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Sample records for intracellular survival mechanisms

  1. Kinetic studies of Candida parapsilosis phagocytosis by macrophages and detection of intracellular survival mechanisms.

    PubMed

    Tóth, Renáta; Tóth, Adél; Papp, Csaba; Jankovics, Ferenc; Vágvölgyi, Csaba; Alonso, Maria F; Bain, Judith M; Erwig, Lars-Peter; Gácser, Attila

    2014-01-01

    Even though the number of Candida infections due to non-albicans species like C. parapsilosis has been increasing, little is known about their pathomechanisms. Certain aspects of C. parapsilosis and host interactions have already been investigated; however we lack information about the innate cellular responses toward this species. The aim of our project was to dissect and compare the phagocytosis of C. parapsilosis to C. albicans and to another Candida species C. glabrata by murine and human macrophages by live cell video microscopy. We broke down the phagocytic process into three stages: macrophage migration, engulfment of fungal cells and host cell killing after the uptake. Our results showed increased macrophage migration toward C. parapsilosis and we observed differences during the engulfment processes when comparing the three species. The engulfment time of C. parapsilosis was comparable to that of C. albicans regardless of the pseudohypha length and spatial orientation relative to phagocytes, while the rate of host cell killing and the overall uptake regarding C. parapsilosis showed similarities mainly with C. glabrata. Furthermore, we observed difference between human and murine phagocytes in the uptake of C. parapsilosis. UV-treatment of fungal cells had varied effects on phagocytosis dependent upon which Candida strain was used. Besides statistical analysis, live cell imaging videos showed that this species similarly to the other two also has the ability to survive in host cells via the following mechanisms: yeast replication, and pseudohypha growth inside of phagocytes, exocytosis of fungal cells and also abortion of host cell mitosis following the uptake. According to our knowledge this is the first study that provides a thorough examination of C. parapsilosis phagocytosis and reports intracellular survival mechanisms associated with this species. PMID:25477874

  2. Invasion and Intracellular Survival by Protozoan Parasites

    PubMed Central

    Sibley, L. David

    2013-01-01

    Summary Intracellular parasitism has arisen only a few times during the long ancestry of protozoan parasites including in diverse groups such as microsporidians, kinetoplastids, and apicomplexans. Strategies used to gain entry differ widely from injection (e.g. microsporidians), active penetration of the host cell (e.g. Toxoplasma), recruitment of lysosomes to a plasma membrane wound (e.g. Trypanosoma cruzi), to host cell-mediated phagocytosis (e.g. Leishmania). The resulting range of intracellular niches is equally diverse ranging from cytosolic (e.g. T. cruzi) to residing within a nonfusigenic vacuole (e.g. Toxoplasma, Encephalitizoon) or a modified phagolysosome (e.g. Leishmania). These lifestyle choices influence access to nutrients, interaction with host cell signaling pathways, and detection by pathogen recognition systems. As such, intracellular life requires a repertoire of adaptations to assure entry-exit from the cell, as well as to thwart innate immune mechanisms and prevent clearance. Elucidating these pathways at the cellular and molecular level may identify key steps that can be targeted to reduce parasite survival or augment immunological responses and thereby prevent disease. PMID:21349087

  3. Strategies for Intracellular Survival of Burkholderia pseudomallei.

    PubMed

    Allwood, Elizabeth M; Devenish, Rodney J; Prescott, Mark; Adler, Ben; Boyce, John D

    2011-01-01

    Burkholderia pseudomallei is the causative agent of melioidosis, a disease with high mortality that is prevalent in tropical regions of the world. A key component of the pathogenesis of melioidosis is the ability of B. pseudomallei to enter, survive, and replicate within mammalian host cells. For non-phagocytic cells, bacterial adhesins have been identified both on the bacterial surface and associated with Type 4 pili. Cell invasion involves components of one or more of the three Type 3 Secretion System clusters, which also mediate, at least in part, the escape of bacteria from the endosome into the cytoplasm, where bacteria move by actin-based motility. The mechanism of actin-based motility is not clearly understood, but appears to differ from characterized mechanisms in other bacterial species. A small proportion of intracellular bacteria is targeted by host cell autophagy, involving direct recruitment of LC3 to endosomes rather than through uptake by canonical autophagosomes. However, the majority of bacterial cells are able to circumvent autophagy and other intracellular defense mechanisms such as the induction of inducible nitric oxide synthase, and then replicate in the cytoplasm and spread to adjacent cells via membrane fusion, resulting in the formation of multi-nucleated giant cells. A potential role for host cell ubiquitin in the autophagic response to bacterial infection has recently been proposed. PMID:22007185

  4. A transcriptomic analysis of Yersinia enterocolitica biovar 1B infecting murine macrophages reveals new mechanisms of intracellular survival

    SciTech Connect

    Bent, Zachary W.; Poorey, Kunal; Brazel, David M.; LaBauve, Annette E.; Sinha, Anupama; Curtis, Deanna Joy; House, Samantha E.; Tew, Karen E.; Hamblin, Rachelle Y.; Williams, Kelly Porter; Branda, Steven S.; Young, Glenn M.; Meagher, Robert J.

    2015-04-20

    Yersinia enterocolitica is typically considered an extracellular pathogen; however, during the course of an infection, a significant number of bacteria are stably maintained within host cell vacuoles. Little is known about this population and the role it plays during an infection. To address this question and to elucidate the spatially and temporally dynamic gene expression patterns of Y. enterocoliticabiovar 1B through the course of an in vitro infection, transcriptome sequencing and differential gene expression analysis of bacteria infecting murine macrophage cells were performed under four distinct conditions. Bacteria were first grown in a nutrient-rich medium at 26°C to establish a baseline of gene expression that is unrelated to infection. The transcriptomes of these bacteria were then compared to bacteria grown in a conditioned cell culture medium at 37°C to identify genes that were differentially expressed in response to the increased temperature and medium but not in response to host cells. Infections were then performed, and the transcriptomes of bacteria found on the extracellular surface and intracellular compartments were analyzed individually. The upregulated genes revealed potential roles for a variety of systems in promoting intracellular virulence, including the Ysa type III secretion system, the Yts2 type II secretion system, and the Tad pilus. It was further determined that mutants of each of these systems had decreased virulence while infecting macrophages. Overall, these results reveal the complete set of genes expressed by Y. enterocolitica in response to infection and provide the groundwork for future virulence studies.

  5. Intracellular Inactivation of Thyroid Hormone Is a Survival Mechanism for Muscle Stem Cell Proliferation and Lineage Progression

    PubMed Central

    Dentice, Monica; Ambrosio, Raffaele; Damiano, Valentina; Sibilio, Annarita; Luongo, Cristina; Guardiola, Ombretta; Yennek, Siham; Zordan, Paola; Minchiotti, Gabriella; Colao, Annamaria; Marsili, Alessandro; Brunelli, Silvia; Del Vecchio, Luigi; Larsen, P. Reed; Tajbakhsh, Shahragim; Salvatore, Domenico

    2014-01-01

    Summary Precise control of the thyroid hormone (T3)-dependent transcriptional program is required by multiple cell systems, including muscle stem cells. Deciphering how this is achieved and how the T3 signal is controlled in stem cell niches is essentially unknown. We report that in response to proliferative stimuli such as acute skeletal muscle injury, type 3 deiodinase (D3), the thyroid hormone-inactivating enzyme, is induced in satellite cells where it reduces intracellular thyroid signaling. Satellite cell-specific genetic ablation of dio3 severely impairs skeletal muscle regeneration. This impairment is due to massive satellite cell apoptosis caused by exposure of activated satellite cells to the circulating TH. The execution of this proapoptotic program requires an intact FoxO3/MyoD axis, both genes positively regulated by intracellular TH. Thus, D3 is dynamically exploited in vivo to chronically attenuate TH signaling under basal conditions while also being available to acutely increase gene programs required for satellite cell lineage progression. PMID:25456740

  6. A transcriptomic analysis of Yersinia enterocolitica biovar 1B infecting murine macrophages reveals new mechanisms of intracellular survival

    DOE PAGESBeta

    Bent, Zachary W.; Poorey, Kunal; Brazel, David M.; LaBauve, Annette E.; Sinha, Anupama; Curtis, Deanna Joy; House, Samantha E.; Tew, Karen E.; Hamblin, Rachelle Y.; Williams, Kelly Porter; et al

    2015-04-20

    Yersinia enterocolitica is typically considered an extracellular pathogen; however, during the course of an infection, a significant number of bacteria are stably maintained within host cell vacuoles. Little is known about this population and the role it plays during an infection. To address this question and to elucidate the spatially and temporally dynamic gene expression patterns of Y. enterocoliticabiovar 1B through the course of an in vitro infection, transcriptome sequencing and differential gene expression analysis of bacteria infecting murine macrophage cells were performed under four distinct conditions. Bacteria were first grown in a nutrient-rich medium at 26°C to establish amore » baseline of gene expression that is unrelated to infection. The transcriptomes of these bacteria were then compared to bacteria grown in a conditioned cell culture medium at 37°C to identify genes that were differentially expressed in response to the increased temperature and medium but not in response to host cells. Infections were then performed, and the transcriptomes of bacteria found on the extracellular surface and intracellular compartments were analyzed individually. The upregulated genes revealed potential roles for a variety of systems in promoting intracellular virulence, including the Ysa type III secretion system, the Yts2 type II secretion system, and the Tad pilus. It was further determined that mutants of each of these systems had decreased virulence while infecting macrophages. Overall, these results reveal the complete set of genes expressed by Y. enterocolitica in response to infection and provide the groundwork for future virulence studies.« less

  7. Macrophage defense mechanisms against intracellular bacteria

    PubMed Central

    Weiss, Günter; Schaible, Ulrich E

    2015-01-01

    Macrophages and neutrophils play a decisive role in host responses to intracellular bacteria including the agent of tuberculosis (TB), Mycobacterium tuberculosis as they represent the forefront of innate immune defense against bacterial invaders. At the same time, these phagocytes are also primary targets of intracellular bacteria to be abused as host cells. Their efficacy to contain and eliminate intracellular M. tuberculosis decides whether a patient initially becomes infected or not. However, when the infection becomes chronic or even latent (as in the case of TB) despite development of specific immune activation, phagocytes have also important effector functions. Macrophages have evolved a myriad of defense strategies to combat infection with intracellular bacteria such as M. tuberculosis. These include induction of toxic anti-microbial effectors such as nitric oxide and reactive oxygen intermediates, the stimulation of microbe intoxication mechanisms via acidification or metal accumulation in the phagolysosome, the restriction of the microbe's access to essential nutrients such as iron, fatty acids, or amino acids, the production of anti-microbial peptides and cytokines, along with induction of autophagy and efferocytosis to eliminate the pathogen. On the other hand, M. tuberculosis, as a prime example of a well-adapted facultative intracellular bacterium, has learned during evolution to counter-balance the host's immune defense strategies to secure survival or multiplication within this otherwise hostile environment. This review provides an overview of innate immune defense of macrophages directed against intracellular bacteria with a focus on M. tuberculosis. Gaining more insights and knowledge into this complex network of host-pathogen interaction will identify novel target sites of intervention to successfully clear infection at a time of rapidly emerging multi-resistance of M. tuberculosis against conventional antibiotics. PMID:25703560

  8. NAD+-Glycohydrolase Promotes Intracellular Survival of Group A Streptococcus

    PubMed Central

    Sharma, Onkar; O’Seaghdha, Maghnus; Velarde, Jorge J.; Wessels, Michael R.

