Regulation of cardiac myocyte cell death.

Cardiac myocyte death, whether through necrotic or apoptotic mechanisms, is a contributing factor to many cardiac pathologies. Although necrosis and apoptosis are the widely accepted forms of cell death, they may utilize the same cell death machinery. The environment within the cell probably dictates the final outcome, producing a spectrum of response between the two extremes. This review examines the probable mechanisms involved in myocyte death. Caspases, the generally accepted executioners of apoptosis, are significant in executing cardiac myocyte death, but other proteases (e.g., calpains, cathepsins) also promote cell death, and these are discussed. The two principal cell death pathways (death receptor- and mitochondrial-mediated) are described in relation to the emerging structural information for the principal proteins, and they are discussed relative to current understanding of myocyte cell death mechanisms. Whereas the mitochondrial pathway is probably a significant factor in myocyte death in both acute and chronic phases of myocardial diseases, the death receptor pathway may prove significant in the longer term. The Bcl-2 family of proteins are key regulators of the mitochondrial death pathway. These proteins are described and their possible functions are discussed. The commitment to cell death is also influenced by protein kinase cascades that are activated in the cell. Whereas certain pathways are cytoprotective (e.g., phosphatidylinositol 3'-kinase), the roles of other kinases are less clear. Since myocyte death is implicated in a number of cardiac pathologies, attenuation of the death pathways may prove important in ameliorating such disease states, and possible therapeutic strategies are explored.

Evidence of a possible role for Lys-gamma 3-MSH in the regulation of adipocyte function

Lys-gamma 3-MSH is a melanocortin peptide derived from the C-terminal of the 16 kDa fragment of POMC. The physiological role of Lys-gamma 3-MSH is unclear, although it has previously been shown that, although not directly steroidogenic, it can act to potentiate the steroidogenic response of adrenal cortical cells to ACTH. This synergistic effect appears to be correlated with an ability to increase the activity of hormone sensitive lipase (HSL) and therefore the rate of cholesterol ester hydrolysis. Ligand binding studies have suggested that high-affinity binding sites for Lys-gamma 3-MSH exist in the adrenal gland and a number of other rat tissues that express HSL, including adipose, skeletal muscle and testes. To investigate the hypothesis that Lys-gamma 3-MSH may play a wider role in cholesterol and lipid metabolism, we tested the effect of Lys-gamma 3-MSH on lipolysis, an HSL-mediated process, in 3T3-L1 adipocytes. In comparison with other melanocortin peptides, Lys-gamma 3-MSH was found to be a potent stimulator of lipolysis. It was also able to phosphorylate HSL at key serine residues and stimulate the hyper-phosphorylation of perilipin A. The receptor through which the lipolytic actions of Lys-gamma 3-MSH are being mediated is not clear. Attempts to characterise this receptor suggest that either the pharmacology of the melanocortin receptor 5 in 3T3-L1 adipocytes is different from that described when expressed in heterologous systems or the possibility that a further, as yet uncharacterised, receptor exists.

Endosomal endothelin-converting enzyme-1: a regulator of beta-arrestin-dependent ERK signaling

Neuropeptide signaling at the cell surface is regulated by metalloendopeptidases, which degrade peptides in the extracellular fluid, and beta-arrestins, which interact with G protein-coupled receptors (GPCRs) to mediate desensitization. beta-Arrestins also recruit GPCRs and mitogen-activated protein kinases to endosomes to allow internalized receptors to continue signaling, but the mechanisms regulating endosomal signaling are unknown. We report that endothelin-converting enzyme-1 (ECE-1) degrades substance P (SP) in early endosomes of epithelial cells and neurons to destabilize the endosomal mitogen-activated protein kinase signalosome and terminate signaling. ECE-1 inhibition caused endosomal retention of the SP neurokinin 1 receptor, beta-arrestins, and Src, resulting in markedly sustained ERK2 activation in the cytosol and nucleus, whereas ECE-1 overexpression attenuated ERK2 activation. ECE-1 inhibition also enhanced SP-induced expression and phosphorylation of the nuclear death receptor Nur77, resulting in cell death. Thus, endosomal ECE-1 attenuates ERK2-mediated SP signaling in the nucleus to prevent cell death. We propose that agonist availability in endosomes, here regulated by ECE-1, controls beta-arrestin-dependent signaling of endocytosed GPCRs.

