Sabine D. Klein

Mitochondrial Nitric-oxide Synthase Stimulation Causes Cytochromec Release from Isolated Mitochondria EVIDENCE FOR INTRAMITOCHONDRIAL PEROXYNITRITE FORMATION

Nitric oxide (NO) is synthesized by members of the NO synthase (NOS) family. Recently the existence of a mitochondrial NOS (mtNOS), its Ca2+ dependence, and its relevance for mitochondrial bioenergetics was reported (Ghafourifar, P., and Richter, C. (1997) FEBS Lett. 418, 291–296; Giulivi, C., Poderoso, J. J., and Boveris, A. (1998) J. Biol. Chem. 273, 11038–11043). Here we report on the possible involvement of mtNOS in apoptosis. We show that uptake of Ca2+ by mitochondria triggers mtNOS activity and causes the release of cytochrome c from isolated mitochondria in a Bcl-2-sensitive manner. mtNOS-induced cytochrome c release was paralleled by increased lipid peroxidation. The release of cytochrome c as well as increase in lipid peroxidation were prevented by NOS inhibitors, a superoxide dismutase mimic, and a peroxynitrite scavenger. We show that mtNOS-induced cytochromec release is not mediated via the mitochondrial permeability transition pore because the release was aggravated by cyclosporin A and abolished by blockade of mitochondrial calcium uptake by ruthenium red. We conclude that, upon Ca2+-induced mtNOS activation, peroxynitrite is formed within mitochondria, which causes the release of cytochrome c from isolated mitochondria, and we propose a mechanism by which elevated Ca2+ levels induce apoptosis.

Ceramide Induces Cytochrome c Release from Isolated Mitochondria IMPORTANCE OF MITOCHONDRIAL REDOX STATE

In the present study we show thatN-acetylsphingosine (C2-ceramide),N-hexanoylsphingosine (C6-ceramide), and, to a much lesser extent, C2-dihydroceramide induce cytochromec (cyto c) release from isolated rat liver mitochondria. Ceramide-induced cyto c release is prevented by preincubation of mitochondria with a low concentration (40 nm) of Bcl-2. The release takes place when cytoc is oxidized but not when it is reduced. Upon cytoc loss, mitochondrial oxygen consumption, mitochondrial transmembrane potential (ΔΨ), and Ca2+ retention are diminished. Incubation with Bcl-2 prevents, and addition of cytoc reverses the alteration of these mitochondrial functions. In ATP-energized mitochondria, ceramides do not alter ΔΨ, neither when cyto c is oxidized nor when it is reduced, ruling out a nonspecific disturbance by ceramides of mitochondrial membrane integrity. Furthermore, ceramides decrease the reducibility of cytoc. We conclude that the apoptogenic properties of ceramides are in part mediated via their interaction with mitochondrial cytoc followed by its release and that the redox state of cytoc influences its detachment by ceramide from the inner mitochondrial membrane.

Mitochondrial Nitric Oxide Synthase, Oxidative Stress and Apoptosis

Nitric oxide (NO) exerts a wide range of its biological properties via its interaction with mitochondria. By competing with O2, physiologically relevant concentrations of NO reversibly inhibit cytochrome oxidase and decrease O2 consumption, in a manner resembling a pharmacological competitive antagonism. The inhibition regulates many cellular functions, by e.g., regulating the synthesis of ATP and the formation of mitochondrial transmembrane potential (ΔΨ). NO regulates the oxygen consumption of both the NO-producing and the neighboring cells; thus, it can serve as autoregulator and paracrine modulator of the respiration. On the other hand, NO reacts avidly with superoxide anion (O2–) to produce the powerful oxidizing agent, peroxynitrite (ONOO–) which affects mitochondrial functions mostly in an irreversible manner. How mitochondria and cells harmonize the reversible effects of NO versus the irreversible effects of ONOO– will be discussed in this review article. The exciting recent finding of mitochondrial NO synthase will also be discussed.