    2016-01-01

    A global increase in invasive infections due to group A Streptococcus (S. pyogenes or GAS) has been observed since the 1980s, associated with emergence of a clonal group of strains of the M1T1 serotype. Among other virulence attributes, the M1T1 clone secretes NAD+-glycohydrolase (NADase). When GAS binds to epithelial cells in vitro, NADase is translocated into the cytosol in a process mediated by streptolysin O (SLO), and expression of these two toxins is associated with enhanced GAS intracellular survival. Because SLO is required for NADase translocation, it has been difficult to distinguish pathogenic effects of NADase from those of SLO. To resolve the effects of the two proteins, we made use of anthrax toxin as an alternative means to deliver NADase to host cells, independently of SLO. We developed a novel method for purification of enzymatically active NADase fused to an amino-terminal fragment of anthrax toxin lethal factor (LFn-NADase) that exploits the avid, reversible binding of NADase to its endogenous inhibitor. LFn-NADase was translocated across a synthetic lipid bilayer in vitro in the presence of anthrax toxin protective antigen in a pH-dependent manner. Exposure of human oropharyngeal keratinocytes to LFn-NADase in the presence of protective antigen resulted in cytosolic delivery of NADase activity, inhibition of protein synthesis, and cell death, whereas a similar construct of an enzymatically inactive point mutant had no effect. Anthrax toxin-mediated delivery of NADase in an amount comparable to that observed during in vitro infection with live GAS rescued the defective intracellular survival of NADase-deficient GAS and increased the survival of SLO-deficient GAS. Confocal microscopy demonstrated that delivery of LFn-NADase prevented intracellular trafficking of NADase-deficient GAS to lysosomes. We conclude that NADase mediates cytotoxicity and promotes intracellular survival of GAS in host cells. PMID:26938870

  9. NAD+-Glycohydrolase Promotes Intracellular Survival of Group A Streptococcus.

    PubMed

    Sharma, Onkar; O'Seaghdha, Maghnus; Velarde, Jorge J; Wessels, Michael R

    2016-03-01

    A global increase in invasive infections due to group A Streptococcus (S. pyogenes or GAS) has been observed since the 1980s, associated with emergence of a clonal group of strains of the M1T1 serotype. Among other virulence attributes, the M1T1 clone secretes NAD+-glycohydrolase (NADase). When GAS binds to epithelial cells in vitro, NADase is translocated into the cytosol in a process mediated by streptolysin O (SLO), and expression of these two toxins is associated with enhanced GAS intracellular survival. Because SLO is required for NADase translocation, it has been difficult to distinguish pathogenic effects of NADase from those of SLO. To resolve the effects of the two proteins, we made use of anthrax toxin as an alternative means to deliver NADase to host cells, independently of SLO. We developed a novel method for purification of enzymatically active NADase fused to an amino-terminal fragment of anthrax toxin lethal factor (LFn-NADase) that exploits the avid, reversible binding of NADase to its endogenous inhibitor. LFn-NADase was translocated across a synthetic lipid bilayer in vitro in the presence of anthrax toxin protective antigen in a pH-dependent manner. Exposure of human oropharyngeal keratinocytes to LFn-NADase in the presence of protective antigen resulted in cytosolic delivery of NADase activity, inhibition of protein synthesis, and cell death, whereas a similar construct of an enzymatically inactive point mutant had no effect. Anthrax toxin-mediated delivery of NADase in an amount comparable to that observed during in vitro infection with live GAS rescued the defective intracellular survival of NADase-deficient GAS and increased the survival of SLO-deficient GAS. Confocal microscopy demonstrated that delivery of LFn-NADase prevented intracellular trafficking of NADase-deficient GAS to lysosomes. We conclude that NADase mediates cytotoxicity and promotes intracellular survival of GAS in host cells. PMID:26938870

  10. Unveiling the Intracellular Survival Gene Kit of Trypanosomatid Parasites

    PubMed Central

    Bartholomeu, Daniella Castanheira; de Paiva, Rita Marcia Cardoso; Mendes, Tiago A. O.; DaRocha, Wanderson D.; Teixeira, Santuza M. R.

    2014-01-01

    Trypanosomatids are unicellular protozoans of medical and economical relevance since they are the etiologic agents of infectious diseases in humans as well as livestock. Whereas Trypanosoma cruzi and different species of Leishmania are obligate intracellular parasites, Trypanosoma brucei and other trypanosomatids develop extracellularly throughout their entire life cycle. After their genomes have been sequenced, various comparative genomic studies aimed at identifying sequences involved with host cell invasion and intracellular survival have been described. However, for only a handful of genes, most of them present exclusively in the T. cruzi or Leishmania genomes, has there been any experimental evidence associating them with intracellular parasitism. With the increasing number of published complete genome sequences of members of the trypanosomatid family, including not only different Trypanosoma and Leishmania strains and subspecies but also trypanosomatids that do not infect humans or other mammals, we may now be able to contemplate a slightly better picture regarding the specific set of parasite factors that defines each organism's mode of living and the associated disease phenotypes. Here, we review the studies concerning T. cruzi and Leishmania genes that have been implicated with cell invasion and intracellular parasitism and also summarize the wealth of new information regarding the mode of living of intracellular parasites that is resulting from comparative genome studies that are based on increasingly larger trypanosomatid genome datasets. PMID:25474314

  11. Membranes, mechanics, and intracellular transport

    NASA Astrophysics Data System (ADS)

    Parthasarathy, Raghuveer

    2012-10-01

    Cellular membranes are remarkable materials -- self-assembled, flexible, two-dimensional fluids. Understanding how proteins manipulate membrane curvature is crucial to understanding the transport of cargo in cells, yet the mechanical activities of trafficking proteins remain poorly understood. Using an optical-trap based assay involving dynamic deformation of biomimetic membranes, we have examined the behavior of Sar1, a key component of the COPII family of transport proteins. We find that Sar1 from yeast (S. cerevisiae) lowers membrane rigidity by up to 100% as a function of its concentration, thereby lowering the energetic cost of membrane deformation. Human Sar1 proteins can also lower the mechanical rigidity of the membranes to which they bind. However, unlike the yeast proteins, the rigidity is not a monotonically decreasing function of concentration but rather shows increased rigidity and decreased mobility at high concentrations that implies interactions between proteins. In addition to describing this study of membrane mechanics, I'll also discuss some topics relevant to a range of biophysical investigations, such as the insights provided by imaging methods and open questions in the dynamics of multicellular systems.

  12. Intracellular survival of Candida glabrata in macrophages: immune evasion and persistence.

    PubMed

    Kasper, Lydia; Seider, Katja; Hube, Bernhard

    2015-08-01

    Candida glabrata is a successful human opportunistic pathogen which causes superficial but also life-threatening systemic infections. During infection, C. glabrata has to cope with cells of the innate immune system such as macrophages, which belong to the first line of defense against invading pathogens. Candida glabrata is able to survive and even replicate inside macrophages while causing surprisingly low damage and cytokine release. Here, we present an overview of recent studies dealing with the interaction of C. glabrata with macrophages, from phagocytosis to intracellular growth and escape. We review the strategies of C. glabrata that permit intracellular survival and replication, including poor host cell activation, modification of phagosome maturation and phagosome pH, adaptation to antimicrobial activities, and mechanisms to overcome the nutrient limitations within the phagosome. In summary, these studies suggest that survival within macrophages may be an immune evasion and persistence strategy of C. glabrata during infection. PMID:26066553

  13. Decreased intracellular survival of an fkpA mutant of Salmonella typhimurium Copenhagen.

    PubMed

    Horne, S M; Kottom, T J; Nolan, L K; Young, K D

    1997-02-01

    The fkpA gene of Salmonella typhimurium encodes a protein similar to the macrophage infectivity potentiator (Mip) proteins of Legionella pneumophila and Chlamydia trachomatis. Because Mip proteins enhance the ability of these intracellular pathogens to survive within macrophages and epithelial cells, we tested whether the product of the fkpA gene would have the same effect on the intracellular growth of a virulent strain of S. typhimurium. By a series of P22 transductions, the fkpA gene of S. typhimurium Copenhagen was replaced with the inactive fkpA1::omega-Cm gene from Escherichia coli, creating the mutant S. typhimurium KY32H1. The Copenhagen and KY32H1 strains were equally able to enter Caco-2 cells (an epithelial cell line) and J774.A1 cells (a macrophage-like cell line). However, compared to the parent, the fkpA mutant survived less well in both types of cells during the first 6 h after infection. The number of viable intracellular S. typhimurium Copenhagen bacteria remained constant 6 h after infection of Caco-2 cells, but the viability of S. typhimurium KY32H1 decreased significantly by 4 h postinfection. The fkpA mutant also exhibited a reduced ability to survive intracellularly in J774.A1 cells as little as 2 h postinfection. Complementation of the fkpA mutation by a plasmid-borne wild-type fkpA gene from E. coli restored the ability of S. typhimurium KY32H1 to grow normally in J774.A1 cells. Thus, expression of the mip-like fkpA gene confers on S. typhimurium Copenhagen properties analogous to those mediated by the Mip proteins in other intracellular pathogens, suggesting that this mechanism may play a role in the virulence and/or intracellular growth of numerous bacteria. PMID:9009347

  14. Intracellular and extracellular ATP coordinately regulate the inverse correlation between osteoclast survival and bone resorption.

    PubMed

    Miyazaki, Tsuyoshi; Iwasawa, Mitsuyasu; Nakashima, Tomoki; Mori, Shuuichi; Shigemoto, Kazuhiro; Nakamura, Hiroaki; Katagiri, Hideki; Takayanagi, Hiroshi; Tanaka, Sakae

    2012-11-01

    Osteoclasts, highly differentiated bone-resorbing cells of hematopoietic origin, have two conflicting tendencies: a lower capacity to survive and a higher capacity to execute energy-consuming activities such as bone resorption. Here, we report that when compared with their precursors, mature mitochondria-rich osteoclasts have lower levels of intracellular ATP, which is associated with receptor activator of nuclear factor κ-B ligand (RANKL)-induced Bcl-x(L) down-regulation. Severe ATP depletion, caused by disrupting mitochondrial transcription factor A (Tfam) gene, leads to increased bone-resorbing activity despite accelerated apoptosis. Although AMP-activated protein kinase (AMPK) activation by ATP depletion is not involved in the regulation of osteoclast function, the release of ATP from intracellular stores negatively regulates bone-resorbing activity through an autocrine/paracrine feedback loop by altering cytoskeletal structures. Furthermore, osteoclasts derived from aged mice exhibit reduced mitochondrial DNA (mtDNA) and intracellular ATP levels with increased bone-resorbing activity, implicating the possible involvement of age-related mitochondrial dysfunction in osteoporosis. Thus, our study provides evidence for a mechanism underlying the control of cellular functions by reciprocal changes in intracellular and extracellular ATP, which regulate the negative correlation between osteoclast survival and bone resorption. PMID:22988253

  15. Intracellular Mechanics of Migrating FibroblastsD⃞

    PubMed Central

    Kole, Thomas P.; Tseng, Yiider; Jiang, Ingjye; Katz, Joseph L.; Wirtz, Denis

    2005-01-01

    Cell migration is a highly coordinated process that occurs through the translation of biochemical signals into specific biomechanical events. The biochemical and structural properties of the proteins involved in cell motility, as well as their subcellular localization, have been studied extensively. However, how these proteins work in concert to generate the mechanical properties required to produce global motility is not well understood. Using intracellular microrheology and a fibroblast scratch-wound assay, we show that cytoskeleton reorganization produced by motility results in mechanical stiffening of both the leading lamella and the perinuclear region of motile cells. This effect is significantly more pronounced in the leading edge, suggesting that the mechanical properties of migrating fibroblasts are spatially coordinated. Disruption of the microtubule network by nocodazole treatment results in the arrest of cell migration and a loss of subcellular mechanical polarization; however, the overall mechanical properties of the cell remain mostly unchanged. Furthermore, we find that activation of Rac and Cdc42 in quiescent fibroblasts elicits mechanical behavior similar to that of migrating cells. We conclude that a polarized mechanics of the cytoskelton is essential for directed cell migration and is coordinated through microtubules. PMID:15483053

  16. Survival of Brevibacterium linens during nutrient starvation and intracellular changes.

    PubMed

    Boyaval, P; Boyaval, E; Desmazeaud, M J

    1985-03-01

    The present work reports the survival capacity of a strain of Brevibacterium linens isolated from a French camembert cheese and the ensuing changes in cell composition. Exponentially growing cells were harvested, washed and resuspended with shaking in pH 8.0 buffer at 21 degrees C in the absence of a carbon source. The viability of this strain, assessed with slide cultures, is much less than that of coryneform bacteria isolated from soil samples, even though no cell lysis was detected. Intracellular RNA was rapidly consumed during the first few days although magnesium levels remained high. The quantity of DNA initially increased by 17% within 24 h and then remained stable during the 30 days of the experiment. During the same period, absorbance of the medium at 260 nm reached 2 absorbance units. Reserve polysaccharides in this strain are less abundant than in Arthrobacter and were rapidly consumed. Proteolysis was regular and thus maintained a pool of free amino acids which was greater than 60% of the initial value. There was a parallel accumulation of ammonia in the medium. Catalase activity decreased regularly during the first 80 h whereas the quantity of Adenosine-5'-triphosphate (ATP) dropped by 47% in 10 h, stabilizing at less than 10% of its initial value. Cell respiration declined very rapidly and was very low after 24 h. PMID:3994487

  17. Burkholderia pseudomallei Differentially Regulates Host Innate Immune Response Genes for Intracellular Survival in Lung Epithelial Cells

    PubMed Central

    Vellasamy, Kumutha Malar; Mariappan, Vanitha; Shankar, Esaki M.; Vadivelu, Jamuna

