Xenotransplantation of mitochondrial electron transfer enzyme, Ndi1, in myocardial reperfusion injury

A significant consequence of ischemia/reperfusion (I/R) is mitochondrial respiratory dysfunction, leading to energetic deficits and cellular toxicity from reactive oxygen species (ROS). Mammalian complex I, a NADH-quinone oxidoreductase enzyme, is a multiple subunit enzyme that oxidizes NADH and pumps protons across the inner membrane. Damage to complex I leads to superoxide production which further damages complex I as well as other proteins, lipids and mtDNA. The yeast, S. cerevisiae, expresses internal rotenone insensitive NADH-quinone oxidoreductase (Ndi1); a single 56 kDa polypeptide which, like the multi-subunit mammalian complex I, serves as the entry site of electrons to the respiratory chain, but without proton pumping. Heterologous expression of Ndi1 in mammalian cells results in protein localization to the inner mitochondrial membrane which can function in parallel with endogenous complex I to oxidize NADH and pass electrons to ubiquinone. Expression of Ndi1 in HL-1 cardiomyocytes and in neonatal rat ventricular myocytes protected the cells from simulated ischemia/reperfusion (sI/R), accompanied by lower ROS production, and preservation of ATP levels and NAD+/NADH ratios. We next generated a fusion protein of Ndi1 and the 11aa protein transduction domain from HIV TAT. TAT-Ndi1 entered cardiomyocytes and localized to mitochondrial membranes. Furthermore, TAT-Ndi1 introduced into Langendorff-perfused rat hearts also localized to mitochondria. Perfusion of TAT-Ndi1 before 30 min no-flow ischemia and up to 2 hr reperfusion suppressed ROS production and preserved ATP stores. Importantly, TAT-Ndi1 infused before ischemia reduced infarct size by 62%; TAT-Ndi1 infused at the onset of reperfusion was equally cardioprotective. These results indicate that restoring NADH oxidation and electron flow at reperfusion can profoundly ameliorate reperfusion injury.     

Mismatch repair proteins are activators of toxic responses to chromium-DNA damage

Chromium(VI) is a toxic and carcinogenic metal that causes the formation of DNA phosphate-based adducts. Cr-DNA adducts are genotoxic in human cells, although they do not block replication in vitro. Here, we report that induction of cytotoxicity in Cr(VI)-treated human colon cells and mouse embryonic fibroblasts requires the presence of all major mismatch repair (MMR) proteins. Cr-DNA adducts lost their ability to block replication of Cr-modified plasmids in human colon cells lacking MLH1 protein. The presence of functional mismatch repair caused induction of p53-independent apoptosis associated with activation of caspases 2 and 7. Processing of Cr-DNA damage by mismatch repair resulted in the extensive formation of gamma-H2AX foci in G(2) phase, indicating generation of double-stranded breaks as secondary toxic lesions. Induction of gamma-H2AX foci was observed at 6 to 12 h postexposure, which was followed by activation of apoptosis in the absence of significant G(2) arrest. Our results demonstrate that mismatch repair system triggers toxic responses to Cr-DNA backbone modifications through stress mechanisms that are significantly different from those for other forms of DNA damage. Selection for Cr(VI) resistant, MMR-deficient cells may explain the very high frequency of lung cancers with microsatellite instability among chromate workers.     

Respiratory syncytial virus nonstructural proteins NS1 and NS2 mediate inhibition of Stat2 expression and alpha/beta interferon responsiveness

Respiratory syncytial virus (RSV) subverts the antiviral interferon (IFN) response, but the mechanism for this evasion was unclear. Here we show that RSV preferentially inhibits IFN-alpha/beta signaling by expression of viral NS1 and NS2. Thus, RSV infection or expression of recombinant NS1 and NS2 in epithelial host cells causes a marked decrease in Stat2 levels and the consequent downstream IFN-alpha/beta response. Similarly, NS1/NS2-deficient RSV no longer decreases Stat2 levels or IFN responsiveness. RSV infection decreased human but not mouse Stat2 levels, so this mechanism of IFN antagonism may contribute to viral host range, as well as immune subversion.     

Ilex paraguariensis extracts are potent inhibitors of nitrosative stress: a comparative study with green tea and wines using a protein nitration model and mammalian cell cytotoxicity

Due to the increasing importance of nitrosative stress in pathology and the efficacy displayed by flavonoids in cancelling the effects of peroxynitrite, we decided to conduct a comparative study of three commonly used beverages with the highest polyphenol contents and proven antioxidant properties: mate (Ilex paraguariensis); green tea (Camelia sinensis) extracts and white and red wines of the main varietals. We directly evaluated and compared the extracts and wines as protein nitration inhibitors using 3-nitrotyrosine as a biomarker, we studied the extracts as protectors from OONO-induced cytotoxicity in two mammalian cell lines. Both green tea and mate extracts have a high polyphenol content, in the case of Ip, its higher concentration and higher free radical quenching activity on the DPPH assay may be mainly due to the sui generis extraction procedure. When BSA was incubated in the presence of SIN-1, a time and dose dependent nitration of the protein is clearly shown. Co-incubation of BSA with Ip, green tea or red wines led to a dose dependent inhibition of the effect. Ip displayed the highest inhibitory activity, followed by red wines and the green tea. Dilutions as low as 1/1500 produced more than 80% inhibition of albumin nitration. When we studied peroxynitrite-induced cytotoxicity in murine RAW 264.7 macrophages and 31EG4 mammary cells., we found a potent, dose-dependent protective effect that was Ilex paraguariensis > red wines > green tea. Taken together, our results indicate that when the herbal preparations studied here are prepared the way they are usually drunk, Ip displays the highest inhibition of protein nitration, and the highest promotion of cell survival, whereas green tea or red wines display significant but lesser effects at the same concentrations. Further studies aiming at isolation of the active principles and assessment of their bioavailability are warranted.     