Quantitative visualization of colloidal and intracellular gold nanoparticles by confocal microscopy

Gold nanoparticles (AuNPs) have the potential to become a versatile biomarker. For further use of AuNPs labeled with functionalized molecules, their visualization in biological systems by routine laboratory tools such as light microscopy is crucial. However, the size far below the diffraction limit affords specialized parameters for microscopical detection, which stimulated the current study, aimed to determine from which size onward AuNPs, either in dispersion or cell-associated, can be reliably detected by standard confocal microscopy. First, gold colloids of size-restricted fractions are examined in dispersion. At a minimum particle size of 60 nm, detection appears to be reliable. Particle counts in dilution series confirm these results by revealing single particle detection of 60-nm colloids. Second, AuNPs are visualized and quantified in cells, which interestingly cause a phase shift in the reflection of AuNPs. Gold mass spectroscopy confirms the number of AuNPs counted microscopically inside cells. Furthermore, it demonstrates for the first time a very high diffusion rate of 15-nm particles into the cells. In conclusion, the results back the suitability of confocal microscopy for the quantitative tracking of colloidal and intracellular gold nanoparticles sized 60 nm.

Heat-shock protein 70 attenuates nitric oxide-induced apoptosis in RAW macrophages by preventing cytochrome c release

Heat-shock protein (Hsp) 70 is an inhibitor of apoptosis and has been shown to protect against nitric oxide-mediated toxicity. To gain mechanistic insights into the actions of Hsp70, we stably transfected RAW 264.7 mouse macrophages with the human Hsp70 gene and investigated critical steps in the progression towards cell demise. Incubation of control and Hsp70-transfected macrophages with S-nitrosoglutathione induced accumulation of the tumour suppressor p53, expression of p21(WAF1/CIP1) (where WAF1 corresponds to wild-type p53-activated fragment 1 and CIP1 corresponds to cyclin-dependent kinase-interacting protein 1) and G(1) cell-cycle arrest. However, cytochrome c translocation to the cytosol and activation of caspase 9 and caspase 3 were markedly reduced in Hsp70-overexpressing cells. In addition, changes in nuclear morphology, as determined by Hoechst staining, and the appearance of cells in the sub-G(1) phase were diminished in Hsp70-overexpressing cells compared with controls. We conclude that, in macrophages, Hsp70 interferes with cytochrome c release from mitochondria and, thereby, prevents nitric oxide-induced apoptosis, but leaves p53 accumulation and interference in the cell cycle intact.

Toxicity of gold nanoparticles on somatic and reproductive cells.

Along with the number of potential applications for gold nanoparticles (AuNP) especially for medical and scientific purposes, the interest in possible toxic effects of such particles is rising. The general perception views nanosized gold colloids as relatively inert towards biological systems. However, a closer analysis of pertinent studies reveals a more complex picture. While the chemical compound of which the nanoparticles consists plays an important role, further biocompatibility determining aspects have been made out. The vast majority of trials concerning AuNP-toxicity were performed using somatic cell culture lines. The results show a considerable dependency of toxic effects on size, zeta potential and surface functionalisation. In vivo studies on this subject are still rare. Based on the existing data it can be assumed, that a dosage of under <400 µg Au/kg showed no untoward effects. If higher amounts were applied toxicity depended on route of administration and particle size. Since nanoparticles have been shown to cross reproduction-relevant biological barriers such as the blood-testicle and the placental barrier the question of their reprotoxicity arises. Yet data concerning this subject is far from adequate. Regarding gametes, recent experiments showed a dose-dependent sensitivity of spermatozoa towards AuNP. Oocytes have not yet been tested in that respect. Interestingly, so far no effects were detected on embryos after gold nanoparticle exposure. In conclusion, the biocompatibility of gold nanoparticles depends on a range of particle specific aspects as well as the choice of target tissue. Further clarification of such matters are subject to ongoing research.

Cerebral hemodynamic and oxygenation changes induced by inner and heard speech: a study combining functional near-infrared spectroscopy and capnography

Abstract. The aim of this study was to investigate the effects of inner and heard speech on cerebral hemodynamics and oxygenation in the anterior prefrontal cortex (PFC) using functional near-infrared spectroscopy and to test whether potential effects were caused by alterations in the arterial carbon dioxide pressure (PaCO2). Twenty-nine healthy adult volunteers performed six different tasks of inner and heard speech according to a randomized crossover design. During the tasks, we generally found a decrease in PaCO2 (only for inner speech), tissue oxygen saturation (StO2), oxyhemoglobin ([O2Hb]), total hemoglobin ([tHb]) concentration and an increase in deoxyhemoglobin concentration ([HHb]). Furthermore, we found significant relations between changes in [O2Hb], [HHb], [tHb], or StO2 and the participants’ age, the baseline PETCO2, or certain speech tasks. We conclude that changes in breathing during the tasks led to lower PaCO2 (hypocapnia) for inner speech. During heard speech, no significant changes in PaCO2 occurred, but the decreases in StO2, [O2Hb], and [tHb] suggest that changes in PaCO2 were also involved here. Different verse types (hexameter and alliteration) led to different changes in [tHb], implying different brain activations. In conclusion, StO2, [O2Hb], [HHb], and [tHb] are affected by interplay of both PaCO2 reactivity and functional brain activity.

