Bleomycin induces alveolar epithelial cell death through JNK-dependent activation of the mitochondrial death pathway

Exposure to bleomycin in rodents induces lung injury and fibrosis. Alveolar epithelial cell death has been hypothesized as an initiating mechanism underlying bleomycin-induced lung injury and fibrosis. In the present study we evaluated the contribution of mitochondrial and receptor-meditated death pathways in bleomycin-induced death of mouse alveolar epithelial cells (MLE-12 cells) and primary rat alveolar type II cells. Control MLE-12 cells and primary rat alveolar type II cells died after 48 h of exposure to bleomycin. Both MLE-12 cells and rat alveolar type II cells overexpressing Bcl-X(L) did not undergo cell death in response to bleomycin. Dominant negative Fas-associating protein with a death domain failed to prevent bleomycin-induced cell death in MLE-12 cells. Caspase-8 inhibitor CrmA did not prevent bleomycin-induced cell death in primary rat alveolar type II cells. Furthermore, fibroblast cells deficient in Bax and Bak, but not Bid, were resistant to bleomycin-induced cell death. To determine whether the stress kinase JNK was an upstream regulator of Bax activation, MLE-12 cells were exposed to bleomycin in the presence of an adenovirus encoding a dominant negative JNK. Bleomycin-induced Bax activation was prevented by the expression of a dominant negative JNK in MLE-12 cells. Dominant negative JNK prevented cell death in MLE-12 cells and in primary rat alveolar type II cells exposed to bleomycin. These data indicate that bleomycin induces cell death through a JNK-dependent mitochondrial death pathway in alveolar epithelial cells.     

Campanian-Maastricthian (Late Cretaceous) veneroids (Bivalvia: Heterodonta) from the Ariyalur Group,South India

Eleven veneroid (Bivalvia) species are systematically described from the Campanian-Maastrichtian (Late Cretaceous) sediments of Ariyalur sub-basin of South India. Among these, only Crassatella (Crassatella) macrodonta (Sowerby) and Arctica lacianata (Stoliczka) were earlier recorded by Stoliczka. Protocardia (Pachycardium) madagascariensis (Colignon), Protocardia (Pachycardium) pauli (Cocquand), Epicyprina angulata (Sowerby), Venilicardia truncata (Sowerby), and Calva (Egelicalva) buttensis (Anderson) were previously unknown from the Late Cretaceous horizons of the Indian sub-continent. The present record also includes a new species Palaeomoera stoliczkai n. sp. erected on the basis of its unique hinge characters and surface features. Lucina (Lucina) cf. fallax Forbes, Nicaniella (Nicaniella) aff. trigonoides (Stoliczka), and Corbicula? sp. indet., are tentatively identified because of their imperfect preservation. The present record may add useful information to the understanding of the Late Cretaceous palaeobiology of the region.     

Loss of Nek11 Prevents G2/M Arrest and Promotes Cell Death in HCT116 Colorectal Cancer Cells Exposed to Therapeutic DNA Damaging Agents

The Nek11 kinase is a potential mediator of the DNA damage response whose expression is upregulated in early stage colorectal cancers (CRCs). Here, using RNAi-mediated depletion, we examined the role of Nek11 in HCT116 WT and p53-null CRC cells exposed to ionizing radiation (IR) or the chemotherapeutic drug, irinotecan. We demonstrate that depletion of Nek11 prevents the G2/M arrest induced by these genotoxic agents and promotes p53-dependent apoptosis both in the presence and absence of DNA damage. Interestingly, Nek11 depletion also led to long-term loss of cell viability that was independent of p53 and exacerbated following IR exposure. CRC cells express four splice variants of Nek11 (L/S/C/D). These are predominantly cytoplasmic, but undergo nucleocytoplasmic shuttling mediated through adjacent nuclear import and export signals in the C-terminal non-catalytic domain. In HCT116 cells, Nek11S in particular has an important role in the DNA damage response. These data provide strong evidence that Nek11 contributes to the response of CRC cells to genotoxic agents and is essential for survival either with or without exposure to DNA damage.     