The integration of lipid-sensing and anti-inflammatory effects: how the PPARs play a role in metabolic balance

The peroxisomal proliferating-activated receptors (PPARs) are lipid-sensing transcription factors that have a role in embryonic development, but are primarily known for modulating energy metabolism, lipid storage, and transport, as well as inflammation and wound healing. Currently, there is no consensus as to the overall combined function of PPARs and why they evolved. We hypothesize that the PPARs had to evolve to integrate lipid storage and burning with the ability to reduce oxidative stress, as energy storage is essential for survival and resistance to injury/infection, but the latter increases oxidative stress and may reduce median survival (functional longevity). In a sense, PPARs may be an evolutionary solution to something we call the 'hypoxia-lipid' conundrum, where the ability to store and burn fat is essential for survival, but is a 'double-edged sword', as fats are potentially highly toxic. Ways in which PPARs may reduce oxidative stress involve modulation of mitochondrial uncoupling protein (UCP) expression (thus reducing reactive oxygen species, ROS), optimising forkhead box class O factor (FOXO) activity (by improving whole body insulin sensitivity) and suppressing NFkB (at the transcriptional level). In light of this, we therefore postulate that inflammation-induced PPAR downregulation engenders many of the signs and symptoms of the metabolic syndrome, which shares many features with the acute phase response (APR) and is the opposite of the phenotype associated with calorie restriction and high FOXO activity. In genetically susceptible individuals (displaying the naturally mildly insulin resistant 'thrifty genotype'), suboptimal PPAR activity may follow an exaggerated but natural adipose tissue-related inflammatory signal induced by excessive calories and reduced physical activity, which normally couples energy storage with the ability to mount an immune response. This is further worsened when pancreatic decompensation occurs, resulting in gluco-oxidative stress and lipotoxicity, increased inflammatory insulin resistance and oxidative stress. Reactivating PPARs may restore a metabolic balance and help to adapt the phenotype to a modern lifestyle.

Analysis of molecular determinants of affinity and relative efficacy of a series of R- and S-2-(dipropylamino)tetralins at the 5-HT1A serotonin receptor

1 Factors influencing agonist affinity and relative efficacy have been studied for the 5-HT1A serotonin receptor using membranes of CHO cells expressing the human form of the receptor and a series of R-and S-2-(dipropylamino)tetralins (nonhydroxylated and monohydroxylated (5-OH, 6-OH, 7-OH, 8-OH) species). 2 Ligand binding studies were used to determine dissociation constants for agonist binding to the 5HT(1A) receptor: (a) K-i values for agonists were determined in competition versus the binding of the agonist [H-3]-8-OH DPAT. Competition data were all fitted best by a one-binding site model. (b) K-i values for agonists were also determined in competition versus the binding of the antagonist [H-3]-NAD-199. Competition data were all fitted best by a two-binding site model, and agonist affinities for the higher (K-h) and lower affinity (K-1) sites were determined. 3 The ability of the agonists to activate the 5-HT1A receptor was determined using stimulation of [S-35]-GTPgammaS binding. Maximal effects of agonists (E-max) and their potencies (EC50) were determined from concentration/response curves for stimulation of [S-35]-GTPgammaS binding. 4 K-1/K-h determined from ligand binding assays correlated with the relative efficacy (relative Em) of agonists determined in [S-35]-GTPgammaS binding assays. There was also a correlation between K-1/K-h and K-1/EC50 for agonists determined from ligand binding and [S-35]-GTPgammaS binding assays. 5 Simulations of agonist binding and effect data were performed using the Ternary Complex Model in order to assess the use of K-1/K-h for predicting the relative efficacy of agonists. British Journal of Pharmacology (2003) 138, 1129-1139. doi: 10. 1038/sj.bjp.705085.

Association between serotonin 5-HT-2C receptor gene (HTR2C) polymorphisms and obesity- and mental health-related phenotypes in a large population-based cohort