    2016-01-01

    Background Burkholderia pseudomallei, the causative agent of melioidosis poses a serious threat to humankind. B. pseudomallei secretes numerous virulence proteins that alter host cell functions to escape from intracellular immune sensors. However, the events underlying disease pathogenesis are poorly understood. Methods We determined the ability of B. pseudomallei to invade and survive intracellularly in A549 human lung epithelial cells, and also investigated the early transcriptional responses using an Illumina HumanHT-12 v4 microarray platform, after three hours of exposure to live B. pseudomallei (BCMS) and its secreted proteins (CCMS). Results We found that the ability of B. pseudomallei to invade and survive intracellularly correlated with increase of multiplicity of infection and duration of contact. Activation of host carbohydrate metabolism and apoptosis as well as suppression of amino acid metabolism and innate immune responses both by live bacteria and its secreted proteins were evident. These early events might be linked to initial activation of host genes directed towards bacterial dissemination from lungs to target organs (via proposed in vivo mechanisms) or to escape potential sensing by macrophages. Conclusion Understanding the early responses of A549 cells toward B. pseudomallei infection provide preliminary insights into the likely pathogenesis mechanisms underlying melioidosis, and could contribute to development of novel intervention strategies to combat B. pseudomallei infections. PMID:27367858

  18. Intracellular Uropathogenic E. coli Exploits Host Rab35 for Iron Acquisition and Survival within Urinary Bladder Cells

    PubMed Central

    Dikshit, Neha; Bist, Pradeep; Fenlon, Shannon N.; Pulloor, Niyas Kudukkil; Chua, Christelle En Lin; Scidmore, Marci A.; Carlyon, Jason A.; Tang, Bor Luen; Chen, Swaine L.; Sukumaran, Bindu

    2015-01-01

    Recurrent urinary tract infections (UTIs) caused by uropathogenic E. coli (UPEC) are common and morbid infections with limited therapeutic options. Previous studies have demonstrated that persistent intracellular infection of bladder epithelial cells (BEC) by UPEC contributes to recurrent UTI in mouse models of infection. However, the mechanisms employed by UPEC to survive within BEC are incompletely understood. In this study we aimed to understand the role of host vesicular trafficking proteins in the intracellular survival of UPEC. Using a cell culture model of intracellular UPEC infection, we found that the small GTPase Rab35 facilitates UPEC survival in UPEC-containing vacuoles (UCV) within BEC. Rab35 plays a role in endosomal recycling of transferrin receptor (TfR), the key protein responsible for transferrin–mediated cellular iron uptake. UPEC enhance the expression of both Rab35 and TfR and recruit these proteins to the UCV, thereby supplying UPEC with the essential nutrient iron. Accordingly, Rab35 or TfR depleted cells showed significantly lower intracellular iron levels and reduced ability to support UPEC survival. In the absence of Rab35, UPEC are preferentially trafficked to degradative lysosomes and killed. Furthermore, in an in vivo murine model of persistent intracellular infection, Rab35 also colocalizes with intracellular UPEC. We propose a model in which UPEC subverts two different vesicular trafficking pathways (endosomal recycling and degradative lysosomal fusion) by modulating Rab35, thereby simultaneously enhancing iron acquisition and avoiding lysosomal degradation of the UCV within bladder epithelial cells. Our findings reveal a novel survival mechanism of intracellular UPEC and suggest a potential avenue for therapeutic intervention against recurrent UTI. PMID:26248231

  19. Mechanisms of Obligatory Intracellular Infection with Anaplasma phagocytophilum

    PubMed Central

    Rikihisa, Yasuko

    2011-01-01

    Summary: Anaplasma phagocytophilum persists in nature by cycling between mammals and ticks. Human infection by the bite of an infected tick leads to a potentially fatal emerging disease called human granulocytic anaplasmosis. A. phagocytophilum is an obligatory intracellular bacterium that replicates inside mammalian granulocytes and the salivary gland and midgut cells of ticks. A. phagocytophilum evolved the remarkable ability to hijack the regulatory system of host cells. A. phagocytophilum alters vesicular traffic to create an intracellular membrane-bound compartment that allows replication in seclusion from lysosomes. The bacterium downregulates or actively inhibits a number of innate immune responses of mammalian host cells, and it upregulates cellular cholesterol uptake to acquire cholesterol for survival. It also upregulates several genes critical for the infection of ticks, and it prolongs tick survival at freezing temperatures. Several host factors that exacerbate infection have been identified, including interleukin-8 (IL-8) and cholesterol. Host factors that overcome infection include IL-12 and gamma interferon (IFN-γ). Two bacterial type IV secretion effectors and several bacterial proteins that associate with inclusion membranes have been identified. An understanding of the molecular mechanisms underlying A. phagocytophilum infection will foster the development of creative ideas to prevent or treat this emerging tick-borne disease. PMID:21734244

  20. Salmonella Engages Host MicroRNAs To Modulate SUMOylation: a New Arsenal for Intracellular Survival.

    PubMed

    Verma, Smriti; Mohapatra, Gayatree; Ahmad, Salman Mustfa; Rana, Sarika; Jain, Swati; Khalsa, Jasneet Kaur; Srikanth, C V

    2015-09-01

    Posttranslational modifications (PTMs) can alter many fundamental properties of a protein. One or combinations of them have been known to regulate the dynamics of many cellular pathways and consequently regulate all vital processes. Understandably, pathogens have evolved sophisticated strategies to subvert these mechanisms to achieve instantaneous control over host functions. Here, we present the first report of modulation by intestinal pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) of host SUMOylation, a PTM pathway central to all fundamental cellular processes. Both in cell culture and in a mouse model, we observed that S. Typhimurium infection led to a dynamic SUMO-conjugated proteome alteration. The intracellular survival of S. Typhimurium was dependent on SUMO status as revealed by reduced infection and Salmonella-induced filaments (SIFs) in SUMO-upregulated cells. S. Typhimurium-dependent SUMO modulation was seen as a result of depletion of crucial SUMO pathway enzymes Ubc-9 and PIAS1, at both the protein and the transcript levels. Mechanistically, depletion of Ubc-9 relied on upregulation of small noncoding RNAs miR30c and miR30e during S. Typhimurium infection. This was necessary and sufficient for both down-modulation of Ubc-9 and a successful infection. Thus, we demonstrate a novel strategy of pathogen-mediated perturbation of host SUMOylation, an integral mechanism underlying S. Typhimurium infection and intracellular survival. PMID:26100020

  1. Ehrlichia chaffeensis TRP32 Interacts with Host Cell Targets That Influence Intracellular Survival

    PubMed Central

    Luo, Tian

    2012-01-01

    Ehrlichia chaffeensis is an obligately intracellular bacterium that exhibits tropism for mononuclear phagocytes and survives by evading host cell defense mechanisms. Recently, molecular interactions of E. chaffeensis tandem repeat proteins 47 and 120 (TRP47 and -120) and the eukaryotic host cell have been described. In this investigation, yeast two-hybrid analysis demonstrated that an E. chaffeensis type 1 secretion system substrate, TRP32, interacts with a diverse group of human proteins associated with major biological processes of the host cell, including protein synthesis, trafficking, degradation, immune signaling, cell signaling, iron metabolism, and apoptosis. Eight target proteins, including translation elongation factor 1 alpha 1 (EF1A1), deleted in azoospermia (DAZ)-associated protein 2 (DAZAP2), ferritin light polypeptide (FTL), CD63, CD14, proteasome subunit beta type 1 (PSMB1), ring finger and CCCH-type domain 1 (RC3H1), and tumor protein p53-inducible protein 11 (TP53I11) interacted with TRP32 as determined by coimmunoprecipitation assays, colocalization with TRP32 in HeLa and THP-1 cells, and/or RNA interference. Interactions between TRP32 and host targets localized to the E. chaffeensis morulae or in the host cell cytoplasm adjacent to morulae. Common or closely related interacting partners of E. chaffeensis TRP32, TRP47, and TRP120 demonstrate a molecular convergence on common cellular processes and molecular cross talk between Ehrlichia TRPs and host targets. These findings further support the role of TRPs as effectors that promote intracellular survival. PMID:22547548

  2. The opportunistic pathogen Enterococcus faecalis resists phagosome acidification and autophagy to promote intracellular survival in macrophages.

    PubMed

    Zou, Jun; Shankar, Nathan

    2016-06-01

    While many strains of Enterococcus faecalis have been reported to be capable of surviving within macrophages for extended periods, the exact mechanisms involved are largely unknown. In this study, we found that after phagocytosis by macrophages, enterococci-containing vacuoles resist acidification, and E. faecalis is resistant to low pH. Ultrastructural examination of the enterococci-containing vacuole by transmission electron microscopy revealed a single membrane envelope, with no evidence of the classical double-membraned autophagosomes. Western blot analysis further confirmed that E. faecalis could trigger inhibition of the production of LC3-II during infection. By employing cells transfected with RFP-LC3 plasmid and infected with GFP-labelled E. faecalis, we also observed that E. faecalis was not delivered into autophagosomes during macrophage infection. While these observations indicated no role for autophagy in elimination of intracellular E. faecalis, enhanced production of reactive oxygen species and nitric oxide were keys to this process. Stimulation of autophagy suppressed the intracellular survival of E. faecalis in macrophages in vitro and decreased the burden of E. faecalis in vivo. In summary, the results from this study offer new insights into the interaction of E. faecalis with host cells and may provide a new approach to treatment of enterococcal infections. PMID:26663775

  3. The role of autophagy in the intracellular survival of Campylobacter concisus

    PubMed Central

    Burgos-Portugal, Jose A.; Mitchell, Hazel M.; Castaño-Rodríguez, Natalia; Kaakoush, Nadeem O.

    2014-01-01

    Campylobacter concisus is an emerging pathogen that has been associated with gastrointestinal diseases. Given the importance of autophagy for the elimination of intracellular bacteria and the subversion of this process by pathogenic bacteria, we investigated the role of autophagy in C. concisus intracellular survival. Gentamicin protection assays were employed to assess intracellular levels of C. concisus within Caco-2 cells, following autophagy induction and inhibition. To assess the interaction between C. concisus and autophagosomes, confocal microscopy, scanning electron microscopy, and transmission electron microscopy were employed. Expression levels of 84 genes involved in the autophagy process were measured using qPCR. Autophagy inhibition resulted in two- to four-fold increases in intracellular levels of C. concisus within Caco-2 cells, while autophagy induction resulted in a significant reduction in intracellular levels or bacterial clearance. C. concisus strains with low intracellular survival levels showed a dramatic increase in these levels upon autophagy inhibition. Confocal microscopy showed co-localization of the bacterium with autophagosomes, while transmission electron microscopy identified intracellular bacteria persisting within autophagic vesicles. Further, qPCR showed that following infection, 13 genes involved in the autophagy process were significantly regulated, and a further five showed borderline results, with an overall indication towards a dampening effect exerted by the bacterium on this process. Our data collectively indicates that while autophagy is important for the clearance of C. concisus, some strains may manipulate this process to benefit their intracellular survival. PMID:24918042

  4. Proline Mechanisms of Stress Survival

    PubMed Central

    Liang, Xinwen; Zhang, Lu; Natarajan, Sathish Kumar

    2013-01-01

    Abstract Significance: The imino acid proline is utilized by different organisms to offset cellular imbalances caused by environmental stress. The wide use in nature of proline as a stress adaptor molecule indicates that proline has a fundamental biological role in stress response. Understanding the mechanisms by which proline enhances abiotic/biotic stress response will facilitate agricultural crop research and improve human health. Recent Advances: It is now recognized that proline metabolism propels cellular signaling processes that promote cellular apoptosis or survival. Studies have shown that proline metabolism influences signaling pathways by increasing reactive oxygen species (ROS) formation in the mitochondria via the electron transport chain. Enhanced ROS production due to proline metabolism has been implicated in the hypersensitive response in plants, lifespan extension in worms, and apoptosis, tumor suppression, and cell survival in animals. Critical Issues: The ability of proline to influence disparate cellular outcomes may be governed by ROS levels generated in the mitochondria. Defining the threshold at which proline metabolic enzyme expression switches from inducing survival pathways to cellular apoptosis would provide molecular insights into cellular redox regulation by proline. Are ROS the only mediators of proline metabolic signaling or are other factors involved? Future Directions: New evidence suggests that proline biosynthesis enzymes interact with redox proteins such as thioredoxin. An important future pursuit will be to identify other interacting partners of proline metabolic enzymes to uncover novel regulatory and signaling networks of cellular stress response. Antioxid. Redox Signal. 19, 998–1011. PMID:23581681

  5. Ehrlichia chaffeensis Exploits Host SUMOylation Pathways To Mediate Effector-Host Interactions and Promote Intracellular Survival