Accumulation of Oxidized LDL in the Tendon Tissues of C57BL/6 or Apolipoprotein E Knock-Out Mice That Consume a High Fat Diet: Potential Impact on Tendon Health

ObjectiveClinical studies have suggested an association between dyslipidemia and tendon injuries or chronic tendon pain; the mechanisms underlying this association are not yet known. The objectives of this study were (1) to evaluate the impact of a high fat diet on the function of load-bearing tendons and on the distribution in tendons of oxidized low density lipoprotein (oxLDL), and (2) to examine the effect of oxLDL on tendon fibroblast proliferation and gene expression.MethodsGene expression (Mmp2, Tgfb1, Col1a1, Col3a1), fat content (Oil Red O staining), oxLDL levels (immunohistochemistry) and tendon biomechanical properties were examined in mice (C57Bl/6 or ApoE -/-) receiving a standard or a high fat diet. Human tendon fibroblast proliferation and gene expression (COL1A1, COL3A1, MMP2) were examined following oxLDL exposure.ResultsIn both types of mice (C57Bl/6 or ApoE -/-), consumption of a high fat diet led to a marked increase in oxLDL deposition in the load-bearing extracellular matrix of the tendon. The consumption of a high fat diet also reduced the failure stress and load of the patellar tendon in both mouse types, and increased Mmp2 expression. ApoE -/- mice exhibited more pronounced reductions in tendon function than wild-type mice, and decreased expression of Col1a1 compared to wild type mice. Human tendon fibroblasts responded to oxLDL by increasing their proliferation and their mRNA levels of MMP2, while decreasing their mRNA levels for COL1A1 and COL3A1.ConclusionThe consumption of a high fat diet resulted in deleterious changes in tendon function, and these changes may be explained in part by the effects of oxLDL, which induced a proliferative, matrix-degrading phenotype in human tenocytes.     

Honey bee PTEN--description, developmental knockdown, and tissue-specific expression of splice-variants correlated with alternative social phenotypes

Background

Phosphatase and TENsin (PTEN) homolog is a negative regulator that takes part in IIS (insulin/insulin-like signaling) and Egfr (epidermal growth factor receptor) activation in Drosophila melanogaster. IIS and Egfr signaling events are also involved in the developmental process of queen and worker differentiation in honey bees (Apis mellifera). Here, we characterized the bee PTEN gene homologue for the first time and begin to explore its potential function during bee development and adult life.

Results

Honey bee PTEN is alternatively spliced, resulting in three splice variants. Next, we show that the expression of PTEN can be down-regulated by RNA interference (RNAi) in the larval stage, when female caste fate is determined. Relative to controls, we observed that RNAi efficacy is dependent on the amount of PTEN dsRNA that is delivered to larvae. For larvae fed queen or worker diets containing a high amount of PTEN dsRNA, PTEN knockdown was significant at a whole-body level but lethal. A lower dosage did not result in a significant gene down-regulation. Finally, we compared same-aged adult workers with different behavior: nursing vs. foraging. We show that between nurses and foragers, PTEN isoforms were differentially expressed within brain, ovary and fat body tissues. All isoforms were expressed at higher levels in the brain and ovaries of the foragers. In fat body, isoform B was expressed at higher level in the nurse bees.

Conclusion

Our results suggest that PTEN plays a central role during growth and development in queen- and worker-destined honey bees. In adult workers, moreover, tissue-specific patterns of PTEN isoform expression are correlated with differences in complex division of labor between same-aged individuals. Therefore, we propose that knowledge on the roles of IIS and Egfr activity in developmental and behavioral control may increase through studies of how PTEN functions can impact bee social phenotypes.     

Efferocytosis Fuels Requirements of Fatty Acid Oxidation and the Electron Transport Chain to Polarize Macrophages for Tissue Repair

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Identification of a Putative Protein Profile Associated with Tamoxifen Therapy Resistance in Breast Cancer