Suicides in the Canton of Zurich (Switzerland)

QUESTIONS The Swiss Health Observatory recently reported that the Canton of Zurich ranked fourth highest among the Swiss cantons in regard to suicide rates. The aim of this study was to identify sociodemographic risk factors (such as sex, age, marital status, religion and place of residence) in relation to suicide rates in the Canton of Zurich, and to explore suicide methods and co-morbidities from all suicides in this area between 1995 and 2007. METHOD Data from Swiss statistics on causes of death were analysed using statistical modelling. RESULTS Suicides were found to be more frequent among men than women, and suicide was more prevalent in older or single people than in younger or married people, respectively. Age groups over 70 had increasingly high suicide rates compared to lower age groups. Investigating geographical distribution, highest suicide risks were found in the cities and high-income municipalities. The chosen suicide methods differed among age and sex groups. Poisoning was more than twice as frequent among women, and more common with increasing age in both sexes. Usage of firearms was about ten times as frequent among men. For train suicides, the age group of 10 to 19 years predominated. In 36% of suicide cases, mental disorders were listed as co-morbidities; in 12% malignant tumours were reported. CONCLUSIONS Relatively low rates of mental illness were reported in older suicide victims, despite a massively rising suicide rate with increase of age, indicating that undiagnosed depression could contribute to the high suicide rates in the elderly. Demographic changes in the coming years may result in larger numbers of suicides.

Injection of ligand-free gold and silver nanoparticles into murine embryos does not impact pre-implantation development

Summary Intended exposure to gold and silver nanoparticles has increased exponentially over the last decade and will continue to rise due to their use in biomedical applications. In particular, reprotoxicological aspects of these particles still need to be addressed so that the potential impacts of this development on human health can be reliably estimated. Therefore, in this study the toxicity of gold and silver nanoparticles on mammalian preimplantation development was assessed by injecting nanoparticles into one blastomere of murine 2 cell-embryos, while the sister blastomere served as an internal control. After treatment, embryos were cultured and embryo development up to the blastocyst stage was assessed. Development rates did not differ between microinjected and control groups (gold nanoparticles: 67.3%, silver nanoparticles: 61.5%, sham: 66.2%, handling control: 79.4%). Real-time PCR analysis of six developmentally important genes (BAX, BCL2L2, TP53, OCT4, NANOG, DNMT3A) did not reveal an influence on gene expression in blastocysts. Contrary to silver nanoparticles, exposure to comparable Ag+-ion concentrations resulted in an immediate arrest of embryo development. In conclusion, the results do not indicate any detrimental effect of colloidal gold or silver nanoparticles on the development of murine embryos.

Usage of complementary medicine across Switzerland Results of the Swiss Health Survey 2007

QUESTIONS UNDER STUDY This study investigated the use among the Swiss adult population and regional dissemination of various methods of complementary medicine (CM) provided by physicians or therapists in Switzerland. METHODS Data of the Swiss Health Survey 2007 were used, which comprised a telephone interview followed by a written questionnaire (18,760 and 14,432 respondents, respectively) and included questions about peoples state of health, health insurance and usage of health services. Users and non-users of CM were compared using logistic regression models. RESULTS The most popular CM methods were homeopathy, osteopathy, acupuncture and shiatsu/foot reflexology. 30.5% of women and 15.2% of men used at least one CM method in the 12 months preceding the survey. Lake Geneva region and central Switzerland had more CM users than the other regions. Women, people between 25 and 64 years of age and people with higher levels of education were more likely to use CM. 53.5% of the adult population had a supplemental health insurance for CM treatments. 32.9% of people with such an insurance used CM during the 12 months preceding the survey, and so did 12.0% of people without additional insurance. CONCLUSIONS Almost one fourth of the Swiss adult population had used CM within the past 12 months. User profiles were comparable to those in other countries. Despite a generally lower self-perceived health status, elderly people were less likely to use CM.