    PubMed Central

    Dunphy, Paige Selvy; Luo, Tian

    2014-01-01

    Ehrlichia chaffeensis is an obligately intracellular Gram-negative bacterium that selectively infects mononuclear phagocytes. We recently reported that E. chaffeensis utilizes a type 1 secretion (T1S) system to export tandem repeat protein (TRP) effectors and demonstrated that these effectors interact with a functionally diverse array of host proteins. By way of these interactions, TRP effectors modulate host cell functions; however, the molecular basis of these interactions and their roles in ehrlichial pathobiology are not well defined. In this study, we describe the first bacterial protein posttranslational modification (PTM) by the small ubiquitin-like modifier (SUMO). The E. chaffeensis T1S effector TRP120 is conjugated to SUMO at a carboxy-terminal canonical consensus SUMO conjugation motif in vitro and in human cells. In human cells, TRP120 was selectively conjugated with SUMO2/3 isoforms. Disruption of TRP120 SUMOylation perturbed interactions with known host proteins, through predicted SUMO interaction motif-dependent and -independent mechanisms. E. chaffeensis infection did not result in dramatic changes in the global host SUMOylated protein profile, but a robust colocalization of predominately SUMO1 with ehrlichial inclusions was observed. Inhibiting the SUMO pathway with a small-molecule inhibitor had a significant impact on E. chaffeensis replication and recruitment of the TRP120-interacting protein polycomb group ring finger protein 5 (PCGF5) to the inclusion, indicating that the SUMO pathway is critical for intracellular survival. This study reveals the novel exploitation of the SUMO pathway by Ehrlichia, which facilitates effector-eukaryote interactions necessary to usurp the host and create a permissive intracellular niche. PMID:25047847

  6. Identification of a Novel Small Non-Coding RNA Modulating the Intracellular Survival of Brucella melitensis.

    PubMed

    Wang, Yufei; Ke, Yuehua; Xu, Jie; Wang, Ligui; Wang, Tongkun; Liang, Hui; Zhang, Wei; Gong, Chunli; Yuan, Jiuyun; Zhuang, Yubin; An, Chang; Lei, Shuangshuang; Du, Xinying; Wang, Zhoujia; Li, Wenna; Yuan, Xitong; Huang, Liuyu; Yang, Xiaoli; Chen, Zeliang

    2015-01-01

    Bacterial small non-coding RNAs (sRNAs) are gene expression modulators respond to environmental changes, stressful conditions, and pathogenesis. In this study, by using a combined bioinformatic and experimental approach, eight novel sRNA genes were identified in intracellular pathogen Brucella melitensis. BSR0602, one sRNA that was highly induced in stationary phase, was further examined and found to modulate the intracellular survival of B. melitensis. BSR0602 was present at very high levels in vitro under stresses similar to those encountered during infection in host macrophages. Furthermore, BSR0602 was found to be highly expressed in the spleens of infected mice, suggesting its potential role in the control of pathogenesis. BSR0602 targets the mRNAs coding for gntR, a global transcriptional regulator, which is required for B. melitensis virulence. Overexpression of BSR0602 results in distinct reduction in the gntR mRNA level. B. melitensis with high level of BSR0602 is defective in bacteria intracellular survival in macrophages and defective in growth in the spleens of infected mice. Therefore, BSR0602 may directly inhibit the expression of gntR, which then impairs Brucellae intracellular survival and contributes to Brucella infection. Our findings suggest that BSR0602 is responsible for bacterial adaptation to stress conditions and thus modulate B. melitensis intracellular survival. PMID:25852653

  7. Identification of a Novel Small Non-Coding RNA Modulating the Intracellular Survival of Brucella melitensis

    PubMed Central

    Wang, Yufei; Ke, Yuehua; Xu, Jie; Wang, Ligui; Wang, Tongkun; Liang, Hui; Zhang, Wei; Gong, Chunli; Yuan, Jiuyun; Zhuang, Yubin; An, Chang; Lei, Shuangshuang; Du, Xinying; Wang, Zhoujia; Li, Wenna; Yuan, Xitong; Huang, Liuyu; Yang, Xiaoli; Chen, Zeliang

    2015-01-01

    Bacterial small non-coding RNAs (sRNAs) are gene expression modulators respond to environmental changes, stressful conditions, and pathogenesis. In this study, by using a combined bioinformatic and experimental approach, eight novel sRNA genes were identified in intracellular pathogen Brucella melitensis. BSR0602, one sRNA that was highly induced in stationary phase, was further examined and found to modulate the intracellular survival of B. melitensis. BSR0602 was present at very high levels in vitro under stresses similar to those encountered during infection in host macrophages. Furthermore, BSR0602 was found to be highly expressed in the spleens of infected mice, suggesting its potential role in the control of pathogenesis. BSR0602 targets the mRNAs coding for gntR, a global transcriptional regulator, which is required for B. melitensis virulence. Overexpression of BSR0602 results in distinct reduction in the gntR mRNA level. B. melitensis with high level of BSR0602 is defective in bacteria intracellular survival in macrophages and defective in growth in the spleens of infected mice. Therefore, BSR0602 may directly inhibit the expression of gntR, which then impairs Brucellae intracellular survival and contributes to Brucella infection. Our findings suggest that BSR0602 is responsible for bacterial adaptation to stress conditions and thus modulate B. melitensis intracellular survival. PMID:25852653

  8. M2 Polarization of Human Macrophages Favors Survival of the Intracellular Pathogen Chlamydia pneumoniae.

    PubMed

    Buchacher, Tanja; Ohradanova-Repic, Anna; Stockinger, Hannes; Fischer, Michael B; Weber, Viktoria

    2015-01-01

    Intracellular pathogens have developed various strategies to escape immunity to enable their survival in host cells, and many bacterial pathogens preferentially reside inside macrophages, using diverse mechanisms to penetrate their defenses and to exploit their high degree of metabolic diversity and plasticity. Here, we characterized the interactions of the intracellular pathogen Chlamydia pneumoniae with polarized human macrophages. Primary human monocytes were pre-differentiated with granulocyte macrophage colony-stimulating factor or macrophage colony-stimulating factor for 7 days to yield M1-like and M2-like macrophages, which were further treated with interferon-γ and lipopolysaccharide or with interleukin-4 for 48 h to obtain fully polarized M1 and M2 macrophages. M1 and M2 cells exhibited distinct morphology with round or spindle-shaped appearance for M1 and M2, respectively, distinct surface marker profiles, as well as different cytokine and chemokine secretion. Macrophage polarization did not influence uptake of C. pneumoniae, since comparable copy numbers of chlamydial DNA were detected in M1 and M2 at 6 h post infection, but an increase in chlamydial DNA over time indicating proliferation was only observed in M2. Accordingly, 72±5% of M2 vs. 48±7% of M1 stained positive for chlamydial lipopolysaccharide, with large perinuclear inclusions in M2 and less clearly bordered inclusions for M1. Viable C. pneumoniae was present in lysates from M2, but not from M1 macrophages. The ability of M1 to restrict chlamydial replication was not observed in M1-like macrophages, since chlamydial load showed an equal increase over time for M1-like and M2-like macrophages. Our findings support the importance of macrophage polarization for the control of intracellular infection, and show that M2 are the preferred survival niche for C. pneumoniae. M1 did not allow for chlamydial proliferation, but failed to completely eliminate chlamydial infection, giving further evidence

  9. M2 Polarization of Human Macrophages Favors Survival of the Intracellular Pathogen Chlamydia pneumoniae

    PubMed Central

    Buchacher, Tanja; Ohradanova-Repic, Anna; Stockinger, Hannes

    2015-01-01

    Intracellular pathogens have developed various strategies to escape immunity to enable their survival in host cells, and many bacterial pathogens preferentially reside inside macrophages, using diverse mechanisms to penetrate their defenses and to exploit their high degree of metabolic diversity and plasticity. Here, we characterized the interactions of the intracellular pathogen Chlamydia pneumoniae with polarized human macrophages. Primary human monocytes were pre-differentiated with granulocyte macrophage colony-stimulating factor or macrophage colony-stimulating factor for 7 days to yield M1-like and M2-like macrophages, which were further treated with interferon-γ and lipopolysaccharide or with interleukin-4 for 48 h to obtain fully polarized M1 and M2 macrophages. M1 and M2 cells exhibited distinct morphology with round or spindle-shaped appearance for M1 and M2, respectively, distinct surface marker profiles, as well as different cytokine and chemokine secretion. Macrophage polarization did not influence uptake of C. pneumoniae, since comparable copy numbers of chlamydial DNA were detected in M1 and M2 at 6 h post infection, but an increase in chlamydial DNA over time indicating proliferation was only observed in M2. Accordingly, 72±5% of M2 vs. 48±7% of M1 stained positive for chlamydial lipopolysaccharide, with large perinuclear inclusions in M2 and less clearly bordered inclusions for M1. Viable C. pneumoniae was present in lysates from M2, but not from M1 macrophages. The ability of M1 to restrict chlamydial replication was not observed in M1-like macrophages, since chlamydial load showed an equal increase over time for M1-like and M2-like macrophages. Our findings support the importance of macrophage polarization for the control of intracellular infection, and show that M2 are the preferred survival niche for C. pneumoniae. M1 did not allow for chlamydial proliferation, but failed to completely eliminate chlamydial infection, giving further evidence

  10. Ferrochelatase Is Present in Brucella abortus and Is Critical for Its Intracellular Survival and Virulence

    PubMed Central

    Almirón, Marta; Martínez, Marcela; Sanjuan, Norberto; Ugalde, Rodolfo A.

    2001-01-01

    Brucella spp. are pathogenic bacteria that cause brucellosis, an animal disease which can also affect humans. Although understanding the pathogenesis is important for the health of animals and humans, little is known about virulence factors associated with it. In order for chronic disease to be established, Brucella spp. have developed the ability to survive inside phagocytes by evading cell defenses. It hides inside vacuoles, where it then replicates, indicating that it has an active metabolism. The purpose of this work was to obtain better insight into the intracellular metabolism of Brucella abortus. During a B. abortus genomic sequencing project, a clone coding a putative gene homologous to hemH was identified and sequenced. The amino acid sequence revealed high homology to members of the ferrochelatase family. A knockout mutant displayed auxotrophy for hemin, defective intracellular survival inside J774 and HeLa cells, and lack of virulence in BALB/c mice. This phenotype was overcome by complementing the mutant strain with a plasmid harboring wild-type hemH. These data demonstrate that B. abortus synthesizes its own heme and also has the ability to use an external source of heme; however, inside cells, there is not enough available heme to support its intracellular metabolism. It is concluded that ferrochelatase is essential for the multiplication and intracellular survival of B. abortus and thus for the establishment of chronic disease as well. PMID:11553564

  11. Ferrochelatase is present in Brucella abortus and is critical for its intracellular survival and virulence.

    PubMed

    Almirón, M; Martínez, M; Sanjuan, N; Ugalde, R A

    2001-10-01

    Brucella spp. are pathogenic bacteria that cause brucellosis, an animal disease which can also affect humans. Although understanding the pathogenesis is important for the health of animals and humans, little is known about virulence factors associated with it. In order for chronic disease to be established, Brucella spp. have developed the ability to survive inside phagocytes by evading cell defenses. It hides inside vacuoles, where it then replicates, indicating that it has an active metabolism. The purpose of this work was to obtain better insight into the intracellular metabolism of Brucella abortus. During a B. abortus genomic sequencing project, a clone coding a putative gene homologous to hemH was identified and sequenced. The amino acid sequence revealed high homology to members of the ferrochelatase family. A knockout mutant displayed auxotrophy for hemin, defective intracellular survival inside J774 and HeLa cells, and lack of virulence in BALB/c mice. This phenotype was overcome by complementing the mutant strain with a plasmid harboring wild-type hemH. These data demonstrate that B. abortus synthesizes its own heme and also has the ability to use an external source of heme; however, inside cells, there is not enough available heme to support its intracellular metabolism. It is concluded that ferrochelatase is essential for the multiplication and intracellular survival of B. abortus and thus for the establishment of chronic disease as well. PMID:11553564

  12. Ehrlichia chaffeensis TRP120 Activates Canonical Notch Signaling To Downregulate TLR2/4 Expression and Promote Intracellular Survival

    PubMed Central

    Lina, Taslima T.; Dunphy, Paige S.; Luo, Tian

    2016-01-01

    ABSTRACT Ehrlichia chaffeensis preferentially targets mononuclear phagocytes and survives through a strategy of subverting innate immune defenses, but the mechanisms are unknown. We have shown E. chaffeensis type 1 secreted tandem repeat protein (TRP) effectors are involved in diverse molecular pathogen-host interactions, such as the TRP120 interaction with the Notch receptor-cleaving metalloprotease ADAM17. In the present study, we demonstrate E. chaffeensis, via the TRP120 effector, activates the canonical Notch signaling pathway to promote intracellular survival. We found that nuclear translocation of the transcriptionally active Notch intracellular domain (NICD) occurs in response to E. chaffeensis or recombinant TRP120, resulting in upregulation of Notch signaling pathway components and target genes notch1, adam17, hes, and hey. Significant differences in canonical Notch signaling gene expression levels (>40%) were observed during early and late stages of infection, indicating activation of the Notch pathway. We linked Notch pathway activation specifically to the TRP120 effector, which directly interacts with the Notch metalloprotease ADAM17. Using pharmacological inhibitors and small interfering RNAs (siRNAs) against γ-secretase enzyme, Notch transcription factor complex, Notch1, and ADAM17, we demonstrated that Notch signaling is required for ehrlichial survival. We studied the downstream effects and found that E. chaffeensis TRP120-mediated activation of the Notch pathway causes inhibition of the extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) pathways required for PU.1 and subsequent Toll-like receptor 2/4 (TLR2/4) expression. This investigation reveals a novel mechanism whereby E. chaffeensis exploits the Notch pathway to evade the host innate immune response for intracellular survival. PMID:27381289

  13. Host Lipid Bodies as Platforms for Intracellular Survival of Protozoan Parasites

    PubMed Central

    Toledo, Daniel A. M.; D’Avila, Heloísa; Melo, Rossana C. N.