Tamoxifen resistance is a major cause of death in patients with recurrent breast cancer. Current clinical factors can correctly predict therapy response in only half of the treated patients. Identification of proteins that are associated with tamoxifen resistance is a first step toward better response prediction and tailored treatment of patients. In the present study we intended to identify putative protein biomarkers indicative of tamoxifen therapy resistance in breast cancer using nano-LC coupled with FTICR MS. Comparative proteome analysis was performed on ∼5,500 pooled tumor cells (corresponding to ∼550 ng of protein lysate/analysis) obtained through laser capture microdissection (LCM) from two independently processed data sets (n = 24 and n = 27) containing both tamoxifen therapy-sensitive and therapy-resistant tumors. Peptides and proteins were identified by matching mass and elution time of newly acquired LC-MS features to information in previously generated accurate mass and time tag reference databases. A total of 17,263 unique peptides were identified that corresponded to 2,556 non-redundant proteins identified with ≥2 peptides. 1,713 overlapping proteins between the two data sets were used for further analysis. Comparative proteome analysis revealed 100 putatively differentially abundant proteins between tamoxifen-sensitive and tamoxifen-resistant tumors. The presence and relative abundance for 47 differentially abundant proteins were verified by targeted nano-LC-MS/MS in a selection of unpooled, non-microdissected discovery set tumor tissue extracts. ENPP1, EIF3E, and GNB4 were significantly associated with progression-free survival upon tamoxifen treatment for recurrent disease. Differential abundance of our top discriminating protein, extracellular matrix metalloproteinase inducer, was validated by tissue microarray in an independent patient cohort (n = 156). Extracellular matrix metalloproteinase inducer levels were higher in therapy-resistant tumors and significantly associated with an earlier tumor progression following first line tamoxifen treatment (hazard ratio, 1.87; 95% confidence interval, 1.25–2.80; p = 0.002). In summary, comparative proteomics performed on laser capture microdissection-derived breast tumor cells using nano-LC-FTICR MS technology revealed a set of putative biomarkers associated with tamoxifen therapy resistance in recurrent breast cancer.Tamoxifen is an antiestrogenic agent that has been widely and successfully used in the treatment of breast cancer over the past decades (1). Tamoxifen targets and inhibits the estrogen receptor-α, which is expressed in ∼70% of all primary breast tumors and is known to be important in the development and course of the disease. When diagnosed at an early stage, adjuvant systemic tamoxifen therapy can cure ∼10% of the patients (1). In recurrent disease, ∼50% of patients have no benefit from tamoxifen (intrinsic resistance). From the other half of patients who initially respond to therapy with an objective response (OR)¹ or no change (NC), a majority eventually develop progressive disease (PD) due to acquired tamoxifen resistance (2, 3). With the markers available to date we can insufficiently predict therapy response. Therefore, identification of new biomarkers that can more effectively predict response to treatment and that can potentially function as drug targets is a major focus of research.The search for new biomarkers has been enhanced by the introduction of microarray technology. Gene expression studies have resulted in a whole spectrum of profiles for e.g. molecular subtypes, prognosis, and therapy prediction in breast cancer (4–10). Corresponding studies at the protein level are lagging behind because of immature technology. However, protein-level information is crucial for the functional understanding and the ultimate translation of molecular knowledge into clinical practice, and proteomics technologies continue to progress at a rapid pace.Proteomics studies reported so far have mainly been performed with breast cancer cell lines using either two-dimensional gel electrophoresis (11–14) or LC-MS for protein separation (15–17). However, it is known that the proteomic makeup of a cultured cell is rather different from that of a tumor cell surrounded by its native microenvironment (18). Furthermore cell lines lack the required follow-up information for answering important clinical questions. In addition, tumor tissues in general and breast cancer tissues in particular are very heterogeneous in the sense that they harbor many different cell types, such as stroma, normal epithelium, and tumor cells. LCM technology has emerged as an ideal tool for selectively extracting cells of interest from their natural environment (19) and has therefore been an important step forward in the context of genomics and proteomics cancer biomarker discovery research. LCM-derived breast cancer tumor cells have been used for comparative proteomics analyses in the past using both two-dimensional gel electrophoresis (20, 21) and LC-MS (22). This has resulted in the identification of proteins involved in breast cancer prognosis (21) and metastasis (20, 22). Although these studies demonstrated that proteomics technology has advanced to the level where it can contribute to biomarker discovery, major drawbacks, such as large sample requirements (42–700 μg) and low proteome coverage (50–76 proteins), for small amounts of starting material (∼1 μg) persist. Because clinical samples are often available in limited quantities, in-depth analysis of minute amounts of material (<1 μg) necessitates advanced technologies with sufficient sensitivity and depth of coverage.Recently we demonstrated the applicability of nano-LC-FTICR MS in combination with the accurate mass and time (AMT) tag approach for proteomics characterization of ∼3,000 LCM-derived breast cancer cells (23). This study showed that proteome coverage was improved compared with conventional techniques. The AMT tag approach initially utilizes conventional LC-MS/MS measurements to establish a reference database of AMT tags specific for a particular proteome sample (e.g. breast cancer tissue). Each tag consists of a theoretical mass calculated from the peptide sequence, an LC normalized elution time (NET) value, and an indicator of quality. The AMT tag database serves as a “lookup table” for identifying peptides in subsequent quantitative LC-MS analyses. Substituting routine LC-MS/MS analyses (shotgun approach) with LC-FTICR MS analyses (AMT tag approach) significantly increases overall throughput and sensitivity while reducing sample requirements. Additionally quantitative intensity information related to the abundance of the protein can be discerned from these MS analyses (24). In the present study, we used the same strategy to analyze eight pools of tumor cells in duplicate or triplicate (resulting in 19 samples) derived from 51 fresh frozen primary invasive breast carcinomas that appeared to be either sensitive or resistant to tamoxifen treatment after recurrence. This work resulted in the identification of a putative protein profile associated with tamoxifen therapy resistance. In addition, the top discriminating protein of the putative profile, extracellular matrix metalloproteinase inducer (EMMPRIN), was validated in an independent patient cohort and was significantly associated with resistance to tamoxifen therapy and shorter time to progression upon tamoxifen treatment in recurrent breast cancer.     

