Matrix Metalloproteinase (MMP)-1 Induces Lung Alveolar Epithelial Cell Migration and Proliferation,Protects from Apoptosis,and Represses Mitochondrial Oxygen Consumption

Idiopathic pulmonary fibrosis is a devastating lung disorder of unknown etiology. Although its pathogenesis is unclear, considerable evidence supports an important role of aberrantly activated alveolar epithelial cells (AECs), which produce a large variety of mediators, including several matrix metalloproteases (MMPs), which participate in fibroblast activation and lung remodeling. MMP-1 has been shown to be highly expressed in AECs from idiopathic pulmonary fibrosis lungs although its role is unknown. In this study, we explored the role of MMP-1 in several AECs functions. Mouse lung epithelial cells (MLE12) transfected with human Mmp-1 showed significantly increased cell growth and proliferation at 36 and 48 h of culture (p < 0.01). Also, MMP-1 promoted MLE12 cell migration through collagen I, accelerated wound closing, and protected cells from staurosporine- and bleomycin-induced apoptosis compared with mock cells (p < 0.01). MLE12 cells expressing human MMP-1 showed a significant repression of oxygen consumption ratio compared with the cells with the empty vector. As under hypoxic conditions hypoxia-inducible factor-1α (HIF-1α) mediates a transition from oxidative to glycolytic metabolism, we analyzed activation of HIF-1α. Ηigher activation of this factor was detected in MMP-1-transfected cells under normoxia and hypoxia. Likewise, a significant decrease of both total and mitochondrial reactive oxygen species was observed in MMP-1-transfected cells. Paralleling these findings, attenuation of MMP-1 expression by shRNA in A549 (human) AECs markedly reduced proliferation and migration (p < 0.01) and increased the oxygen consumption ratio. These findings indicate that epithelial expression of MMP-1 inhibits mitochondrial function, increases HIF-1α expression, decreases reactive oxygen species production, and contributes to a proliferative, migratory, and anti-apoptotic AEC phenotype.     

Differential Expression of Ecdysone Receptor Leads to Variation in Phenotypic Plasticity across Serial Homologs

Bodies are often made of repeated units, or serial homologs, that develop using the same core gene regulatory network. Local inputs and modifications to this network allow serial homologs to evolve different morphologies, but currently we do not understand which modifications allow these repeated traits to evolve different levels of phenotypic plasticity. Here we describe variation in phenotypic plasticity across serial homologous eyespots of the butterfly Bicyclus anynana, hypothesized to be under selection for similar or different functions in the wet and dry seasonal forms. Specifically, we document the presence of eyespot size and scale brightness plasticity in hindwing eyespots hypothesized to vary in function across seasons, and reduced size plasticity and absence of brightness plasticity in forewing eyespots hypothesized to have the same function across seasons. By exploring the molecular and physiological causes of this variation in plasticity across fore and hindwing serial homologs we discover that: 1) temperature experienced during the wandering stages of larval development alters titers of an ecdysteroid hormone, 20-hydroxyecdysone (20E), in the hemolymph of wet and dry seasonal forms at that stage; 2) the 20E receptor (EcR) is differentially expressed in the forewing and hindwing eyespot centers of both seasonal forms during this critical developmental stage; and 3) manipulations of EcR signaling disproportionately affected hindwing eyespots relative to forewing eyespots. We propose that differential EcR expression across forewing and hindwing eyespots at a critical stage of development explains the variation in levels of phenotypic plasticity across these serial homologues. This finding provides a novel signaling pathway, 20E, and a novel molecular candidate, EcR, for the regulation of levels of phenotypic plasticity across body parts or serial homologs.     

The intrinsic apoptotic pathway is required for lipopolysaccharide-induced lung endothelial cell death

LPS has been implicated in the pathogenesis of endothelial cell death associated with Gram-negative bacterial sepsis. The binding of LPS to the TLR-4 on the surface of endothelial cells initiates the formation of a death-inducing signaling complex at the cell surface. The subsequent signaling pathways that result in apoptotic cell death remain unclear and may differ among endothelial cells in different organs. We sought to determine whether LPS and cycloheximide-induced cell death in human lung microvascular endothelial cells (HmVECs) was dependent upon activation of the intrinsic apoptotic pathway and the generation of reactive oxygen species. We found that cells overexpressing the anti-apoptotic protein Bcl-X(L) were resistant to LPS and cycloheximide-induced death and that the proapoptotic Bcl-2 protein Bid was cleaved following treatment with LPS. The importance of Bid was confirmed by protection of Bid-deficient (bid(-/-)) mice from LPS-induced lung injury. Neither HmVECs treated with the combined superoxide dismutase/catalase mimetic EUK-134 nor HmVECs depleted of mitochondrial DNA (rho(0) cells) were protected against LPS and cycloheximide-induced death. We conclude that LPS and cycloheximide-induced death in HmVECs requires the intrinsic cell death pathway, but not the generation of reactive oxygen species.     

