Dystrophic cardiomyopathy: amplification of cellular damage by Ca2+ signalling and reactive oxygen species-generating pathways

AIMS: Cardiac myopathies are the second leading cause of death in patients with Duchenne and Becker muscular dystrophy, the two most common and severe forms of a disabling striated muscle disease. Although the genetic defect has been identified as mutations of the dystrophin gene, very little is known about the molecular and cellular events leading to progressive cardiac muscle damage. Dystrophin is a protein linking the cytoskeleton to a complex of transmembrane proteins that interact with the extracellular matrix. The fragility of the cell membrane resulting from the lack of dystrophin is thought to cause an excessive susceptibility to mechanical stress. Here, we examined cellular mechanisms linking the initial membrane damage to the dysfunction of dystrophic heart. METHODS AND RESULTS: Cardiac ventricular myocytes were enzymatically isolated from 5- to 9-month-old dystrophic mdx and wild-type (WT) mice. Cells were exposed to mechanical stress, applied as osmotic shock. Stress-induced cytosolic and mitochondrial Ca(2+) signals, production of reactive oxygen species (ROS), and mitochondrial membrane potential were monitored with confocal microscopy and fluorescent indicators. Pharmacological tools were used to scavenge ROS and to identify their possible sources. Osmotic shock triggered excessive cytosolic Ca(2+) signals, often lasting for several minutes, in 82% of mdx cells. In contrast, only 47% of the WT cardiomyocytes responded with transient and moderate intracellular Ca(2+) signals. On average, the reaction was 6-fold larger in mdx cells. Removal of extracellular Ca(2+) abolished these responses, implicating Ca(2+) influx as a trigger for abnormal Ca(2+) signalling. Our further experiments revealed that osmotic stress in mdx cells produced an increase in ROS production and mitochondrial Ca(2+) overload. The latter was followed by collapse of the mitochondrial membrane potential, an early sign of cell death. CONCLUSION: Overall, our findings reveal that excessive intracellular Ca(2+) signals and ROS generation link the initial sarcolemmal injury to mitochondrial dysfunctions. The latter possibly contribute to the loss of functional cardiac myocytes and heart failure in dystrophy. Understanding the sequence of events of dystrophic cell damage and the deleterious amplification systems involved, including several positive feed-back loops, may allow for a rational development of novel therapeutic strategies.

Cellular telephone use and time trends in brain tumour mortality in Switzerland from 1969 to 2002

A rising concern exists that with the widespread use of mobile communication technologies, the incidence of brain tumours may increase. On the basis of data from the Swiss national mortality registry from 1969 to 2002, annual age-standardized brain tumour mortality rates per 100,000 person-years were calculated using the European standard population. Time trend analyses were performed by the Poisson regression for six different age groups in men and women separately. The study period was divided into two intervals: before and after 1987, when the analogue mobile technology was introduced in Switzerland. Age-standardized brain tumour mortality rates ranged between 3.7 and 6.7 for men and 2.5 and 4.4 for women per 100,000 person-years. For the whole study period, a significant increase in brain tumour mortality was observed for men and women in the older age groups (60-74 and 75+ years) but not in the younger ones in whom mobile phone use was more prevalent. Time trend analyses restricted to data from 1987 onwards revealed relatively stable brain tumour mortality rates in all age groups. For instance, the annual change in brain tumour mortality rate for the 45-59-year age group was -0.3% (95% confidence interval: -1.7; 1.1) for men and -0.4% (95% confidence interval:-2.2; 1.3) for women. We conclude that after the introduction of mobile phone technology in Switzerland, brain tumour mortality rates remained stable in all age groups. Our results suggest that mobile phone use is not a strong risk factor in the short term for mortality from brain tumours. Ecological analyses like this, however, are limited in their ability to reveal potentially small increases in risk for diseases with a long latency period.