Characterization of the structural and functional changes in the myocardium following focal ischemia-reperfusion injury

High-resolution (11.7 T) cardiac magnetic resonance imaging (MRI) and histological approaches have been employed in tandem to characterize the secondary damage suffered by the murine myocardium following the initial insult caused by ischemia-reperfusion (I/R). I/R-induced changes in the myocardium were examined in five separate groups at the following time points after I/R: 1 h, day 1, day 3, day 7, and day 14. The infarct volume increased from 1 h to day 1 post-I/R. Over time, the loss of myocardial function was observed to be associated with increased infarct volume and worsened regional wall motion. In the infarct region, I/R caused a decrease in end-systolic thickness coupled with small changes in end-diastolic thickness, leading to massive wall thickening abnormalities. In addition, compromised wall thickening was also observed in left ventricular regions adjacent to the infarct region. A tight correlation (r2 = 0.85) between measured MRI and triphenyltetrazolium chloride (TTC) infarct volumes was noted. Our observation that until day 3 post-I/R the infarct size as measured by TTC staining and MRI was much larger than that of the myocyte-silent regions in trichrome- or hematoxylin-eosin-stained sections is consistent with the literature and leads to the conclusion that at such an early phase, the infarct site contains structurally intact myocytes that are functionally compromised. Over time, such affected myocytes were noted to structurally disappear, resulting in consistent infarct sizes obtained from MRI and TTC as well as trichrome and hematoxylin-eosin analyses on day 7 following I/R. Myocardial remodeling following I/R includes secondary myocyte death followed by the loss of cardiac function over time.     

Assessment of wound-site redox environment and the significance of Rac2 in cutaneous healing

We have previously reported that H(2)O(2) is actively generated by cells at the wound site and that H(2)O(2)-driven redox signaling supports wound angiogenesis and healing. In this study, we have standardized a novel and effective electron paramagnetic resonance spectroscopy-based approach to assess the redox environment of the dermal wound site in vivo. Rac2 regulates inducible NADPH oxidase activation and other functional responses in neutrophils. Using Rac2-deficient mice we sought to investigate the significance of Rac2 in the wound-site redox environment and healing responses. Noninvasive measurements of metabolism of topically applied nitroxide (15)N-perdeuterated tempone in murine excisional dermal wounds demonstrated that the wound site is rich in oxidants, the levels of which peak 2 days postwounding in the inflammatory phase. Rac2-deficient mice had threefold lower production of superoxide compared to controls with similar wounds. In these mice, a lower wound-site superoxide level was associated with compromised wound closure. Immunostaining of wound edges harvested during the inflammatory phase showed that the numbers of phagocytic cells recruited to the wound site in Rac2-deficient and control mice were similar, but the amount of lipid peroxidation was significantly lower in Rac2-deficient mice, indicating compromised NADPH oxidase activity. Taken together, the findings of this study support that the wound site is rich in oxidants. Rac2 significantly contributes to oxidant production at the wound site and supports the healing process.