    2016-01-01

    Pathogens induce several changes in the host cell signaling and trafficking mechanisms in order to evade and manipulate the immune response. One prominent pathogen-mediated change is the formation of lipid-rich organelles, termed lipid bodies (LBs) or lipid droplets, in the host cell cytoplasm. Protozoan parasites, which contribute expressively to the burden of infectious diseases worldwide, are able to induce LB genesis in non-immune and immune cells, mainly macrophages, key players in the initial resistance to the infection. Under host–parasite interaction, LBs not only accumulate in the host cytoplasm but also relocate around and move into parasitophorous vacuoles. There is increasing evidence that protozoan parasites may target host-derived LBs either for gaining nutrients or for escaping the host immune response. Newly formed, parasite-induced LBs may serve as lipid sources for parasite growth and also produce inflammatory mediators that potentially act in the host immune response deactivation. In this mini review, we summarize current knowledge on the formation and role of host LBs as sites exploited by intracellular protozoan parasites as a strategy to maintain their own survival. PMID:27199996

  14. Cryptococcus neoformans Thermotolerance to Avian Body Temperature Is Sufficient For Extracellular Growth But Not Intracellular Survival In Macrophages

    PubMed Central

    Johnston, Simon A.; Voelz, Kerstin; May, Robin C.

    2016-01-01

    Cryptococcus neoformans is a fatal fungal pathogen of humans that efficiently parasitises macrophages. Birds can be colonised by cryptococci and can transmit cryptococcosis to humans via inhalation of inoculated bird excreta. However, colonisation of birds appears to occur in the absence of symptomatic infection. Here, using a pure population of primary bird macrophages, we demonstrate a mechanism for this relationship. We find that bird macrophages are able to suppress the growth of cryptococci seen in mammalian cells despite C. neoformans being able to grow at bird body temperature, and are able to escape from bird macrophages by vomocytosis. A small subset of cryptococci are able to adapt to the inhibitory intracellular environment of bird macrophages, exhibiting a large cell phenotype that rescues growth suppression. Thus, restriction of intracellular growth combined with survival at bird body temperature explains the ability of birds to efficiently spread C. neoformans in the environment whilst avoiding systemic disease. PMID:26883088

  15. Mir-30d increases intracellular survival of Helicobacter pylori through inhibition of autophagy pathway

    PubMed Central

    Yang, Xiao-Jun; Si, Ruo-Huang; Liang, Yu-He; Ma, Bing-Qiang; Jiang, Ze-Bin; Wang, Bin; Gao, Peng

    2016-01-01

    AIM: To determine if mir-30d inhibits the autophagy response to Helicobacter pylori (H. pylori) invasion and increases H. pylori intracellular survival. METHODS: The expression of mir-30d was detected by quantitative polymerase chain reaction (PCR), and autophagy level was examined by transmission electron microscopy, western blot, and GFP-LC3 puncta assay in human AGS cells and GES-1 cells. Luciferase reporter assay was applied to confirm the specificity of mir-30d regulation on the expression of several core molecules involved in autophagy pathway. The expression of multiple core proteins were analyzed at both the mRNA and protein level, and the intracellular survival of H. pylori after different treatments was detected by gentamicin protection assay. RESULTS: Autophagy level was increased in AGS and GES-1 cells in response to H. pylori infection, which was accompanied by upregulation of mir-30d expression (P < 0.05, vs no H. pylori infection). In the two gastric epithelial cell lines, mimic mir-30d was found to repress the autophagy process, whereas mir-30d inhibitor increased autophagy response to H. pylori invasion. mir-30d mimic decreased the luciferase activity of wild type reporter plasmids carrying the 3′ untranslated region (UTR) of all five tested genes (ATG2B, ATG5, ATG12, BECN1, and BNIP3L), whereas it had no effect on the mutant reporter plasmids. These five genes are core genes of autophagy pathway, and their expression was reduced significantly after mir-30d mimic transfection (P < 0.05, vs control cells without mir-30d mimic treatment). Mir-30d mimic transfection and direct inhibition of autophagy increased the intracellular survival of H. pylori in AGS cells. CONCLUSION: Mir-30d increases intracellular survival of H. pylori in gastric epithelial cells through inhibition of multiple core proteins in the autophagy pathway. PMID:27099441

  16. Relevant Genes Linked to Virulence Are Required for Salmonella Typhimurium to Survive Intracellularly in the Social Amoeba Dictyostelium discoideum

    PubMed Central

    Riquelme, Sebastián; Varas, Macarena; Valenzuela, Camila; Velozo, Paula; Chahin, Nicolás; Aguilera, Paulina; Sabag, Andrea; Labra, Bayron; Álvarez, Sergio A.; Chávez, Francisco P.; Santiviago, Carlos A.

    2016-01-01

    The social amoeba Dictyostelium discoideum has proven to be a useful model for studying relevant aspects of the host-pathogen interaction. In this work, D. discoideum was used as a model to study the ability of Salmonella Typhimurium to survive in amoebae and to evaluate the contribution of selected genes in this process. To do this, we performed infection assays using axenic cultures of D. discoideum co-cultured with wild-type S. Typhimurium and/or defined mutant strains. Our results confirmed that wild-type S. Typhimurium is able to survive intracellularly in D. discoideum. In contrast, mutants ΔaroA and ΔwaaL are defective in intracellular survival in this amoeba. Next, we included in our study a group of mutants in genes directly linked to Salmonella virulence. Of note, mutants ΔinvA, ΔssaD, ΔclpV, and ΔphoPQ also showed an impaired ability to survive intracellularly in D. discoideum. This indicates that S. Typhimurium requires a functional biosynthetic pathway of aromatic compounds, a lipopolysaccharide containing a complete O-antigen, the type III secretion systems (T3SS) encoded in SPI-1 and SPI-2, the type VI secretion system (T6SS) encoded in SPI-6 and PhoP/PhoQ two-component system to survive in D. discoideum. To our knowledge, this is the first report on the requirement of O-antigen and T6SS in the survival of Salmonella within amoebae. In addition, mutants ΔinvA and ΔssaD were internalized in higher numbers than the wild-type strain during competitive infections, suggesting that S. Typhimurium requires the T3SS encoded in SPI-1 and SPI-2 to evade phagocytosis by D. discoideum. Altogether, these results indicate that S. Typhimurium exploits a common set of genes and molecular mechanisms to survive within amoeba and animal host cells. The use of D. discoideum as a model for host–pathogen interactions will allow us to discover the gene repertoire used by Salmonella to survive inside the amoeba and to study the cellular processes that are affected

  17. Relevant Genes Linked to Virulence Are Required for Salmonella Typhimurium to Survive Intracellularly in the Social Amoeba Dictyostelium discoideum.

    PubMed

    Riquelme, Sebastián; Varas, Macarena; Valenzuela, Camila; Velozo, Paula; Chahin, Nicolás; Aguilera, Paulina; Sabag, Andrea; Labra, Bayron; Álvarez, Sergio A; Chávez, Francisco P; Santiviago, Carlos A

    2016-01-01

    The social amoeba Dictyostelium discoideum has proven to be a useful model for studying relevant aspects of the host-pathogen interaction. In this work, D. discoideum was used as a model to study the ability of Salmonella Typhimurium to survive in amoebae and to evaluate the contribution of selected genes in this process. To do this, we performed infection assays using axenic cultures of D. discoideum co-cultured with wild-type S. Typhimurium and/or defined mutant strains. Our results confirmed that wild-type S. Typhimurium is able to survive intracellularly in D. discoideum. In contrast, mutants ΔaroA and ΔwaaL are defective in intracellular survival in this amoeba. Next, we included in our study a group of mutants in genes directly linked to Salmonella virulence. Of note, mutants ΔinvA, ΔssaD, ΔclpV, and ΔphoPQ also showed an impaired ability to survive intracellularly in D. discoideum. This indicates that S. Typhimurium requires a functional biosynthetic pathway of aromatic compounds, a lipopolysaccharide containing a complete O-antigen, the type III secretion systems (T3SS) encoded in SPI-1 and SPI-2, the type VI secretion system (T6SS) encoded in SPI-6 and PhoP/PhoQ two-component system to survive in D. discoideum. To our knowledge, this is the first report on the requirement of O-antigen and T6SS in the survival of Salmonella within amoebae. In addition, mutants ΔinvA and ΔssaD were internalized in higher numbers than the wild-type strain during competitive infections, suggesting that S. Typhimurium requires the T3SS encoded in SPI-1 and SPI-2 to evade phagocytosis by D. discoideum. Altogether, these results indicate that S. Typhimurium exploits a common set of genes and molecular mechanisms to survive within amoeba and animal host cells. The use of D. discoideum as a model for host-pathogen interactions will allow us to discover the gene repertoire used by Salmonella to survive inside the amoeba and to study the cellular processes that are affected

  18. Gamma Band Activity in the RAS-intracellular mechanisms

    PubMed Central

    Garcia-Rill, E.; Kezunovic, N.; D’Onofrio, S.; Luster, B.; Hyde, J.; Bisagno, V.; Urbano, F.J.

    2014-01-01

    Gamma band activity participates in sensory perception, problem solving, and memory. This review considers recent evidence showing that cells in the reticular activating system (RAS) exhibit gamma band activity, and describes the intrinsic membrane properties behind such manifestation. Specifically, we discuss how cells in the mesopontine pedunculopontine nucleus (PPN), intralaminar parafascicular nucleus (Pf), and pontine Subcoeruleus nucleus dorsalis (SubCD) all fire in the gamma band range when maximally activated, but no higher. The mechanisms involve high threshold, voltage-dependent P/Q-type calcium channels or sodium-dependent subthreshold oscillations. Rather than participating in the temporal binding of sensory events as in the cortex, gamma band activity in the RAS may participate in the processes of preconscious awareness, and provide the essential stream of information for the formulation of many of our actions. We address three necessary next steps resulting from these discoveries, an intracellular mechanism responsible for maintaining gamma band activity based on persistent G-protein activation, separate intracellular pathways that differentiate between gamma band activity during waking vs during REM sleep, and an intracellular mechanism responsible for the dysregulation in gamma band activity in schizophrenia. These findings open several promising research avenues that have not been thoroughly explored. What are the effects of sleep or REM sleep deprivation on these RAS mechanisms? Are these mechanisms involved in memory processing during waking and/or during REM sleep? Does gamma band processing differ during waking vs REM sleep after sleep or REM sleep deprivation? PMID:24309750

  19. The ability to survive intracellular freezing in nematodes is related to the pattern and distribution of ice formed.

    PubMed

    Raymond, Méliane R; Wharton, David A

    2016-07-01

    A few species of nematodes can survive extensive intracellular freezing throughout all their tissues, an event that is usually thought to be fatal to cells. How are they able to survive in this remarkable way? The pattern and distribution of ice formed, after freezing at -10°C, can be observed using freeze substitution and transmission electron microscopy, which preserves the former position of ice as white spaces. We compared the pattern and distribution of ice formed in a nematode that survives intracellular freezing well (Panagrolaimus sp. DAW1), one that survives poorly (Panagrellus redivivus) and one with intermediate levels of survival (Plectus murrayi). We also examined Panagrolaimus sp. in which the survival of freezing had been compromised by starvation. Levels of survival were as expected and the use of vital dyes indicated cellular damage in those that survived poorly (starved Panagrolaimus sp. and P. murrayi). In fed Panagrolaimus sp. the intracellular ice spaces were small and uniform, whereas in P. redivivus and starved Panagrolaimus sp. there were some large spaces that may be causing cellular damage. The pattern and distribution of ice formed was different in P. murrayi, with a greater number of individuals having no ice or only small intracellular ice spaces. Control of the size of the ice formed is thus important for the survival of intracellular freezing in nematodes. PMID:27143749