Evidence that osteogenic progenitor cells in the human tunica albuginea may originate from stem cells: implications for peyronie disease

Tissue ossification in Peyronie disease (commonly known as Peyronie's disease [PD]), a localized fibrotic lesion within the tunica albuginea (TA) of the penis, may result from osteogenic differentiation of fibroblasts, myofibroblasts, and/or adult stem cells in the TA, and may be triggered by chronic inflammation, oxidative stress, and profibrotic factors like transforming growth factor beta 1 (TGFB1). In this study, we have investigated whether cultures of cells from normal TA and PD plaques undergo osteogenesis, express markers for stem cells, and originate other cell lineages via processes modulated by TGFB1. We found that TA and PD cells in osteogenic medium (OM) expressed osteogenic markers, alkaline phosphatase, and osteopontin and underwent calcification. PD cells, but not TA cells, formed foci in soft agar that were positive for alkaline phosphatase and calcification and expressed the mRNAs for osteoblast-specific factors pleiotrophin and periostin and bone morphogenic protein 2. Both cultures expressed stem cell marker CD34 antigen but not protein tyrosine phosphatase, receptor type c. TA and PD cells expressed smooth-muscle cell markers smoothelin and transgelin. None of the cultures underwent adipogenesis in adipogenic medium. Incubation with TGFB1 increased osteogenesis and myofibroblast differentiation and reduced CD34 antigen expression in both cultures. TA and PD cells modulated the differentiation of the multipotent C3H 10T(1/2) cells in dual cultures, into osteoblasts and myofibroblasts. In conclusion, both TA and PD cultures contain cells, presumably stem cells, that undergo osteogenic and myofibroblast differentiation, and may induce these processes by paracrine interactions. This may explain progression of fibrosis in the PD plaque and its eventual calcification.     

Differences in expression of cell surface co-stimulatory molecules, Toll-like receptor genes and secretion of IL-12 by bone marrow-derived dendritic cells from susceptible and resistant mouse strains in response to Coccidioides posadasii

Coccidioides posadasii is a soil fungus that causes coccidioidomycosis or Valley Fever in the endemic regions of the southwestern US and Central America. Persons with decreased T cells reactivity and immune deficiency are at increased risk of developing severe disseminated infection. Among different mouse strains, DBA/2 mice are relatively resistant to C. posadasii whereas BALB/c mice are highly susceptible, and this discrepancy has been attributed to the difference in the development and expression of their Th1 cellular response. Dendritic cells (DC) are the most potent antigen-presenting cells that are activated after taking up pathogens or pathogens-derived antigens and regulate the immune response in the host, including Th1 cellular response. However, the DC responses against C. posadasii are not characterized. In the present study, we cultured bone-marrow derived DC (BMDC) from BALB/c and DBA/2 mice and infected with C. posadasii arthroconidia. The activation of BMDC was characterized by studying expression of cell surface co-stimulatory molecules (CD11c, MHC class II, CD40, CD80, and CD86), expression of genes encoding Toll-like receptors and release of IL-12. We found that the BMDC from DBA/2 mice showed significant upregulation of Toll-like receptor-2 and 4 genes expression, secretion of IL-12 (p<0.05) and modest increase in T cell co-stimulatory molecules as compared to BMDC from BALB/c mice. The data suggest that the differences in the activation status of DC in DBA/2 and BALB/c mice may be responsible for the discrepancy in their susceptibility to C. posadasii.     

Human nucleotide excision repair efficiently removes chromium-DNA phosphate adducts and protects cells against chromate toxicity

Intracellular reduction of carcinogenic Cr(VI) leads to the extensive formation of Cr(III)-DNA phosphate adducts. Repair mechanisms for chromium and other DNA phosphate-based adducts are currently unknown in human cells. We found that nucleotide excision repair (NER)-proficient human cells rapidly removed chromium-DNA adducts, with an average t((1/2)) of 7.1 h, whereas NER-deficient XP-A, XP-C, and XP-F cells were severely compromised in their ability to repair chromium-DNA lesions. Activation of NER in Cr(VI)-treated human fibroblasts or lung epithelial H460 cells was manifested by XPC-dependent binding of the XPA protein to the nuclear matrix, which was also observed in UV light-treated (but not oxidant-stressed) cells. Intracellular replication of chromium-modified plasmids demonstrated increased mutagenicity of binary Cr(III)-DNA and ternary cysteine-Cr(III)-DNA adducts in cells with inactive NER. NER deficiency created by the loss of XPA in fibroblasts or by knockdown of this protein by stable expression of small interfering RNA in H460 cells increased apoptosis and clonogenic death by Cr(VI), providing genetic evidence for the role of monofunctional chromium-DNA adducts in the toxic effects of this metal. The rate of NER of chromium-DNA adducts under saturating conditions was calculated to be approximately 50,000 lesions/min/cell. Because chromium-DNA adducts cause only small changes in the DNA helix, rapid repair of these modifications in human cells indicates that the presence of major structural distortions in DNA is not required for the efficient detection of the damaged sites by NER proteins in vivo.