Mitochondria are devoid of poly(ADP-ribose)polymerase-1, but harbor its product oligo(ADP-ribose).

There are conflicting data about localization of poly(ADP-ribose)polymerase-1 and its product poly(ADP-ribose) in mitochondria. To finally clarify the discussion, we investigated with biochemical and cell biological methods the potential presence of poly(ADP-ribose) polymerase-1 in these organelles. Our data show that endogenous and overexpressed poly(ADP-ribose)polymerase 1 is only localized to the nucleus with a clear exclusion of cytosolic compartments. In addition, highly purified mitochondria devoid of nuclear contaminations do not contain poly(ADP-ribose)polymerase-1. Although no poly(ADP-ribose)polymerase-1 enzyme is detectable in mitochondria, a shorter variant of its product poly(ADP-ribose) is present, associated specifically with a small subset of mitochondrial proteins as revealed by immunoprecipitation and protein fingerprint analysis. These proteins are located at key-points of the Krebs-cycle, are chaperones involved in mitochondrial functionality and quality-control, and are RNA-binding proteins important for transcript stability, respectively. Of note, despite the fact that especially poly(ADP-ribose)polymerase-1 is its own major target for modification, we could not detect this enzyme by mass spectrometry in these organelles. These data suggests a new way of targeted nuclear-mitochondrial signaling, mediated by nuclear poly(ADP-ribosyl)ation dependent on poly(ADP-ribose)polymerase-1.

Cytosolic Ca2+ shifts as early markers of cytotoxicity.

The determination of the cytotoxic potential of new and so far unknown compounds as well as their metabolites is fundamental in risk assessment. A variety of strategic endpoints have been defined to describe toxin-cell interactions, leading to prediction of cell fate. They involve measurement of metabolic endpoints, bio-energetic parameters or morphological cell modifications. Here, we evaluated alterations of the free cytosolic Ca2+ homeostasis using the Fluo-4 dye and compared results with the metabolic cell viability assay Alamar Blue. We investigated a panel of toxins (As2O3, gossypol, H2O2, staurosporine, and titanium(IV)-salane complexes) in four different mammalian cell lines covering three different species (human, mouse, and African green monkey). All tested compounds induced an increase in free cytosolic Ca2+ within the first 5 s after toxin application. Cytosolic Ca2+ shifts occurred independently of the chemical structure in all tested cell systems and were persistent up to 3 h. The linear increase of free cytosolic Ca2+ within the first 5 s of drug treatment correlates with the EC25 and EC75 values obtained in Alamar Blue assays one day after toxin exposure. Moreover, a rise of cytosolic Ca2+ was detectable independent of induced cell death mode as assessed by caspase and poly(ADP-ribose) polymerase (PARP) activity in HeLa versus MCF-7 cells at very low concentrations. In conclusion, a cytotoxicity assay based on Ca2+ shifts has a low limit of detection (LOD), is less time consuming (at least 24 times faster) compared to the cell viability assay Alamar Blue and is suitable for high-troughput-screening (HTS).

The ups and downs of tannins as inhibitors of poly(ADP-ribose)glycohydrolase.

DNA damage to cells activates nuclear poly(ADP-ribose)polymerases (PARPs) and the poly(ADP-ribose) (PAR) synthesized is rapidly cleaved into ADP-ribose (ADPR) by PAR glycohydrolase (PARG) action. Naturally appearing tannin-like molecules have been implicated in specific inhibition of the PARG enzyme. This review deals with the in vitro and in vivo effects of tannins on PAR metabolism and their downstream actions in DNA damage signaling.

Water Filtered Infrared A and Visible Light (wIRA/VIS) Irradiation Reduces Chlamydia trachomatis Infectivity Independent of Targeted Cytokine Inhibition.

