Hydrogen Sulfide Protects HUVECs against Hydrogen Peroxide Induced Mitochondrial Dysfunction and Oxidative Stress

10.1371/journal.pone.0053147PLoS ONE82

Nucleocytoplasmic transport: a thermodynamic mechanism

The nuclear pore supports molecular communication between cytoplasm and nucleus in eukaryotic cells. Selective transport of proteins is mediated by soluble receptors, whose regulation by the small GTPase Ran leads to cargo accumulation in, or depletion from the nucleus, i.e., nuclear import or nuclear export. We consider the operation of this transport system by a combined analytical and experimental approach. Provocative predictions of a simple model were tested using cell-free nuclei reconstituted in Xenopus egg extract, a system well suited to quantitative studies. We found that accumulation capacity is limited, so that introduction of one import cargo leads to egress of another. Clearly, the pore per se does not determine transport directionality. Moreover, different cargo reach a similar ratio of nuclear to cytoplasmic concentration in steady-state. The model shows that this ratio should in fact be independent of the receptor-cargo affinity, though kinetics may be strongly influenced. Numerical conservation of the system components highlights a conflict between the observations and the popular concept of transport cycles. We suggest that chemical partitioning provides a framework to understand the capacity to generate concentration gradients by equilibration of the receptor-cargo intermediary.Comment: in press at HFSP Journal, vol 3 16 text pages, 1 table, 4 figures, plus Supplementary Material include

Features of programmed cell death in intact Xenopus oocytes and early embryos revealed by near-infrared fluorescence and real-time monitoring.

Factors influencing apoptosis of vertebrate eggs and early embryos have been studied in cell-free systems and in intact embryos by analyzing individual apoptotic regulators or caspase activation in static samples. A novel method for monitoring caspase activity in living Xenopus oocytes and early embryos is described here. The approach, using microinjection of a near-infrared caspase substrate that emits fluorescence only after its proteolytic cleavage by active effector caspases, has enabled the elucidation of otherwise cryptic aspects of apoptotic regulation. In particular, we show that brief caspase activity (10 min) is sufficient to cause apoptotic death in this system. We illustrate a cytochrome c dose threshold in the oocyte, which is lowered by Smac, a protein that binds thereby neutralizing the inhibitor of apoptosis proteins. We show that meiotic oocytes develop resistance to cytochrome c, and that the eventual death of oocytes arrested in meiosis is caspase-independent. Finally, data acquired through imaging caspase activity in the Xenopus embryo suggest that apoptosis in very early development is not cell-autonomous. These studies both validate this assay as a useful tool for apoptosis research and reveal subtleties in the cell death program during early development. Moreover, this method offers a potentially valuable screening modality for identifying novel apoptotic regulators

Active nuclear import and cytoplasmic retention of activation-induced deaminase

The enzyme activation-induced deaminase (AID) triggers antibody diversification in B cells by catalyzing deamination and consequently mutation of immunoglobulin genes. To minimize off-target deamination, AID is restrained by several regulatory mechanisms including nuclear exclusion, thought to be mediated exclusively by active nuclear export. Here we identify two other mechanisms involved in controlling AID subcellular localization. AID is unable to passively diffuse into the nucleus, despite its small size, and its nuclear entry requires active import mediated by a conformational nuclear localization signal. We also identify in its C terminus a determinant for AID cytoplasmic retention, which hampers diffusion to the nucleus, competes with nuclear import and is crucial for maintaining the predominantly cytoplasmic localization of AID in steady-state conditions. Blocking nuclear import alters the balance between these processes in favor of cytoplasmic retention, resulting in reduced isotype class switching.This work was supported by the Canadian Institutes of Health Research (MOP 84543) and a Canada Research Chair (to J.M.D.). A.O. was supported by a fellowship from the Canadian Institutes of Health Research Cancer Training Program at the IRCM. V.A.C. was supported in part by a Michel Saucier fellowship from the Louis-Pasteur Canadian Fund through the University of Montreal

Pro-apoptotic Bax is the major and Bak an auxiliary effector in cytokine deprivation-induced mast cell apoptosis

The process of apoptosis in immune cells like mast cells is essential to regain homeostasis after an inflammatory response. The intrinsic pathway of apoptosis is ultimately controlled by the pro-apoptotic Bcl-2 family members Bax and Bak, which upon activation oligomerize to cause increased permeabilization of the mitochondria outer membrane leading to cell death. We examined the role of Bax and Bak in cytokine deprivation-induced apoptosis in mast cells using connective tissue-like mast cells and mucosal-like mast cells derived from bax−/−, bak−/− and bax−/−bak−/− mice. Although both Bax and Bak were expressed at readily detectable protein levels, we found a major role for Bax in mediating mast cell apoptosis induced by cytokine deprivation. We analyzed cell viability by propidium iodide exclusion and flow cytometry after deprivation of vital cytokines for each mast cell population. Upon cytokine withdrawal, bak−/− mast cells died at a similar rate as wild type, whereas bax−/− and bax−/−bak−/− mast cells were partially or completely resistant to apoptosis, respectively. The total resistance seen in bax−/−bak−/− mast cells is comparable with mast cells deficient of both pro-apoptotic Bim and Puma or mast cells overexpressing anti-apoptotic Bcl-2. These results show that Bax has a predominant and Bak a minor role in cytokine deprivation-induced apoptosis in both connective tissue-like and mucosal-like mast cells

Immunohistochemical expression of mitochondrial membrane complexes (MMCs) I, III, IV and V in malignant and benign periampullary epithelium: a potential target for drug therapy of periampullary cancer?