Interactions of nanoparticles with pulmonary structures and cellular responses

Combustion-derived and synthetic nano-sized particles (NSP) have gained considerable interest among pulmonary researchers and clinicians for two main reasons: 1) Inhalation exposure to combustion-derived NSP was associated with increased pulmonary and cardiovascular morbidity and mortality as suggested by epidemiological studies. Experimental evidence has provided a mechanistic picture of the adverse health effects associated with inhalation of combustion-derived and synthetic NSP. 2) The toxicological potential of NSP contrasts with the potential application of synthetic NSP in technological as well as medicinal settings with the latter including the use of NSP as diagnostics or therapeutics. In order to shed light on this paradox, this article aims to highlight recent findings about the interaction of inhaled NSP with the structures of the respiratory tract including surfactant and alveolar macrophages and epithelial cells. Cellular responses to NSP exposure include the generation of reactive oxygen species and the induction of an inflammatory response. Furthermore, this review places special emphasis on methodological differences between experimental studies and the caveats associated with the dose metrics and points out ways to overcome inherent methodological problems. Key words: electron tomography, surfactant, translocation, oxidative stress, inflammation.

Cellular immune response, stress resistance and competitiveness in nestling great tits in relation to maternally transmitted carotenoids

1. Egg yolks contain carotenoids that protect biological molecules against free-radical damage and promote maturation of the immune system. Availability of carotenoids to birds is often limited. Trade-offs can thus arise in the allocation of carotenoids to different physiological functions, and mothers may influence the immunocompetence of nestlings by modulating the transfer of carotenoid to the yolk.;2. In the great tit Parus major, we experimentally manipulated the dietary supply of carotenoid to mothers, and partially cross-fostered hatchlings to investigate the effect of an increased availability of carotenoids during egg laying on immunocompetence of nestlings.;3. In addition, we infested half of the nests with hen fleas Ceratophyllus gallinae to investigate the relationship between carotenoid availability, resistance to ectoparasites and immunocompetence.;4. We found that the procedure of cross-fostering can reduce the immune response of nestlings, but this effect can be compensated by the maternally transferred carotenoids. Cross-fostered nestlings of carotenoid-supplemented females show a similar immune response to non-cross-fostered nestlings, while cross-fostered nestlings of control females mounted a weaker cell-mediated immune response. This suggests that yolk carotenoids may help nestlings to cope with stress, for example the one generated by cross-fostering and/or they may enhance nestling competitiveness.;5. There was no statistically significant interaction between parasite and carotenoid treatments, as would be expected if carotenoids helped nestlings to fight parasites. Under parasite pressure, however, lighter nestlings raised a lower immune response, while the immune response was only weakly correlated with body mass in uninfested nests.

Influence of ABCB1, ABCC1, ABCC2, and ABCG2 haplotypes on the cellular exposure of nelfinavir in vivo

OBJECTIVES: The human immunodeficiency virus protease inhibitor nelfinavir is substrate of polyspecific drug transporters encoded by ABCB1 (P-glycoprotein), ABCC1 (MRP1) and ABCC2 (MRP2), and an inhibitor of BCRP, encoded by ABCG2. Genetic polymorphism in these genes may be associated with changes in transport function. METHODS: A comprehensive evaluation of single nucleotide polymorphisms (39 SNPs in ABCB1, 7 in ABCC1, 27 in ABCC2, and 16 in ABCG2), and inferred haplotypes was done to assess possible associations of genetic variants with cellular exposure of nelfinavir in vivo. Analysis used peripheral mononuclear cells from individuals receiving nelfinavir (n=28). Key results were re-examined in a larger sample size (n=129) contributing data on plasma drug levels. RESULTS AND CONCLUSIONS: There was no significant association between cellular nelfinavir area under the curve (AUC) and SNPs or haplotypes at ABCC1, ABCC2, ABCG2. There was an association with cellular exposure for two loci in strong linkage disequilibrium: ABCB1 3435C>T; AUCTT>AUCCT>AUCCC (ratio 2.1, 1.4, 1, Ptrend=0.01), and intron 26 +80T>C; AUCCC> AUCCT > AUCTT (ratio 2.4, 1.3, 1, Ptrend=0.006). Haplotypic analysis using tagging SNPs did not improve the single SNP association values.