Detection of Seizure Event and Its Onset/Offset Using Orthonormal Triadic Wavelet Based Features

Background

Epileptic seizures are unpredictable in nature and its quick detection is important for immediate treatment of patients. In last few decades researchers have proposed different algorithms for onset and offset detection of seizure using Electroencephalogram (EEG) signals.

Analysis of genetic stability of in vitro propagated potato microtubers using DNA markers

The genetic stability of in vitro propagated potato microtubers was assessed using random amplified polymorphic DNA (RAPD), inter simple sequence repeat (ISSR), simple sequence repeat (SSR) and amplified fragment length polymorphism (AFLP) markers. Microtubers were developed through in vitro from potato microplants using standardized protocols. The microtubers were conserved for 1 year under three different culture media and consequently microplants were regenerated for the DNA analyses. During the study, a total of 38 (10 RAPD, 11 ISSR, 12 SSR and 5 AFLP) primers produced a total of 407 (58 RAPD, 56 ISSR, 96 SSR and 197 AFLP) clear, distinct and reproducible amplicons. Cluster analysis revealed 100 % genetic similarity among the mother plant and its derivatives within the clusters by SSR, ISSR and RAPD analyses, whereas AFLP analysis revealed from 85 to 100 % genetic similarity. Dendrogram analysis based on the Jaccard’s coefficient classified the genotypes into five clusters (I–V), each cluster consisting of mother plant and its derivatives. Principal component analysis (PCA) also plotted mother plant and its genotypes of each cluster together. Based on our results, it is concluded that AFLP is the best method followed by SSR, ISSR and RAPD to detect genetic stability of in vitro conserved potato microtubers. The in vitro conservation medium (T2) is a safe method for conservation of potato microtubers to produce true-to-type plans.     

Mitochondrial-derived free radicals mediate asbestos-induced alveolar epithelial cell apoptosis

Asbestos causes pulmonary toxicity by mechanisms that in part involve reactive oxygen species (ROS). However, the precise source of ROS is unclear. We showed that asbestos induces alveolar epithelial cell (AEC) apoptosis by a mitochondrial-regulated death pathway. To determine whether mitochondrial-derived ROS are necessary for causing asbestos-induced AEC apoptosis, we utilized A549-rho(omicron) cells that lack mitochondrial DNA and a functional electron transport. As expected, antimycin, which induces an oxidative stress by blocking mitochondrial electron transport at complex III, increased dichlorofluoroscein (DCF) fluorescence in A549 cells but not in A549-rho(omicron) cells. Compared with A549 cells, rho(omicron) cells have less asbestos-induced ROS production, as assessed by DCF fluorescence, and reductions in total glutathione levels as well as less caspase-9 activation and apoptosis, as assessed by TdT-mediated dUTP nick end labeling staining and DNA fragmentation. A mitochondrial anion channel inhibitor that prevents ROS release from the mitochondria to the cytoplasm also blocked asbestos-induced A549 cell caspase-9 activation and apoptosis. Finally, a role for nonmitochondrial-derived ROS with exposure to high levels of asbestos (50 microg/cm(2)) was suggested by our findings that an iron chelator (phytic acid or deferoxamine) or a free radical scavenger (sodium benzoate) provided additional protection against asbestos-induced caspase-9 activation and DNA fragmentation in rho(omicron) cells. We conclude that asbestos fibers affect mitochondrial DNA and functional electron transport, resulting in mitochondrial-derived ROS production that in turn mediates AEC apoptosis. Nonmitochondrial-associated ROS may also contribute to AEC apoptosis, particularly with high levels of asbestos exposure.     