<p>Abstract</p> <p>Background</p> <p>Mitochondrial membrane complexes (MMCs) are key mediators of cellular oxidative phosphorylation, and inhibiting them could lead to cell death. No published data are available on the relative abundance of MMCs in different periampullary cancers. Therefore, we studied the expression profile of MMCs I, III, IV and V in periampullary cancers, reactive pancreatitis, normal pancreas and chronic pancreatitis.</p> <p>Methods</p> <p>This was a retrospective study on tissue microarrays constructed from formalin-fixed paraffin-embedded tissue from 126 consecutive patients (cancer = 104, chronic pancreatitis = 22) undergoing pancreatic resections between June 2001 and June 2006. 78 specimens of chronic pancreatitis tissue were obtained adjacent to areas of cancer. Normal pancreatic tissue was obtained from the resection specimens in a total of 30 patients. Metastatic tumours in 61 regional lymph nodes from 61 patients were also studied.</p> <p>Results</p> <p>MMCs I, III, IV and V were highly expressed (p < 0.05) in all primary periampullary cancers compared with metastatic lymph nodes and adjacent benign pancreas. MMCs III, IV and V were highly expressed in all cancers regardless of type compared with chronic pancreatitis (p < 0.05). Higher expression of MMCs I and V was associated with better survival and may, in part, relate to lower expression of these MMCs in poorly differentiated tumours compared with well and moderately differentiated tumours.</p> <p>Conclusions</p> <p>Differential expression of MMCs III, IV and V in primary periampullary cancers compared with adjacent benign periampullary tissue and chronic pancreatitis is a novel finding, which may render them attractive anticancer targets.</p

Ubiquitous mitochondrial creatine kinase downregulated in oral squamous cell carcinoma

In this study, we performed two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption/ionisation time of fly mass spectrometry to identify the protein(s) associated with the development of oral squamous cell carcinomas (OSCCs) by comparing patterns of OSCC-derived cell lines with normal oral keratinocytes (NOKs), and found that downregulation of ubiquitous mitochondrial creatine kinase (CKMT1) could be a good candidate. Decreased levels of CKMT1 mRNA and protein were detected in all OSCC-derived cell lines examined (n=9) when compared to those in primary normal oral keratinocytes. Although no sequence variation in the coding region of the CKMT1 gene with the exception of a nonsense mutation in exon 8 was identified in these cell lines, we found a frequent hypermethylation in the CpG island region. CKMT1 expression was restored by experimental demethylation. In addition, when we transfected CKMT1 into the cell lines, they showed an apoptotic phenotype but no invasiveness. In clinical samples, high frequencies of CKMT1 downregulation were detected by immunohistochemistry (19 of 52 (37%)) and quantitative real-time RT–PCR (21 of 50 (42%)). Furthermore, the CKMT1 expression status was significantly correlated with tumour differentiation (P<0.0001). These results suggest that the CKMT1 gene is frequently inactivated during oral carcinogenesis and that an epigenetic mechanism may regulate loss of expression, which may lead to block apoptosis

Suppression of Lung Tumorigenesis by Leucine Zipper/EF Hand–Containing Transmembrane-1

Leucine zipper/EF hand-containing transmembrane-1 (LETM1) encodes for the human homologue of yeast Mdm38p, which is a mitochondria-shaping protein of unclear function. However, a previous study demonstrated that LETM1 served as an anchor protein for complex formation between mitochondria and ribosome, and regulated mitochondrial biogenesis.Therefore, we examine the possibility that LETM1 may function to regulate mitochondria and lung tumor growth. In this study, we addressed this question by studying in the effect of adenovirus-mediated LETM1 in the lung cancer cell and lung cancer model mice. To investigate the effects of adenovirus-LETM1 in vitro, we infected with adenovirus-LETM1 in A549 cells. Additionally, in vivo effects of LETM1 were evaluated on K-ras(LA1) mice, human non-small cell lung cancer model mice, by delivering the LETM1 via aerosol through nose-only inhalation system. The effects of LETM1 on lung cancer growth and AMPK related signals were evaluated. Adenovirus-mediated overexpression of LETM1 could induce destruction of mitochondria of lung cancer cells through depleting ATP and AMPK activation. Furthermore, adenoviral-LETM1 also altered Akt signaling and inhibited the cell cycle while facilitating apoptosis. Theses results demonstrated that adenovirus-LETM1 suppressed lung cancer cell growth in vitro and in vivo.Adenovirus-mediated LETM1 may provide a useful target for designing lung tumor prevention and treatment