OBJECTIVE: Studies have shown that common single-nucleotide polymorphisms (SNPs) in the serotonin 5-HT-2C receptor (HTR2C) are associated with antipsychotic agent-induced weight gain and the development of behavioural and psychological symptoms. We aimed to analyse whether variation in the HTR2C is associated with obesity- and mental health-related phenotypes in a large population-based cohort. METHOD: Six tagSNPs, which capture all common genetic variation in the HTR2C gene, were genotyped in 4978 men and women from the European Prospective Investigation into Cancer (EPIC)-Norfolk study, an ongoing prospective population-based cohort study in the United Kingdom. To confirm borderline significant associations, the -759C/T SNP (rs3813929) was genotyped in the remaining 16 003 individuals from the EPIC-Norfolk study. We assessed social and psychological circumstances using the Health and Life Experiences Questionnaire. Genmod models were used to test associations between the SNPs and the outcomes. Logistic regression was performed to test for association of SNPs with obesity- and mental health- related phenotypes. RESULTS: Of the six HTR2C SNPs, only the T allele of the -759C/T SNP showed borderline significant associations with higher body mass index (BMI) (0.23 kg m(-2); (95% confidence interval (CI): 0.01-0.44); P=0.051) and increased risk of lifetime major depressive disorder (MDD) (Odds ratio (OR): 1.13 (95% CI: 1.01-1.22), P=0.02). The associations between the -759C/T and BMI and lifetime MDD were independent. As associations only achieved borderline significance, we aimed to validate our findings on the -759C/T SNP in the full EPIC-Norfolk cohort (n=20 981). Although the association with BMI remained borderline significant (beta=0.20 kg m(-2); 95% CI: 0.04-0.44, P=0.09), that with lifetime MDD (OR: 1.01; 95% CI: 0.94-1.09, P=0.73) was not replicated. CONCLUSIONS: Our findings suggest that common HTR2C gene variants are unlikely to have a major role in obesity- and mental health-related traits in the general population.

Tumour necrosis factor alpha induces rapid reduction in AMPA receptor-mediated calcium entry in motor neurones by increasing cell surface expression of the GluR2 subunit: relevance to neurodegeneration

The AMPA receptor (AMPAR) subunit GluR2, which regulates excitotoxicity and the inflammatory cytokine tumour necrosis factor alpha (TNF alpha) have both been implicated in motor neurone vulnerability in Amyotrophic Lateral Sclerosis/Motor Neurone Disease. TNF alpha has been reported to increase cell surface expression of AMPAR subunits to increase synaptic strength and enhance excitotoxicity, but whether this mechanism occurs in motor neurones is unknown. We used primary cultures of mouse motor neurones and cortical neurones to examine the interaction between TNF alpha receptor activation, GluR2 availability, AMPAR-mediated calcium entry and susceptibility to excitotoxicity. Short exposure to a physiologically relevant concentration of TNFalpha (10 ng/ml, 15 min) caused a marked redistribution of both GluR1 and GluR2 to the cell surface as determined by cell surface biotinylation and immunofluorescence. Using Fura-2 AM microfluorimetry we showed that exposure to TNFalpha caused a rapid reduction in the peak amplitude of AMPA-mediated calcium entry in a PI3-kinase and p38 kinase-dependent manner, consistent with increased insertion of GluR2-containing AMPAR into the plasma membrane. This resulted in a protection of motor neurones against kainate-induced cell death. Our data therefore, suggests that TNF alpha acts primarily as a physiological regulator of synaptic activity in motor neurones rather than a pathological drive in ALS

Modelling seasonally varying data: A case study for sudden infant death syndrome (SIDS)

Many time series are measured monthly, either as averages or totals, and such data often exhibit seasonal variability-the values of the series are consistently larger for some months of the year than for others. A typical series of this type is the number of deaths each month attributed to SIDS (Sudden Infant Death Syndrome). Seasonality can be modelled in a number of ways. This paper describes and discusses various methods for modelling seasonality in SIDS data, though much of the discussion is relevant to other seasonally varying data. There are two main approaches, either fitting a circular probability distribution to the data, or using regression-based techniques to model the mean seasonal behaviour. Both are discussed in this paper.

Life and death in a civitas capital: metabolic disease and trauma in the children from late Roman Dorchester, Dorset

The impact that “Romanization” and the development of urban centers had on the health of the Romano-British population is little understood. A re-examination of the skeletal remains of 364 nonadults from the civitas capital at Roman Dorchester (Durnovaria) in Dorset was carried out to measure the health of the children living in this small urban area. The cemetery population was divided into two groups; the first buried their dead organized within an east–west alignment with possible Christian-style graves, and the second with more varied “pagan” graves, aligned north–south. A higher prevalence of malnutrition and trauma was evident in the children from Dorchester than in any other published Romano-British group, with levels similar to those seen in postmedieval industrial communities. Cribra orbitalia was present in 38.5% of the children, with rickets and/or scurvy at 11.2%. Twelve children displayed fractures of the ribs, with 50% of cases associated with rickets and/or scurvy, suggesting that rib fractures should be considered during the diagnosis of these conditions. The high prevalence of anemia, rickets, and scurvy in the Poundbury children, and especially the infants, indicates that this community may have adopted child-rearing practices that involved fasting the newborn, a poor quality weaning diet, and swaddling, leading to general malnutrition and inadequate exposure to sunlight. The Pagan group showed no evidence of scurvy or rib fractures, indicating difference in religious and child-rearing practices but that both burial groups were equally susceptible to rickets and anemia suggests a shared poor standard of living in this urban environment.