Chlamydia trachomatis is the major cause of infectious blindness and represents the most common bacterial sexually transmitted infection worldwide. Considering the potential side effects of antibiotic therapy and increasing threat of antibiotic resistance, alternative therapeutic strategies are needed. Previous studies showed that water filtered infrared A alone (wIRA) or in combination with visible light (wIRA/VIS) reduced C. trachomatis infectivity. Furthermore, wIRA/VIS irradiation led to secretion of pro-inflammatory cytokines similar to that observed upon C. trachomatis infection. We confirmed the results of previous studies, namely that cytokine secretion (IL-6, IL-8, and RANTES/CCL5) upon wIRA/VIS treatment, and the subsequent reduction of chlamydial infectivity, are independent of the addition of cycloheximide, a host protein synthesis inhibitor. Reproducible cytokine release upon irradiation indicated that cytokines might be involved in the anti-chlamydial mechanism of wIRA/VIS. This hypothesis was tested by inhibiting IL-6, IL-8, and RANTES secretion in C. trachomatis or mock-infected cells by gene silencing or pharmaceutical inhibition. Celastrol, a substance derived from Trypterygium wilfordii, used in traditional Chinese medicine and known for anti-cancer and anti-inflammatory effects, was used for IL-6 and IL-8 inhibition, while Maraviroc, a competitive CCR5 antagonist and anti-HIV drug, served as a RANTES/CCL5 inhibitor. HeLa cell cytotoxicity and impact on chlamydial morphology, size and inclusion number was evaluated upon increasing inhibitor concentration, and concentrations of 0.1 and 1 µM Celastrol and 10 and 20 µM Maraviroc were subsequently selected for irradiation experiments. Celastrol at any concentration reduced chlamydial infectivity, an effect only observed for 20 µM Maraviroc. Triple dose irradiation (24, 36, 40 hpi) significantly reduced chlamydial infectivity regardless of IL-6, IL-8, or RANTES/CCL5 gene silencing, Celastrol or Maraviroc treatment. Neither gene silencing nor pharmaceutical cytokine inhibition provoked the chlamydial stress response. The anti-chlamydial effect of wIRA/VIS is independent of cytokine inhibition under all conditions evaluated. Thus, factors other than host cell cytokines must be involved in the working mechanism of wIRA/VIS. This study gives a first insight into the working mechanism of wIRA/VIS in relation to an integral component of the host immune system and supports the potential of wIRA/VIS as a promising new tool for treatment in trachoma.

Poly(ADP-ribose) glycohydrolase silencing protects against H2O2-induced cell death.

PAR [poly(ADP-ribose)] is a structural and regulatory component of multiprotein complexes in eukaryotic cells. PAR catabolism is accelerated under genotoxic stress conditions and this is largely attributable to the activity of a PARG (PAR glycohydrolase). To overcome the early embryonic lethality of parg-knockout mice and gain more insights into the biological functions of PARG, we used an RNA interference approach. We found that as little as 10% of PARG protein is sufficient to ensure basic cellular functions: PARG-silenced murine and human cells proliferated normally through several subculturing rounds and they were able to repair DNA damage induced by sublethal doses of H2O2. However, cell survival following treatment with higher concentrations of H2O2 (0.05-1 mM) was increased. In fact, PARG-silenced cells were more resistant than their wild-type counterparts to oxidant-induced apoptosis while exhibiting delayed PAR degradation and transient accumulation of ADP-ribose polymers longer than 15-mers at early stages of drug treatment. No difference was observed in response to the DNA alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine, suggesting a specific involvement of PARG in the cellular response to oxidative DNA damage.

Water-filtered infrared A reduces chlamydial infectivity in vitro without causing ex vivo eye damage in pig and mouse models.

Repeated ocular infections with Chlamydia trachomatis trigger the development of trachoma, the most common cause of infectious blindness worldwide. Water-filtered infrared A (wIRA) has shown positive effects on cultured cells and human skin. Our aim was to evaluate the potential of wIRA as a possible non-chemical treatment for trachoma patients. We both modeled ocular chlamydial infections using C. trachomatis B to infect human conjunctival epithelial cells (HCjE) and studied the effects of wIRA on non-infected ocular structures with two ex vivo eye models. We focused on the temperature development during wIRA irradiation in cell culture and perfused pig eyes to exclude potentially harmful side effects. Furthermore, cell viability of HCjE and cytotoxicity in mouse retina explants was analyzed. We demonstrated a significant wIRA-dependent reduction of chlamydial infectivity in HCjE cells. Moreover, we observed that wIRA treatment of HCjE prior to infection was sufficient to inhibit chlamydial infectivity and that visible light enhances the effect of wIRA. Irradiation did not reduce cell viability and there was no indication of retinal damage post treatment. Additionally, temperatures during wIRA exposure did not markedly exceed physiological eye temperatures, suggesting that hyperthermia-related lesions are unlikely. For clinical applications, further exploration of wIRA as a non-chemical treatment device in an experimental animal model is essential.

The contribution of temperature, exposure intensity and visible light to the inhibitory effect of irradiation on acute chlamydial infection.

Water-filtered infrared A (wIRA) is radiation with a spectrum ranging from 780 to 1400 nm. Chlamydiaceae are obligate intracellular bacteria associated with various diseases in both animals and humans. A recent in vitro study demonstrated that wIRA combined with visible light (wIRA/VIS) has potential as a non-chemical method for the treatment of chlamydial infections without adversely affecting the cell viability. The aim of this study was to investigate the influence of various factors on the effect of wIRA/VIS on acute chlamydial infection, namely the impact of temperature, exposure intensity and infectious dose (multiplicity of infection) as well as the efficacy of the visible light component.We demonstrate that non-thermal effects contribute to the inhibition of acute chlamydial infection. Visible light enhances the inhibitory effect of wIRA on extracellular bacteria (elementary bodies or EBs).Moreover, the inhibitory effect of wIRA/VIS following treatment of EBs prior to infection correlated with increased irradiation intensity. The infectivity of mature chlamydial inclusions was significantly reduced upon wIRA/VIS exposure at all irradiation intensities investigated, suggesting the contribution of host cell factors to the anti-chlamydial effect of wIRA/VIS in the late stage of the developmental cycle. The effect of irradiation was not influenced by the infectious dose.