Biological mediators and periodontal regeneration: a review of enamel matrix proteins at the cellular and molecular levels

BACKGROUND: Despite a large body of clinical and histological data demonstrating beneficial effects of enamel matrix proteins (EMPs) for regenerative periodontal therapy, it is less clear how the available biological data can explain the mechanisms underlying the supportive effects of EMPs. OBJECTIVE: To analyse all available biological data of EMPs at the cellular and molecular levels that are relevant in the context of periodontal wound healing and tissue formation. METHODS: A stringent systematic approach was applied using the key words "enamel matrix proteins" OR "enamel matrix derivative" OR "emdogain" OR "amelogenin". The literature search was performed separately for epithelial cells, gingival fibroblasts, periodontal ligament cells, cementoblasts, osteogenic/chondrogenic/bone marrow cells, wound healing, and bacteria. RESULTS: A total of 103 papers met the inclusion criteria. EMPs affect many different cell types. Overall, the available data show that EMPs have effects on: (1) cell attachment, spreading, and chemotaxis; (2) cell proliferation and survival; (3) expression of transcription factors; (4) expression of growth factors, cytokines, extracellular matrix constituents, and other macromolecules; and (5) expression of molecules involved in the regulation of bone remodelling. CONCLUSION: All together, the data analysis provides strong evidence for EMPs to support wound healing and new periodontal tissue formation.

Translocation and cellular entering mechanisms of nanoparticles in the respiratory tract

Anthropogenic nano-sized particles (NSP), ie, particles with a diameter of less than 100 nm, are generated with or without purpose as chemically and physically well-defined materials or as a consequence of combustion processes respectively. Inhalation of NSP occurs on a regular basis due to air pollution and is associated with an increase in respiratory and cardiovascular morbidity and mortality. Manufactured NSP may intentionally be inhaled as pharmaceuticals or unintentionally during production at the workplace. Hence the interactions of NSP with the respiratory tract are currently under intensive investigation. Due to special physicochemical features of NSP, its biological behaviour may differ from that of larger sized particles. Here we review two important themes of current research into the effects of NSP on the lungs: 1) The potential of NSP to cross the blood-air barrier of the lungs, thus gaining access to the circulation and extrapulmonary organs. It is currently accepted that a small fraction of inhaled NSP may translocate to the circulation. The significance of this translocation requires further research. 2) The entering mechanisms of NSP into different cell types. There is evidence that NSP are taken up by cells via well-known pathways of endocytosis but also via different mechanisms not well understood so far. Knowledge of the quantitative relationship between the different entering mechanisms and cellular responses is not yet available but is urgently needed in order to understand the effects of intentionally or unintentionally inhaled NSP on the respiratory tract.

Electron microscopy of high pressure frozen samples: bridging the gap between cellular ultrastructure and atomic resolution

Transmission electron microscopy has provided most of what is known about the ultrastructural organization of tissues, cells, and organelles. Due to tremendous advances in crystallography and magnetic resonance imaging, almost any protein can now be modeled at atomic resolution. To fully understand the workings of biological "nanomachines" it is necessary to obtain images of intact macromolecular assemblies in situ. Although the resolution power of electron microscopes is on the atomic scale, in biological samples artifacts introduced by aldehyde fixation, dehydration and staining, but also section thickness reduces it to some nanometers. Cryofixation by high pressure freezing circumvents many of the artifacts since it allows vitrifying biological samples of about 200 mum in thickness and immobilizes complex macromolecular assemblies in their native state in situ. To exploit the perfect structural preservation of frozen hydrated sections, sophisticated instruments are needed, e.g., high voltage electron microscopes equipped with precise goniometers that work at low temperature and digital cameras of high sensitivity and pixel number. With them, it is possible to generate high resolution tomograms, i.e., 3D views of subcellular structures. This review describes theory and applications of the high pressure cryofixation methodology and compares its results with those of conventional procedures. Moreover, recent findings will be discussed showing that molecular models of proteins can be fitted into depicted organellar ultrastructure of images of frozen hydrated sections. High pressure freezing of tissue is the base which may lead to precise models of macromolecular assemblies in situ, and thus to a better understanding of the function of complex cellular structures.