Expression analysis of steroid pathway genes revealed positive correlation with diosgenin biosynthesis in <Emphasis Type="Italic">Trillium govanianum</Emphasis>

Trillium govanianum Wall. ex D. Don, popularly known as Nagchhatri, is an important medicinal plant of northwestern Himalayas. No data exist on molecular characteristics of diosgenin biosynthesis, the main chemical component of T. govanianum. HPLC analysis revealed diosgenin content of 2.4 and 0.7% in hydrolyzed rhizome extracts of Chamba and Lahaul and Spiti, respectively. Quantitative real-time PCR analysis of five genes of steroid pathway in the rhizomes of two locations, differing for diosgenin content showed up to 3.2-fold increase in expression level of genes viz. hydroxymethylglutaryl Co-A reductase (HMGR), farnesyl pyrophosphate synthase (FPPS), squalene synthase (SQS), 26-O-beta-glucosidase (BETA) and cycloartenol synthase (CAS) in rhizomes of Chamba (2.4%) as compared to Lahaul and Spiti (0.7%), thus inferring their role in diosgenin biosynthesis. The present study delivers the first report where an elite chemotype of T. govanianum for diosgenin content was identified and partial sequences of five genes of steroid pathway were cloned and investigated for their potential role in diosgenin biosynthesis in T. govanianum. The result of this study has prospective applications in the genetic improvement of this medicinally important plant species.     

Bioconversion of Saccharum spontaneum (wild sugarcane) hemicellulosic hydrolysate into ethanol by mono and co-cultures of Pichia stipitis NCIM3498 and thermotolerant Saccharomyces cerevisiae-VS₃

The lignocellulosic biomass is a low-cost renewable resource for eco-benign liquid fuel 'ethanol'. To resolve the hydrolysis of mixed sugars in lignocellulosic substrate Saccharum spontaneum, the microbial co-cultures of Pichia stipitis NCIM 3498 and thermotolerant Saccharomyces cerevisiae-VS(3) were analyzed for efficient bioconversion of mixed sugars into ethanol. Among the hydrolysis conditions, the acid hydrolysis released maximum sugars along with furans, phenolics and acetic acid. The acidic hydrolysate was detoxified and fermented by monocultures of P. stipitis NCIM3498 (P.S.) and thermotolerant S. cerevisiae VS(3) (S.C.), and co-culture of P.S. (7.5 mL) and S.C. (2.5 mL). Before the fermentation of hemicellulose acid hydrolysate, both the monocultures (P.S. and S.C.), and varying ratios of P.S. and S.C. microorganisms in co-cultures #1, #2 and #3 were grown on simulated synthetic medium. The ethanol yield from monocultures of P.S. (0.44 ± 0.021 g/g), S.C. (0.22 ± 0.01 g/g) and co-culture #3 (0.49 ± 0.02 g/g) revealed unique characteristics of each mono and co-culture technology. The fermentation of hemicellulose acid hydrolysate with monocultures of P.S., S.C. and co-culture #3 produced 12.08 ± 0.72 g/L, 1.40 ± 0.07 g/L, and 15.0 ± -0.92 g/L ethanol, respectively.     

Bcl-2 Family Members and Functional Electron Transport Chain Regulate Oxygen Deprivation-Induced Cell Death

The mechanisms underlying cell death during oxygen deprivation are unknown. We report here a model for oxygen deprivation-induced apoptosis. The death observed during oxygen deprivation involves a decrease in the mitochondrial membrane potential, followed by the release of cytochrome c and the activation of caspase-9. Bcl-X(L) prevented oxygen deprivation-induced cell death by inhibiting the release of cytochrome c and caspase-9 activation. The ability of Bcl-X(L) to prevent cell death was dependent on allowing the import of glycolytic ATP into the mitochondria to generate an inner mitochondrial membrane potential through the F(1)F(0)-ATP synthase. In contrast, although activated Akt has been shown to inhibit apoptosis induced by a variety of apoptotic stimuli, it did not prevent cell death during oxygen deprivation. In addition to Bcl-X(L), cells devoid of mitochondrial DNA (rho degrees cells) that lack a functional electron transport chain were resistant to oxygen deprivation. Further, murine embryonic fibroblasts from bax(-/-) bak(-/-) mice did not die in response to oxygen deprivation. These data suggest that when subjected to oxygen deprivation, cells die as a result of an inability to maintain a mitochondrial membrane potential through the import of glycolytic ATP. Proapoptotic Bcl-2 family members and a functional electron transport chain are required to initiate cell death in response to oxygen deprivation.