  20. Intracellular survival of Staphylococcus aureus during persistent infection in the insect Tenebrio molitor.

    PubMed

    McGonigle, John E; Purves, Joanne; Rolff, Jens

    2016-06-01

    Survival of bacteria within host cells and tissues presents a challenge to the immune systems of higher organisms. Escape from phagocytic immune cells compounds this issue, as immune cells become potential vehicles for pathogen dissemination. However, the duration of persistence within phagocytes and its contribution to pathogen load has yet to be determined. We investigate the immunological significance of intracellular persistence within the insect model Tenebrio molitor, assessing the extent, duration and location of bacterial recovery during a persistent infection. Relative abundance of Staphylococcus aureus in both intracellular and extracellular fractions was determined over 21 days, and live S. aureus were successfully recovered from both the hemolymph and within phagocytic immune cells across the entire time course. The proportion of bacteria recovered from within phagocytes also increased over time. Our results show that to accurately estimate pathogen load it is vital to account for bacteria persisting within immune cells. PMID:26778297

  1. A mechanism of intracellular P2X receptor activation.

    PubMed

    Sivaramakrishnan, Venketesh; Fountain, Samuel J

    2012-08-17

    P2X receptors (P2XRs) are ATP-activated calcium-permeable ligand-gated ion channels traditionally viewed as sensors of extracellular ATP during diverse physiological processes including pain, inflammation, and taste. However, in addition to a cell surface residency P2XRs also populate the membranes of intracellular compartments, including mammalian lysosomes, phagosomes, and the contractile vacuole (CV) of the amoeba Dictyostelium. The function of intracellular P2XRs is unclear and represents a major gap in our understanding of ATP signaling. Here, we exploit the genetic versatility of Dictyostelium to investigate the effects of physiological concentrations of ATP on calcium signaling in isolated CVs. Within the CV, an acidic calcium store, P2XRs are orientated to sense luminal ATP. Application of ATP to isolated vacuoles leads to luminal translocation of ATP and release of calcium. Mechanisms of luminal ATP translocation and ATP-evoked calcium release share common pharmacology, suggesting that they are linked processes. The ability of ATP to mobilize stored calcium is reduced in vacuoles isolated from P2X(A)R knock-out amoeba and ablated in cells devoid of P2XRs. Pharmacological inhibition of luminal ATP translocation or depletion of CV calcium attenuates CV function in vivo, manifesting as a loss of regulatory cell volume decrease following osmotic swelling. We propose that intracellular P2XRs regulate vacuole activity by acting as calcium release channels, activated by translocation of ATP into the vacuole lumen. PMID:22736763

  2. Characterization of New Virulence Factors Involved in the Intracellular Growth and Survival of Burkholderia pseudomallei.

    PubMed

    Moule, Madeleine G; Spink, Natasha; Willcocks, Sam; Lim, Jiali; Guerra-Assunção, José Afonso; Cia, Felipe; Champion, Olivia L; Senior, Nicola J; Atkins, Helen S; Clark, Taane; Bancroft, Gregory J; Cuccui, Jon; Wren, Brendan W

    2015-01-01

    Burkholderia pseudomallei, the causative agent of melioidosis, has complex and poorly understood extracellular and intracellular lifestyles. We used transposon-directed insertion site sequencing (TraDIS) to retrospectively analyze a transposon library that had previously been screened through a BALB/c mouse model to identify genes important for growth and survival in vivo. This allowed us to identify the insertion sites and phenotypes of negatively selected mutants that were previously overlooked due to technical constraints. All 23 unique genes identified in the original screen were confirmed by TraDIS, and an additional 105 mutants with various degrees of attenuation in vivo were identified. Five of the newly identified genes were chosen for further characterization, and clean, unmarked bpsl2248, tex, rpiR, bpsl1728, and bpss1528 deletion mutants were constructed from the wild-type strain K96243. Each of these mutants was tested in vitro and in vivo to confirm their attenuated phenotypes and investigate the nature of the attenuation. Our results confirm that we have identified new genes important to in vivo virulence with roles in different stages of B. pseudomallei pathogenesis, including extracellular and intracellular survival. Of particular interest, deletion of the transcription accessory protein Tex was shown to be highly attenuating, and the tex mutant was capable of providing protective immunity against challenge with wild-type B. pseudomallei, suggesting that the genes identified in our TraDIS screen have the potential to be investigated as live vaccine candidates. PMID:26712202

  3. Swedish isolates of Vibrio cholerae enhance their survival when interacted intracellularly with Acanthamoeba castellanii

    PubMed Central

    Shanan, Salah; Bayoumi, Magdi; Saeed, Amir; Sandström, Gunnar; Abd, Hadi

    2016-01-01

    Vibrio cholerae is a Gram-negative bacterium that occurs naturally in aquatic environment. Only V. cholerae O1 and V. cholerae O139 produce cholera toxin and cause cholera, other serogroups can cause gastroenteritis, open wounds infection, and septicaemia. V. cholerae O1 and V. cholerae O139 grow and survive inside Acanthamoeba castellanii. The aim of this study is to investigate the interactions of the Swedish clinical isolates V. cholerae O3, V. cholerae O4, V. cholerae O5, V. cholerae O11, and V. cholerae O160 with A. castellanii. The interaction between A. castellanii and V. cholerae strains was studied by means of amoeba cell counts, viable counts of the bacteria in the absence or presence of amoebae, and of the intracellularly growing bacteria, visualised by electron microscopy. These results show that all V. cholerae can grow and survive outside and inside the amoebae, disclosing that V. cholerae O3, V. cholerae O4, V. cholerae O5, V. cholerae O11, and V. cholerae O160 all can be considered as facultative intracellular bacteria. PMID:27118300

  4. The β-Hemolysin and Intracellular Survival of Streptococcus agalactiae in Human Macrophages

    PubMed Central

    Sagar, Anubha; Klemm, Carolin; Hartjes, Lara; Mauerer, Stefanie; van Zandbergen, Ger; Spellerberg, Barbara

    2013-01-01

    S. agalactiae (group B streptococci, GBS) is a major microbial pathogen in human neonates and causes invasive infections in pregnant women and immunocompromised individuals. The S. agalactiae β-hemolysin is regarded as an important virulence factor for the development of invasive disease. To examine the role of β-hemolysin in the interaction with professional phagocytes, the THP-1 monocytic cell line and human granulocytes were infected with a serotype Ia S. agalactiae wild type strain and its isogenic nonhemolytic mutant. We could show that the nonhemolytic mutants were able to survive in significantly higher numbers than the hemolytic wild type strain, in THP-1 macrophage-like cells and in assays with human granulocytes. Intracellular bacterial multiplication, however, could not be observed. The hemolytic wild type strain stimulated a significantly higher release of Tumor Necrosis Factor-α than the nonhemolytic mutant in THP-1 cells, while similar levels of the chemokine Interleukin-8 were induced. In order to investigate bacterial mediators of IL-8 release in this setting, purified cell wall preparations from both strains were tested and found to exert a potent proinflammatory stimulus on THP-1 cells. In conclusion, our results indicate that the β-hemolysin has a strong influence on the intracellular survival of S. agalactiae and that a tightly controlled regulation of β-hemolysin expression is required for the successful establishment of S. agalactiae in different host niches. PMID:23593170

  5. Early Events in Phagosome Establishment Are Required for Intracellular Survival of Legionella pneumophila

    PubMed Central

    Wiater, Lawrence A.; Dunn, Kenneth; Maxfield, Frederick R.; Shuman, Howard A.

    1998-01-01

    During infection, the Legionnaires’ disease bacterium, Legionella pneumophila, survives and multiplies within a specialized phagosome that is near neutral pH and does not fuse with host lysosomes. In order to understand the molecular basis of this organism’s ability to control its intracellular fate, we have isolated and characterized a group of transposon-generated mutants which were unable to kill macrophages and were subsequently found to be defective in intracellular multiplication. These mutations define a set of 20 genes (19 icm [for intracellular multiplication] genes and dotA [for defect in organelle trafficking]). In this report, we describe a quantitative assay for phagosome-lysosome fusion (PLF) and its use to measure the levels of PLF in cells that have been infected with either wild-type L. pneumophila or one of several mutants defective in different icm genes or dotA. By using quantitative confocal fluorescence microscopy, PLF could be scored on a per-bacterium basis by determining the extent to which fluorescein-labeled L. pneumophila colocalized with host lysosomes prelabeled with rhodamine-dextran. Remarkably, mutations in the six genes that were studied resulted in maximal levels of PLF as quickly as 30 min following infection. These results indicate that several, and possibly all, of the icm and dotA gene products act at an early step during phagosome establishment to determine whether L. pneumophila-containing phagosomes will fuse with lysosomes. Although not ruled out, subsequent activity of these gene products may not be necessary for successful intracellular replication. PMID:9712800

  6. Intracellular survival of wild-type Salmonella typhimurium and macrophage-sensitive mutants in diverse populations of macrophages.

    PubMed

    Buchmeier, N A; Heffron, F

    1989-01-01

    Salmonella typhimurium survives within macrophages and causes a fatal infection in susceptible strains of mice. A number of S. typhimurium mutants that contain Tn10 insertions in genes which are necessary for survival within the macrophage have been isolated. To demonstrate the importance of each gene in intracellular survival, the mutations were transduced into a smooth-strain background and the ability to survive intracellularly was assayed in five different populations of macrophages. The majority of the original macrophage-sensitive mutants retained the macrophage-sensitive phenotype in the smooth-strain background. The ability to survive or grow within macrophages varied with both the source of macrophages and the individual mutants. S. typhimurium grew best in the macrophage-like cell line J774, survived at moderate levels in splenic and bone marrow-derived macrophages, and was killed most efficiently in peritoneal macrophages. Macrophage-sensitive mutants transduced into a smooth background were also less virulent than the parent, with a 50% lethal dose of 2 to 5 logs greater than that of the parental strain. These experiments demonstrate that survival of S. typhimurium within macrophages varies with the source of cells, with a distinct ability to survive in macrophages from mouse spleens, where S. typhimurium grows rapidly. These experiments also demonstrate the heterogeneity in intracellular survival among the various macrophage-sensitive mutants, which may reflect the relative importance of the individual mutated genes in survival within macrophages. PMID:2642463

  7. Mechanisms of Defense against Intracellular Pathogens Mediated by Human Macrophages.

    PubMed

    Bloom, Barry R; Modlin, Robert L

    2016-06-01

    The key question our work has sought to address has been, "What are the necessary and sufficient conditions that engender protection from intracellular pathogens in the human host?" The origins of this work derive from a long-standing interest in the mechanisms of protection against two such paradigmatic intracellular pathogens, Mycobacterium tuberculosis and Mycobacterium leprae, that have brilliantly adapted to the human host. It was obvious that these pathogens, which cause chronic diseases and persist in macrophages, must have acquired subtle strategies to resist host microbicidal mechanisms, yet since the vast majority of individuals infected with M. tuberculosis do not develop disease, there must be some potent human antimicrobial mechanisms. What follows is not a comprehensive review of the vast literature on the role of human macrophages in protection against infectious disease, but a summary of the research in our two laboratories with collaborators that we hope has contributed to some understanding of mechanisms of resistance and pathogenesis. While mouse models revealed some necessary conditions for protection, e.g., innate immunity, Th1 cells and their cytokines, and major histocompatibility complex class I-restricted T cells, here we emphasize multiple antimicrobial mechanisms that exist in human macrophages that differ from those of most experimental animals. Prominent here is the vitamin D-dependent antimicrobial pathway common to human macrophages activated by innate and acquired immune responses, mediated by antimicrobial peptides, e.g., cathelicidin, through an interleukin-15- and interleukin-32-dependent common pathway that is necessary for macrophage killing of M. tuberculosis in vitro. PMID:27337485

  8. Ehrlichia chaffeensis Exploits Canonical and Noncanonical Host Wnt Signaling Pathways To Stimulate Phagocytosis and Promote Intracellular Survival

    PubMed Central

    Luo, Tian; Dunphy, Paige S.; Lina, Taslima T.

    2015-01-01

    Ehrlichia chaffeensis invades and survives in phagocytes by modulating host cell processes and evading innate defenses, but the mechanisms are not fully defined. Recently we have determined that E. chaffeensis tandem repeat proteins (TRPs) are type 1 secreted effectors involved in functionally diverse interactions with host targets, including components of the evolutionarily conserved Wnt signaling pathways. In this study, we demonstrated that induction of host canonical and noncanonical Wnt pathways by E. chaffeensis TRP effectors stimulates phagocytosis and promotes intracellular survival. After E. chaffeensis infection, canonical and noncanonical Wnt signalings were significantly stimulated during early stages of infection (1 to 3 h) which coincided with dephosphorylation and nuclear translocation of β-catenin, a major canonical Wnt signal transducer, and NFATC1, a noncanonical Wnt transcription factor. In total, the expression of ∼44% of Wnt signaling target genes was altered during infection. Knockdown of TRP120-interacting Wnt pathway components/regulators and other critical components, such as Wnt5a ligand, Frizzled 5 receptor, β-catenin, nuclear factor of activated T cells (NFAT), and major signaling molecules, resulted in significant reductions in the ehrlichial load. Moreover, small-molecule inhibitors specific for components of canonical and noncanonical (Ca2+ and planar cell polarity [PCP]) Wnt pathways, including IWP-2, which blocks Wnt secretion, significantly decreased ehrlichial infection. TRPs directly activated Wnt signaling, as TRP-coated microspheres triggered phagocytosis which was blocked by Wnt pathway inhibitors, demonstrating a key role of TRP activation of Wnt pathways to induce ehrlichial phagocytosis. These novel findings reveal that E. chaffeensis exploits canonical and noncanonical Wnt pathways through TRP effectors to facilitate host cell entry and promote intracellular survival. PMID:26712203

  9. Ehrlichia chaffeensis Exploits Canonical and Noncanonical Host Wnt Signaling Pathways To Stimulate Phagocytosis and Promote Intracellular Survival.