Long-term cellular and regional specificity of the photoreceptor toxin, iodoacetic acid (IAA), in the rabbit retina

This study investigated the anatomical consequences of a photoreceptor toxin, iodoacetic acid (IAA), in the rabbit retina. Retinae were examined 2 weeks, 1, 3, and 6 months after systemic IAA injection. The retinae were processed using standard histological methods to assess the gross morphology and topographical distribution of damage, and by immunohistochemistry to examine specific cell populations in the retina. Degeneration was restricted to the photoreceptors and was most common in the ventral retina and visual streak. In damaged regions, the outer nuclear layer was reduced in thickness or eliminated entirely, with a concomitant loss of immunoreactivity for rhodopsin. However, the magnitude of the effect varied between animals with the same IAA dose and survival time, suggesting individual differences in the bioavailability of the toxin. In all eyes, the inner retina remained intact, as judged by the thickness of the inner nuclear layer, and by the pattern of immunoreactivity for protein kinase C-alpha (rod bipolar cells) and calbindin D-28 (horizontal cells). Müller cell stalks became immunoreactive for glial fibrillary acidic protein (GFAP) even in IAA-treated retinae that had no signs of cell loss, indicating a response of the retina to the toxin. However, no marked hypertrophy or proliferation of Müller cells was observed with either GFAP or vimentin immunohistochemistry. Thus the selective, long lasting damage to the photoreceptors produced by this toxin did not lead to a reorganization of the surviving cells, at least with survival as long as 6 months, in contrast to the remodeling of the inner retina that is observed in inherited retinal degenerations such as retinitis pigmentosa and retinal injuries such as retinal detachment.

BIOCHEMICAL AND CELLULAR MECHANISMS OF RETINA AND RETINAL PIGMENT EPITHELIUM APOPTOSIS

Oxidative stress, intense light exposure and oxygen imbalances such as hypoxic or hyperoxic conditions perturb mitochondria, nuclear function and further lead to cellular damage of retina and retinal pigment epithelial (RPE) cells. Our major aim is to understand the various biochemical and proteomic events that occur during the progression of retina and RPE cell death. The comprehensive objectives of this dissertation are to understand the functional aspects of protein expression, posttranslational modifications, protein or lipid binding changes, phenotypic, morphological alterations and their regulation during the retina and RPE apoptosis under oxidative stress. The entire study is divided into four chapters Chapter 1 contains introduction and background on apoptotic signaling in retina and RPE cells. In chapter 2, we demonstrated that the oxidative stress biomarker prohibitin shuttles between mitochondria and nucleus as an anti-apoptotic molecule and acts as a transcriptional regulator by altering its lipid binding affinity and by posttranslational modifications during oxidative damage to the retina and RPE. In chapter 3, we demonstrated that oxidative and photo-oxidative stress induced nitric oxide regulates the RPE apoptosis by altering serine/threonine protein phosphatase 2A (PP2A) catalytic subunit, vimentin phosphorylation and Bcl xL expression regulation in the RPE cells in vitro. In chapter 4, we further analyzed the differential expression of prohibitin in the retina and RPE during oxidative stress, diabetic retinopathy (DR) and age-related macular degeneration (AMD) condition. Our analysis of postmortem retinas reveals that prohibitin is significantly increased in aged and AMD retina, and decreased in retinas of human diabetic retinopathy and RPE of AMD. Our study demonstrates that prohibitin levels determine the apoptotic signaling in the retina and RPE during retinal degenerative disease progression.