Molecule modeling of human pentameric alpha(7) neuronal nicotinic acetylcholine receptor and its interaction with its agonist and competitive antagonist

The nicotinic Acetylcholine Receptor (nAChR) is the major class of neurotransmitter receptors that is involved in many neurodegenerative conditions such as schizophrenia, Alzheimer's and Parkinson's diseases. The N-terminal region or Ligand Binding Domain (LBD) of nAChR is located at pre- and post-synaptic nervous system, which mediates synaptic transmission. nAChR acts as the drug target for agonist and competitive antagonist molecules that modulate signal transmission at the nerve terminals. Based on Acetylcholine Binding Protein (AChBP) from Lymnea stagnalis as the structural template, the homology modeling approach was carried out to build three dimensional model of the N-terminal region of human alpha(7)nAChR. This theoretical model is an assembly of five alpha(7) subunits with 5 fold axis symmetry, constituting a channel, with the binding picket present at the interface region of the subunits. alpha-netlrotoxin is a potent nAChR competitive antagonist that readily blocks the channel resulting in paralysis. The molecular interaction of alpha-Bungarotoxin, a long chain alpha-neurotoxin from (Bungarus multicinctus) and human alpha(7)nAChR seas studied. Agonists such as acetylcholine, nicotine, which are used in it diverse array of biological activities, such as enhancements of cognitive performances, were also docked with the theoretical model of human alpha(7)nAChR. These docked complexes were analyzed further for identifying the crucial residues involved in interaction. These results provide the details of interaction of agonists and competitive antagonists with three dimensional model of the N-terminal region of human alpha(7)nAChR and thereby point to the design of novel lead compounds.

Fas ligand-induced apoptosis is regulated by nitric oxide through the inhibition of fas receptor clustering and the nitrosylation of protein kinase Cepsilon

Apoptosis induced by the death-inducing ligand FasL (CD95L) is a major mechanism of cell death. Trophoblast cells express the Fas receptor yet survive in an environment that is rich in the ligand. We report that basal nitric oxide (NO) production is responsible for the resistance of trophoblasts to FasL-induced apoptosis. In this study we demonstrate that basal NO production resulted in the inhibition of receptor clustering following ligand binding. In addition NO also protected cells through the selective nitrosylation, and inhibition, of protein kinase Cepsilon (PKCepsilon) but not PKCalpha. In the absence of NO production PKCepsilon interacted with, and phosphorylated, the anti-apoptotic protein cFLIP. The interaction is predominantly with the short form of cFLIP and its phosphorylation reduces its recruitment to the death-inducing signaling complex (DISC) that is formed following binding of a death-inducing ligand to its receptor. Inhibition of cFLIP recruitment to the DISC leads to increased activation of caspase 8 and subsequently to apoptosis. Inhibition of PKCepsilon using siRNA significantly reversed the sensitivity to apoptosis induced by inhibition of NO synthesis suggesting that NO-mediated inhibition of PKCepsilon plays an important role in the regulation of Fas-induced apoptosis.

AT(1) receptor ligands: virtual-screening-based design with TOPP descriptors, synthesis, and biological evaluation of pyrrolidine derivatives

As a continuing effort to establish the structure-activity relationships (SARs) within the series of the angiotensin II antagonists (sartans), a pharmacophoric model was built by using novel TOPP 3D descriptors. Statistical values were satisfactory (PC4: r(2)=0.96, q(2) ((5) (random) (groups))=0.84; SDEP=0.26) and encouraged the synthesis and consequent biological evaluation of a series of new pyrrolidine derivatives. SAR together with a combined 3D quantitative SAR and high-throughput virtual screening showed that the newly synthesized 1-acyl-N-(biphenyl-4-ylmethyl)pyrrolidine-2-carboxamides may represent an interesting starting point for the design of new antihypertensive agents. In particular, biological tests performed on CHO-hAT(1) cells stably expressing the human AT(1) receptor showed that the length of the acyl chain is crucial for the receptor interaction and that the valeric chain is the optimal one.