    PubMed

    Luo, Tian; Dunphy, Paige S; Lina, Taslima T; McBride, Jere W

    2016-03-01

    Ehrlichia chaffeensis invades and survives in phagocytes by modulating host cell processes and evading innate defenses, but the mechanisms are not fully defined. Recently we have determined that E. chaffeensis tandem repeat proteins (TRPs) are type 1 secreted effectors involved in functionally diverse interactions with host targets, including components of the evolutionarily conserved Wnt signaling pathways. In this study, we demonstrated that induction of host canonical and noncanonical Wnt pathways by E. chaffeensis TRP effectors stimulates phagocytosis and promotes intracellular survival. After E. chaffeensis infection, canonical and noncanonical Wnt signalings were significantly stimulated during early stages of infection (1 to 3 h) which coincided with dephosphorylation and nuclear translocation of β-catenin, a major canonical Wnt signal transducer, and NFATC1, a noncanonical Wnt transcription factor. In total, the expression of ∼44% of Wnt signaling target genes was altered during infection. Knockdown of TRP120-interacting Wnt pathway components/regulators and other critical components, such as Wnt5a ligand, Frizzled 5 receptor, β-catenin, nuclear factor of activated T cells (NFAT), and major signaling molecules, resulted in significant reductions in the ehrlichial load. Moreover, small-molecule inhibitors specific for components of canonical and noncanonical (Ca(2+) and planar cell polarity [PCP]) Wnt pathways, including IWP-2, which blocks Wnt secretion, significantly decreased ehrlichial infection. TRPs directly activated Wnt signaling, as TRP-coated microspheres triggered phagocytosis which was blocked by Wnt pathway inhibitors, demonstrating a key role of TRP activation of Wnt pathways to induce ehrlichial phagocytosis. These novel findings reveal that E. chaffeensis exploits canonical and noncanonical Wnt pathways through TRP effectors to facilitate host cell entry and promote intracellular survival. PMID:26712203

  10. Oxysterols and mechanisms of survival signaling.

    PubMed

    Vurusaner, Beyza; Leonarduzzi, Gabriella; Gamba, Paola; Poli, Giuseppe; Basaga, Huveyda

    2016-06-01

    Oxysterols, a family of oxidation products of cholesterol, are increasingly drawing attention of scientists to their multifaceted biochemical properties, several of them of clear relevance to human pathophysiology. Taken up by cells through both vesicular and non-vesicular ways or often generated intracellularly, oxysterols contribute to modulate not only the inflammatory and immunological response but also cell viability, metabolism and function by modulating several signaling pathways. Moreover, they have been recognized as elective ligands for the most important nuclear receptors. The outcome of such a complex network of intracellular reactions promoted by these cholesterol oxidation products appears to be largely dependent not only on the type of cells, the dynamic conditions of the cellular and tissue environment but also on the concentration of the oxysterols. Here focus has been given to the cascade of molecular events exerted by relatively low concentrations of certain oxysterols that elicit survival and functional signals in the cells, with the aim to contribute to further expand the knowledge about the biological and physiological potential of the biochemical reactions triggered and modulated by oxysterols. PMID:27017897

  11. Superoxide dismutase C is required for intracellular survival and virulence of Burkholderia pseudomallei.

    PubMed

    Vanaporn, Muthita; Wand, Matthew; Michell, Stephen L; Sarkar-Tyson, Mitali; Ireland, Philip; Goldman, Stan; Kewcharoenwong, Chidchamai; Rinchai, Darawan; Lertmemongkolchai, Ganjana; Titball, Richard W

    2011-08-01

    Burkholderia pseudomallei is an intracellular pathogen and the causative agent of melioidosis, a life-threatening disease of humans. Within host cells, superoxide is an important mediator of pathogen killing. In this study, we have identified the B. pseudomallei K96243 sodC gene, shown that it has superoxide dismutase activity, and constructed an allelic deletion mutant of this gene. Compared with the wild-type, the mutant was more sensitive to killing by extracellular superoxide, but not to superoxide generated intracellularly. The sodC mutant showed a markedly decreased survival in J774A.1 mouse macrophages, and reduced numbers of bacteria were recovered from human polymorphonuclear neutrophils (PMNs) when compared with the wild-type. The numbers of wild-type or mutant bacteria recovered from human diabetic neutrophils were significantly lower than from normal human neutrophils. The sodC mutant was attenuated in BALB/c mice. Our results indicate that SodC plays a key role in the virulence of B. pseudomallei, but that diabetics are not more susceptible to infection because of a reduced ability of PMNs to kill by superoxide. PMID:21659326

  12. Edwardsiella tarda-Induced Inhibition of Apoptosis: A Strategy for Intracellular Survival

    PubMed Central

    Zhou, Ze-jun; Sun, Li

    2016-01-01

    Edwardsiella tarda is a Gram-negative bacterial pathogen that can infect a wide range of freshwater and marine fish. One salient feature of E. tarda is the ability to survive and replicate in various host cells. In this study, we observed that E. tarda replicated robustly in the zebrafish cell line ZF4, and that E. tarda-infected cells exhibited no detectable signs of apoptosis. Global transcriptome analysis and quantitative real-time RT-PCR revealed that E. tarda infection generally significantly downregulated pro-apoptotic genes and upregulated anti-apoptotic genes. To investigate the role of apoptosis in E. tarda infection, two upregulated anti-apoptotic genes (Fech and Prx3) and two downregulated pro-apoptotic genes (Brms1a and Ivns1a) were overexpressed in zebrafish. Subsequent infection study showed that Fech and Prx3 overexpression significantly promoted E. tarda dissemination in and colonization of fish tissues, while Brms1a and Ivns1a overexpression significantly reduced E. tarda dissemination and colonization. Consistently, when Fech and Prx3 were knocked down in zebrafish, E. tarda infection was significantly inhibited, whereas Brms1a and Ivns1a knockdown significantly enhanced E. tarda infection. These results indicate for the first time that E. tarda prevents apoptosis in teleost as a strategy for intracellular survival, and that some putative apoptotic genes of teleost function in the apoptosis pathway probably in a manner similar to that in mammalian systems. PMID:27471679

  13. The Essential Role of Cholesterol Metabolism in the Intracellular Survival of Mycobacterium leprae Is Not Coupled to Central Carbon Metabolism and Energy Production

    PubMed Central

    Marques, Maria Angela M.; Berrêdo-Pinho, Marcia; Rosa, Thabatta L. S. A.; Pujari, Venugopal; Lemes, Robertha M. R.; Lery, Leticia M. S.; Silva, Carlos Adriano M.; Guimarães, Ana Carolina R.; Atella, Georgia C.; Wheat, William H.; Brennan, Patrick J.; Crick, Dean C.; Belisle, John T.

    2015-01-01

    ABSTRACT Mycobacterium leprae induces the formation of lipid droplets, which are recruited to pathogen-containing phagosomes in infected macrophages and Schwann cells. Cholesterol is among the lipids with increased abundance in M. leprae-infected cells, and intracellular survival relies on cholesterol accumulation. The present study investigated the capacity of M. leprae to acquire and metabolize cholesterol. In silico analyses showed that oxidation of cholesterol to cholest-4-en-3-one (cholestenone), the first step of cholesterol degradation catalyzed by the enzyme 3β-hydroxysteroid dehydrogenase (3β-HSD), is apparently the only portion of the cholesterol catabolic pathway seen in Mycobacterium tuberculosis preserved by M. leprae. Incubation of bacteria with radiolabeled cholesterol confirmed the in silico predictions. Radiorespirometry and lipid analyses performed after incubating M. leprae with [4-14C]cholesterol or [26-14C]cholesterol showed the inability of this pathogen to metabolize the sterol rings or the side chain of cholesterol as a source of energy and carbon. However, the bacteria avidly incorporated cholesterol and, as expected, converted it to cholestenone both in vitro and in vivo. Our data indicate that M. leprae has lost the capacity to degrade and utilize cholesterol as a nutritional source but retains the enzyme responsible for its oxidation to cholestenone. Thus, the essential role of cholesterol metabolism in the intracellular survival of M. leprae is uncoupled from central carbon metabolism and energy production. Further elucidation of cholesterol metabolism in the host cell during M. leprae infection will establish the mechanism by which this lipid supports M. leprae intracellular survival and will open new avenues for novel leprosy therapies. IMPORTANCE Our study focused on the obligate intracellular pathogen Mycobacterium leprae and its capacity to metabolize cholesterol. The data make an important contribution for those interested in

  14. Effects of cryoprotectants and ice-seeding temperature on intracellular freezing and survival of human oocytes.

    PubMed

    Trad, F S; Toner, M; Biggers, J D

    1999-06-01

    The accurate determination of the freezing conditions that promote intracellular ice formation (IIF) is crucial for designing cryopreservation protocols for cells. In this paper, the range of temperatures at which IIF occurs in human oocytes was determined. Fresh oocytes with a germinal vesicle, failed-to-fertilize (metaphase I and metaphase II stages) and polyspermic eggs were used for this study. The occurrence of IIF was first visualized at a cooling rate of 120 degrees C/min using a programmable thermal microscope stage connected to a videomicroscope. Then, with a cooling rate of 0.2 degrees C/min, the seeding temperature of the extracellular ice was modified to decrease the incidence of IIF and increase the survival rate of frozen-thawed human oocytes. After adding different cryoprotectants, the median temperature of IIF (TMED) was decreased by approximately 23 degrees C in mouse and only by approximately 6.5 degrees C in human oocytes. Using 1.5 M propylene glycol and seeding temperatures of -8.0, -6.0 and -4.5 degrees C, the incidence of IIF was 22/28 (78%), 8/24 (33%) and 0/33 (0%) and the 24 h post-thaw survival rate was 10/31(32%), 19/34 (56%) and 52/56 (93%) respectively. The results show that IIF occurs more readily in human oocytes, and that ice seeding between -6 degrees C and -8 degrees C triggers IIF in a large number of human oocytes. Undesirable IIF can be prevented and survival rates maximized by raising the seeding temperature as close as possible to the melting point of the solution, which in our instrument was -4.5 degrees C. PMID:10357978

  15. RD-1 encoded EspJ protein gets phosphorylated prior to affect the growth and intracellular survival of mycobacteria

    PubMed Central

    Singh, Pramod K; Saxena, Richa; Tiwari, Sameer; Singh, Diwakar K; Singh, Susmita K; Kumari, Ruma; Srivastava, Kishore K

    2015-01-01

    Mycobacterium tuberculosis (MTB) synchronizes a number of processes and controls a series of events to subvert host defense mechanisms for the sake of residing inside macrophages. Besides these, MTB also possesses a wide range of signal enzyme systems, including eleven serine threonine protein kinases (STPKs). The present study describes STPK modulated modification in one of the hypothetical proteins of the RD1 region; EspJ (ESX-1 secretion associated protein), which is predicted to be involved in virulence of MTB. We have employed knock-out MTB, and M. bovis BCG as a surrogate strain to elaborate the consequence of the phosphorylation of EspJ. The molecular and mass spectrometric analyses in this study, confirmed EspJ as one of the substrates of STPKs. The ectopic expression of phosphoablative mutants of espJ in M. bovis BCG also articulated the effect of phosphorylation on the growth and in survival of mycobacteria. Importantly, the level of phosphorylation of EspJ also differed between pathogenic H37 Rv (Rv) and non pathogenic H37 Ra (Ra) strains of MTB. This further suggested that to a certain extent, the STPKs mediated phosphorylation may be accountable, in determining the growth and in intra-cellular survival of mycobacteria. PMID:26228622

  16. RD-1 encoded EspJ protein gets phosphorylated prior to affect the growth and intracellular survival of mycobacteria.

    PubMed

    Singh, Pramod K; Saxena, Richa; Tiwari, Sameer; Singh, Diwakar K; Singh, Susmita K; Kumari, Ruma; Srivastava, Kishore K

    2015-01-01

    Mycobacterium tuberculosis (MTB) synchronizes a number of processes and controls a series of events to subvert host defense mechanisms for the sake of residing inside macrophages. Besides these, MTB also possesses a wide range of signal enzyme systems, including eleven serine threonine protein kinases (STPKs). The present study describes STPK modulated modification in one of the hypothetical proteins of the RD1 region; EspJ (ESX-1 secretion associated protein), which is predicted to be involved in virulence of MTB. We have employed knock-out MTB, and M. bovis BCG as a surrogate strain to elaborate the consequence of the phosphorylation of EspJ. The molecular and mass spectrometric analyses in this study, confirmed EspJ as one of the substrates of STPKs. The ectopic expression of phosphoablative mutants of espJ in M. bovis BCG also articulated the effect of phosphorylation on the growth and in survival of mycobacteria. Importantly, the level of phosphorylation of EspJ also differed between pathogenic H37 Rv (Rv) and non pathogenic H37 Ra (Ra) strains of MTB. This further suggested that to a certain extent, the STPKs mediated phosphorylation may be accountable, in determining the growth and in intra-cellular survival of mycobacteria. PMID:26228622

  17. Subcellular Localization of the Intracellular Survival-Enhancing Eis Protein of Mycobacterium tuberculosis

    PubMed Central

    Dahl, John L.; Wei, Jun; Moulder, James W.; Laal, Suman; Friedman, Richard L.