Investigating within-canopy variation of functional traits and cellular structure of sugar maple (Acer saccharum) leaves

Patterns of increasing leaf mass per area (LMA), area-based leaf nitrogen (Narea), and carbon isotope composition (δ13C) with increasing height in the canopy have been attributed to light gradients or hydraulic limitation in tall trees. Theoretical optimal distributions of LMA and Narea that scale with light maximize canopy photosynthesis; however, sub-optimal distributions are often observed due to hydraulic constraints on leaf development. Using observational, experimental, and modeling approaches, we investigated the response of leaf functional traits (LMA, density, thickness, and leaf nitrogen), leaf carbon isotope composition (δ13C), and cellular structure to light availability, height, and leaf water potential (Ψl) in an Acer saccharum forest to tease apart the influence of light and hydraulic limitations. LMA, leaf and palisade layer thickness, and leaf density were greater at greater light availability but similar heights, highlighting the strong control of light on leaf morphology and cellular structure. Experimental shading decreased both LMA and area-based leaf nitrogen (Narea) and revealed that LMA and Narea were more strongly correlated with height earlier in the growing season and with light later in the growing season. The supply of CO2 to leaves at higher heights appeared to be constrained by stomatal sensitivity to vapor pressure deficit (VPD) or midday leaf water potential, as indicated by increasing δ13C and VPD and decreasing midday Ψl with height. Model simulations showed that daily canopy photosynthesis was biased during the early growing season when seasonality was not accounted for, and was biased throughout the growing season when vertical gradients in LMA and Narea were not accounted for. Overall, our results suggest that leaves acclimate to light soon after leaf expansion, through an accumulation of leaf carbon, thickening of palisade layers and increased LMA, and reduction in stomatal sensitivity to Ψl or VPD. This period of light acclimation in leaves appears to optimize leaf function over time, despite height-related constraints early in the growing season. Our results imply that vertical gradients in leaf functional traits and leaf acclimation to light should be incorporated in canopy function models in order to refine estimates of canopy photosynthesis.

UPLC-ESI-TOFMS-based metabolomics and gene expression dynamics inspector self-organizing metabolomic maps as tools for understanding the cellular response to ionizing radiation

Global transcriptomic and proteomic profiling platforms have yielded important insights into the complex response to ionizing radiation (IR). Nonetheless, little is known about the ways in which small cellular metabolite concentrations change in response to IR. Here, a metabolomics approach using ultraperformance liquid chromatography coupled with electrospray time-of-flight mass spectrometry was used to profile, over time, the hydrophilic metabolome of TK6 cells exposed to IR doses ranging from 0.5 to 8.0 Gy. Multivariate data analysis of the positive ions revealed dose- and time-dependent clustering of the irradiated cells and identified certain constituents of the water-soluble metabolome as being significantly depleted as early as 1 h after IR. Tandem mass spectrometry was used to confirm metabolite identity. Many of the depleted metabolites are associated with oxidative stress and DNA repair pathways. Included are reduced glutathione, adenosine monophosphate, nicotinamide adenine dinucleotide, and spermine. Similar measurements were performed with a transformed fibroblast cell line, BJ, and it was found that a subset of the identified TK6 metabolites were effective in IR dose discrimination. The GEDI (Gene Expression Dynamics Inspector) algorithm, which is based on self-organizing maps, was used to visualize dynamic global changes in the TK6 metabolome that resulted from IR. It revealed dose-dependent clustering of ions sharing the same trends in concentration change across radiation doses. "Radiation metabolomics," the application of metabolomic analysis to the field of radiobiology, promises to increase our understanding of cellular responses to stressors such as radiation.