Water-filtered infrared a irradiation in combination with visible light inhibits acute chlamydial infection.

New therapeutic strategies are needed to overcome drawbacks in treatment of infections with intracellular bacteria. Chlamydiaceae are Gram-negative bacteria implicated in acute and chronic diseases such as abortion in animals and trachoma in humans. Water-filtered infrared A (wIRA) is short wavelength infrared radiation with a spectrum ranging from 780 to 1400 nm. In clinical settings, wIRA alone and in combination with visible light (VIS) has proven its efficacy in acute and chronic wound healing processes. This is the first study to demonstrate that wIRA irradiation combined with VIS (wIRA/VIS) diminishes recovery of infectious elementary bodies (EBs) of both intra- and extracellular Chlamydia (C.) in two different cell lines (Vero, HeLa) regardless of the chlamydial strain (C. pecorum, C. trachomatis serovar E) as shown by indirect immunofluorescence and titration by subpassage. Moreover, a single exposure to wIRA/VIS at 40 hours post infection (hpi) led to a significant reduction of C. pecorum inclusion frequency in Vero cells and C. trachomatis in HeLa cells, respectively. A triple dose of irradiation (24, 36, 40 hpi) during the course of C. trachomatis infection further reduced chlamydial inclusion frequency in HeLa cells without inducing the chlamydial persistence/stress response, as ascertained by electron microscopy. Irradiation of host cells (HeLa, Vero) neither affected cell viability nor induced any molecular markers of cytotoxicity as investigated by Alamar blue assay and Western blot analysis. Chlamydial infection, irradiation, and the combination of both showed a similar release pattern of a subset of pro-inflammatory cytokines (MIF/GIF, Serpin E1, RANTES, IL-6, IL-8) and chemokines (IL-16, IP-10, ENA-78, MIG, MIP-1α/ß) from host cells. Initial investigation into the mechanism indicated possible thermal effects on Chlamydia due to irradiation. In summary, we demonstrate a non-chemical reduction of chlamydial infection using the combination of water-filtered infrared A and visible light.

The Sound of Silence: RNAi in Poly (ADP-Ribose) Research.

Poly(ADP-ribosyl)-ation is a nonprotein posttranslational modification of proteins and plays an integral part in cell physiology and pathology. The metabolism of poly(ADP-ribose) (PAR) is regulated by its synthesis by poly(ADP-ribose) polymerases (PARPs) and on the catabolic side by poly(ADP-ribose) glycohydrolase (PARG). PARPs convert NAD+ molecules into PAR chains that interact covalently or noncovalently with target proteins and thereby modify their structure and functions. PAR synthesis is activated when PARP1 and PARP2 bind to DNA breaks and these two enzymes account for almost all PAR formation after genotoxic stress. PARG cleaves PAR molecules into free PAR and finally ADP-ribose (ADPR) moieties, both acting as messengers in cellular stress signaling. In this review, we discuss the potential of RNAi to manipulate the levels of PARPs and PARG, and consequently those of PAR and ADPR, and compare the results with those obtained after genetic or chemical disruption.

Cell death and autophagy under oxidative stress: roles of poly(ADP-Ribose) polymerases and Ca(2+).

On the cellular level, oxidative stress may cause various responses, including autophagy and cell death. All of these outcomes involve disturbed Ca(2+) signaling. Here we show that the nuclear enzymes poly(ADP-ribose) polymerase 1 (PARP1) and PARP2 control cytosolic Ca(2+) shifts from extracellular and intracellular sources associated with autophagy or cell death. The different Ca(2+) signals arise from the transient receptor potential melastatin 2 (TRPM2) channels located in the cellular and lysosomal membranes. They induce specific stress kinase responses of canonical autophagy and cell death pathways. Autophagy is under the control of PARP1, which operates as an autophagy suppressor after oxidative stress. Cell death is activated downstream of extracellular signal-regulated kinase 1/2 (ERK1/2) and AKT, whereas cell survival correlates with the phosphorylation of p38, stress-activated protein kinase/Jun amino-terminal kinase (SAPK/JNK), and cyclic AMP response element-binding protein (CREB) with its activating transcription factor (ATF-1). Our results highlight an important role for PARP1 and PARP2 in the epigenetic control of cell death and autophagy pathways.