    2001-01-01

    Mycobacterium tuberculosis is a facultative intracellular pathogen that has evolved the ability to survive and multiply within human macrophages. It is not clear how M. tuberculosis avoids the destructive action of macrophages, but this ability is fundamental in the pathogenicity of tuberculosis. A gene previously identified in M. tuberculosis, designated eis, was found to enhance intracellular survival of Mycobacterium smegmatis in the human macrophage-like cell line U-937 (J. Wei et al., J. Bacteriol. 182:377–384, 2000). When eis was introduced into M. smegmatis on a multicopy vector, sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the appearance of a unique 42-kDa protein band corresponding to the predicted molecular weight of the eis gene product. This band was electroeluted from the gel with a purity of >90% and subjected to N-terminal amino acid sequencing, which demonstrated that the 42-kDa band was indeed the protein product of eis. The Eis protein produced by M. tuberculosis H37Ra had an identical N-terminal amino acid sequence. A synthetic polypeptide corresponding to a carboxyl-terminal region of the deduced eis protein sequence was used to generate affinity-purified rabbit polyclonal antibodies that reacted with the 42-kDa protein in Western blot analysis. Hydropathy profile analysis showed the Eis protein to be predominantly hydrophilic with a potential hydrophobic amino terminus. Phase separation of M. tuberculosis H37Ra lysates by the nonionic detergent Triton X-114 revealed the Eis protein in both the aqueous and detergent phases. After fractionation of M. tuberculosis by differential centrifugation, Eis protein appeared mainly in the cytoplasmic fraction but also in the membrane, cell wall, and culture supernatant fractions as well. Forty percent of the sera from pulmonary tuberculosis patients tested for anti-Eis antibody gave positive reactions in Western blot analysis. Although the function of Eis remains unknown, evidence

  18. GLP-2 rapidly activates divergent intracellular signaling pathways involved in intestinal cell survival and proliferation in neonatal piglets

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We previously demonstrated the dose-dependent glucagon-like peptide (GLP)-2 activation of intracellular signals associated with increased epithelial cell survival and proliferation in the neonatal intestine. Our current aim was to quantify the acute, temporal GLP-2 activation of these key intracellu...

  19. Intracellular mechanisms of hydroquinone toxicity on endotoxin-activated neutrophils.

    PubMed

    Hebeda, Cristina Bichels; Pinedo, Fernanda Júdice; Bolonheis, Simone Marques; Ferreira, Zulma F; Muscará, Marcelo Nicolas; Teixeira, Simone Aparecida; Farsky, Sandra Helena Poliselli

    2012-11-01

    Circulating neutrophils promptly react to different substances in the blood and orchestrate the beginning of the innate inflammatory response. We have shown that in vivo exposure to hydroquinone (HQ), the most oxidative compound of cigarette smoke and a toxic benzene metabolite, affects circulating neutrophils, making them unresponsive to a subsequent bacterial infection. In order to understand the action of toxic molecular mechanisms on neutrophil functions, in vitro HQ actions on pro-inflammatory mediator secretions evoked by Escherichia coli lipopolysaccharide (LPS) were investigated. Neutrophils from male Wistar rats were cultured with vehicle or HQ (5 or 10 μM; 2 h) and subsequently incubated with LPS (5 μg/ml; 18 h). Hydroquinone treatment impaired LPS-induced nitric oxide (NO), tumour necrosis factor α (TNF-α), interleukin (IL)-1β and IL-6 secretions by neutrophils. The toxic effect was not dependent on cell death, reduced expression of the LPS receptor or toll-like receptor-4 (TLR-4) or cell priming, as HQ did not induce reactive oxygen species generation or β(2)integrin membrane expression. The action of toxic mechanisms on cytokine secretion was dependent on reduced gene synthesis, which may be due to decreased nuclear factor κB (NF-κB) nuclear translocation. Conversely, this intracellular pathway was not involved in impaired NO production because HQ treatments only affected inducible nitric oxide synthase protein expression and activity, suggesting posttranscriptional and/or posttranslational mechanisms of action. Altogether, our data show that HQ alters the action of different LPS-activated pathways on neutrophils, which may contribute to the impaired triggering of the host innate immune reaction detected during in vivo HQ exposure. PMID:22717997

  20. Involvement of intracellular calcium in anaerobic gene expression and survival of maize seedlings.

    PubMed Central

    Subbaiah, C C; Zhang, J; Sachs, M M

    1994-01-01

    Ca-mediated processes are known to be involved in transducing many developmental, hormonal, and environmental cues in plant cells. In this study, the role of Ca in the perception of anoxic stress signals by maize (Zea mays L. cv B73) roots was assessed by studying the effect of various Ca antagonists on the induction of alcohol dehydrogenase (ADH) and sucrose synthase mRNA as well as ADH activity under anoxia. The effect of these compounds on the poststress recovery of the seedlings was also monitored. Ruthenium red (RR), an inhibitor of organellar Ca fluxes, repressed the anoxic activation of the alcohol dehydrogenase1 and shrunken1 genes as measured by their transcript levels as well as ADH activity. Furthermore, RR-treated seedlings could not recover even after only 2 h of flooding, in contrast to untreated B73 seedlings that survived 72 h of submergence. Ca, when supplied along with RR, allowed normal anoxic gene expression and also prevented the RR-imposed death of the seedlings from short-term anoxia. Ca (45Ca) fluxes were measured in maize roots to elucidate the mode of action of RR. RR abolished anoxia-stimulated 45Ca influx into maize roots but did not affect aerobic Ca2+ uptake, unlike a few other antagonists that blocked both the aerobic and anoxic fluxes. However, Ca uptake across the plasma membrane was not necessary for the adaptive response to anoxia, since chelation of extracellular Ca by ethyleneglycol-bis(beta-aminoethyl ether)-N,N'-tetraacetic acid or 1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid did not affect the induction of ADH activity or poststress survival of flooded seedlings. The data suggest that RR may act on one of the intracellular stores of Ca and the Ca mobilized from this source is a physiological transducer of anoxic stress signals in maize roots. PMID:7518090

  1. Intracellular Survival of Leishmania major Depends on Uptake and Degradation of Extracellular Matrix Glycosaminoglycans by Macrophages

    PubMed Central

    Saunders, Eleanor C.; Kloehn, Joachim; Rupasinghe, Thusitha W.; Brown, Tracey J.; McConville, Malcolm J.

    2015-01-01

    Abstract Leishmania parasites replicate within the phagolysosome compartment of mammalian macrophages. Although Leishmania depend on sugars as a major carbon source during infections, the nutrient composition of the phagolysosome remains poorly described. To determine the origin of the sugar carbon source in macrophage phagolysosomes, we have generated a N-acetylglucosamine acetyltransferase (GNAT) deficient Leishmania major mutant (∆gnat) that is auxotrophic for the amino sugar, N-acetylglucosamine (GlcNAc). This mutant was unable to grow or survive in ex vivo infected macrophages even when macrophages were cultivated in presence of exogenous GlcNAc. In contrast, the L. major ∆gnat mutant induced normal skin lesions in mice, suggesting that these parasites have access to GlcNAc in tissue macrophages. Intracellular growth of the mutant in ex vivo infected macrophages was restored by supplementation of the macrophage medium with hyaluronan, a GlcNAc-rich extracellular matrix glycosaminoglycan. Hyaluronan is present and constitutively turned-over in Leishmania-induced skin lesions and is efficiently internalized into Leishmania containing phagolysosomes. These findings suggest that the constitutive internalization and degradation of host glycosaminoglycans by macrophages provides Leishmania with essential carbon sources, creating a uniquely favorable niche for these parasites. PMID:26334531

  2. The role of PIP2 and the IP3/DAG pathway in intracellular calcium release and cell survival during nanosecond electric pulse exposures

    NASA Astrophysics Data System (ADS)

    Steelman, Zachary A.; Tolstykh, Gleb P.; Estlack, Larry E.; Roth, Caleb C.; Ibey, Bennett L.

    2015-03-01

    Phosphatidylinositol4,5-biphosphate (PIP2) is a membrane phospholipid of particular importance in cell-signaling pathways. Hydrolysis of PIP2 releases inositol-1,4,5-triphosphate (IP3) from the membrane, activating IP3 receptors on the smooth endoplasmic reticulum (ER) and facilitating a release of intracellular calcium stores and activation of protein kinase C (PKC). Recent studies suggest that nanosecond pulsed electric fields (nsPEF) cause depletion of PIP2 in the cellular membrane, activating the IP3 signaling pathway. However, the exact mechanism(s) causing this observed depletion of PIP2 are unknown. Complicating the matter, nsPEF create nanopores in the plasma membrane, allowing calcium to enter the cell and thus causing an increase in intracellular calcium. While elevated intracellular calcium can cause activation of phospholipase C (PLC) (a known catalyst of PIP2 hydrolysis), PIP2 depletion has been shown to occur in the absence of both extracellular and intracellular calcium. These observations have led to the hypothesis that the high electric field itself may be playing a direct role in the hydrolysis of PIP2 from the plasma membrane. To support this hypothesis, we used edelfosine to block PLC and prevent activation of the IP3/DAG pathway in Chinese Hamster Ovarian (CHO) cells prior to applying nsPEF. Fluorescence microscopy was used to monitor intracellular calcium bursts during nsPEF, while MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) survivability assays were utilized to determine whether edelfosine improved cell survival during nsPEF exposure. This work is critical to refine the role of PIP2 in the cellular response to nsPEF, and also to determine the fundamental biological effects of high electric field exposures.

  3. Sphingosine kinase expression increases intracellular sphingosine-1-phosphate and promotes cell growth and survival.

    PubMed

    Olivera, A; Kohama, T; Edsall, L; Nava, V; Cuvillier, O; Poulton, S; Spiegel, S

    1999-11-01

    Sphingosine-1-phosphate (SPP) is a bioactive lipid that has recently been identified as the ligand for the EDG family of G protein-coupled cell surface receptors. However, the mitogenic and survival effects of exogenous SPP may not correlate with binding to cell-surface receptors (Van Brocklyn, J.R., M.J. Lee, R. Menzeleev, A. Olivera, L. Edsall, O. Cuvillier, D.M. Thomas, P.J.P. Coopman, S. Thangada, T. Hla, and S. Spiegel. 1998. J. Cell Biol. 142:229-240). The recent cloning of sphingosine kinase, a unique lipid kinase responsible for the formation of SPP, has provided a new tool to investigate the role of intracellular SPP. Expression of sphingosine kinase markedly increased SPP levels in NIH 3T3 fibroblasts and HEK293 cells, but no detectable secretion of SPP into the medium was observed. The increased sphingosine kinase activity in NIH 3T3 fibroblasts was sufficient to promote growth in low- serum media, expedite the G(1)/S transition, and increase DNA synthesis and the proportion of cells in the S phase of the cell cycle with a concomitant increase in cell numbers. Transient or stable overexpression of sphingosine kinase in NIH 3T3 fibroblasts or HEK293 cells protected against apoptosis induced by serum deprivation or ceramide elevation. N,N-Dimethylsphingosine, a competitive inhibitor of sphingosine kinase, blocked the effects of sphingosine kinase overexpression on cell proliferation and suppression of apoptosis. In contrast, pertussis toxin did not abrogate these biological responses. In Jurkat T cells, overexpression of sphingosine kinase also suppressed serum deprivation- and ceramide-induced apoptosis and, to a lesser extent, Fas-induced apoptosis, which correlated with inhibition of DEVDase activity, as well as inhibition of the executionary caspase-3. Taken together with ample evidence showing that growth and survival factors activate sphingosine kinase, our results indicate that SPP functions as a second messenger important for growth and survival of

  4. The CovS/CovR acid response regulator is required for intracellular survival of group B Streptococcus in macrophages.

    PubMed

    Cumley, