also cleaved during apoptosis induced by a variety of stimuli including Fas-ligation, gamma-radiation, tumor necrosis factor-alpha, C-8 ceramide, and etoposide treatment. Extracts from apoptotic cells also generated cleavage patterns similar to that seen in vivo, and this cleavage was inhibited by an inhibitor of caspase-3-like proteases. In vitro, Nedd4 was cleaved by a number of caspases, including caspase-1, -3, -6, and -7. By site-directed mutagenesis, one of the in vitro caspase cleavage sites in mouse Nedd4 was mapped to a DQPD237 downward arrow sequence, which is conserved between mouse, rat, and human proteins. This is the first report demonstrating that an enzyme of the ubiquitin pathway is cleaved by caspases during apoptosis.

Ischemic brain injury (stroke) is a major cause of status epilepticus (SE). In our database of 529 adult SE cases, acute or remote cerebrovascular accidents (CVA) were a primary cause of SE for 41% of the patients overall and for 61% of the elderly patients. SE in the setting of acute CVA has a very high mortality, approaching 35%. The degree to which mortality can be attributed to the severity of the underlying CVA etiology vs. the effect of SE has not been evaluated. To address this issue, we prospectively studied patients with SE and acute CVA and compared them to control populations with acute CVA alone or with SE and remote CVA. The groups did not significantly differ with regard to age, sex, or radiographic lesion size. Mortality was unrelated to lesion size in the CVA and SE group. Overall, acute CVA and SE patients had an almost three-fold increase in mortality compared to the CVA group and an eight-fold increase compared to the SE and the non acute (remote) CVA group. Logistic regression analysis demonstrated a statistically significant synergistic effect of SE and CVA on mortality. This is the first study to document that the high mortality of SE and acute CVA is not solely due to the severity of the underlying CVA etiology, but due to the synergistic effect of combined injuries from SE and cerebral vascular ischemia.

Apoptosis is characterised by a precisely orchestrated sequence of morphological changes which result in the engulfment of the dying cell by macrophages in the absence of inflammation. These changes include cell shrinkage, plasma membrane blebbing, chromatin condensation, nuclear segmentation and formation of apoptotic bodies. The morphological features of apoptosis are highly conserved in multicellular organisms; however, the cellular signalling pathways involved in controlling apoptosis remain poorly defined and little is known about the biochemical mechanisms underlying the dramatic changes that accompany cell death. The importance of proteases in the execution of apoptosis has become increasingly apparent, in particular the cysteine proteases of the interleukin lβ-converting enzyme (ICE)-like family (caspases) (Martin and Green 1995; Kumar and Lavin 1996) and the serine proteases of the granzyme B family (Greenberg 1996). A role for calpain (Squier and Cohen 1996) and the proteasome has also been described (Grimm et al. 1996).