Gonadotrophins modulate hormone secretion and steady-state mRNA levels for activin receptors (type I, IIA, IIB) and inhibin co-receptor (betaglycan) in granulosa and theca cells from chicken prehierarchical and preovulatory follicles

Ovarian follicle development is regulated through endocrine and local mechanisms. Increasing evidence indicates roles for transforming growth factor beta superfamily members, including inhibins and activins. We recently identified divergent expression of mRNAs encoding activin receptors (ActR) and inhibin co-receptor betaglycan in chicken follicles at different stages of maturation. Here, we compare the actions of LH and FSH (0, 1, 10, 100 ng/ml) on levels of mRNA for ActRI, ActRIIA, ActRIIB and betaglycan in chicken granulosa and theca cells (GC and TC) from preovulatory (F1) and prehierarchical (6-8 mm) follicles. The expression of mRNAs for LH-R and FSH-R and production of inhibin A, oestradiol and progesterone were also quantified. FSH decreased ActRIIB and ActRI mRNA levels in 6-8 mm GC, whereas LH increased the mRNA levels. Both LH and FSH enhanced ActRIIA (5- and 8.5-fold) and betaglycan mRNA expression (2- and 3.5-fold) in 6-8 mm GC. In 6-8 mm TC, LH and FSH both increased the betaglycan mRNA level (7- and 3.5-fold respectively) but did not affect ActRI, ActRIIA and ActRIIB transcript levels. In F1 GC, both LH and FSH stimulated ActRI (2- and 2.4-fold), ActRIIB (3.2- and 2.7-fold) and betaglycan (7- and 4-fold) mRNA levels, while ActRIIA mRNA was unaffected. In F1 TC, LH and FSH reduced ActRIIA (35-50%) and increased (4.5- and 7.6-fold) betaglycan mRNA, but had no effect on ActRI and ActRIIB transcript levels. Results support the hypothesis that expression of ActR and betaglycan are differentially regulated by gonadotrophins during follicle maturation in the hen. This may represent an important mechanism for fine-tuning follicle responsiveness to local and systemic activins and inhibins.

Differential expression of mRNAs encoding co-receptor (betaglycan) and activin type-I preovulatory and prehierarchical follicles of the putative inhibin and type-II receptors in the laying hen ovary

Ovarian follicle development is primarily regulated by an interplay between the pituitary gonadotrophins, LH and FSH, and ovary-derived steroids. Increasing evidence implicates regulatory roles of transforming growth factor-beta (TGF beta) superfamily members, including inhibins and activins. The aim of this study was to identify the expression of mRNAs encoding key receptors of the inhibin/activin system in ovarian follicles ranging from 4 mm in diameter to the dominant F1 follicle (similar to 40 turn). Ovaries were collected (n=16) from inid-sequence hens maintained on a long-day photoschedule (16h of light:8 h of darkness). All follicles removed were dissected into individual granulosa and thecal layers. RNA was extracted and cDNA synthesized. Real-time quantitative PCR was used to quantify the expression of niRNA encoding betaglycan, activin receptor (ActR) subtypes (type-I, -IIA and -IIB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH); receptor expression data were normalized to GAPDH expression. Detectable levels of ActRI, -IIA and -IIB and the inhibin co-receptor (betaglycan) expression were found in all granulosa and thecal layers analysed. Granulosa ActRI mRNA peaked (P < 0(.)05) in 8-9(.)9 mm follicles, whereas ActRIIA rose significantly from 6-7(.)9 mm to 8-9(.)9 nun, before filling to F3/2; levels then rose sharply (3-fold) to F1 levels. Granulosa betaglycan niRNA expression rose 3-fold from 4-5(.)9 min to 8-9(.)9 mm, before falling 4-fold to F3/2; levels then rose sharply (4-fold) to F1 levels. ActRIIB levels did not vary significantly during follicular development. Thecal ActRI mRNA expression was similar from 4-7(.)9 mm then decreased significantly to a nadir at the F4 position, before increasing 2-fold to the F1 (P < 0(.)05). Although thecal ActRIIB and -IIA expression did not vary significantly from 4 nim to F3, ActRIIB expression increased significantly (2-fold) from F3 to F1 and ActIIA, increased 22-fold from F2 to F1 (P < 0(.)05). Thecal betaglycan fell to a nadir at F6 after follicle selection; levels then increased significantly to F2, before filling similar to 50% in the F I. In all follicles studied expression of betaglycan and ActRI (granulosa: 1-0(.)65, P < 0-001, n=144/group; theca: r=0(.)49, P < 0-001, n=144/group) was well correlated. No significant correlations were identified between betaglycan and ActRIIA or -IIB. Considering all follicles analysed, granulosa mRNA expression of betaglycan, ActRI ActRIIA and ActRIIB were all significantly lower than in corresponding thecal tissue (betaglycan, 11(.)4-fold; ActRIIB, 5(.)1-fold; ActR(.) 3-8-fold: ActRIIA, 2(.)8-fold). The co-localization of type-I and -II activin receptors and betaglycan on granulosa and thecal cells are consistent with a local auto/paracrine role of inhibins and activins in modulating ovarian follicle development, selection and progression in the domestic fowl.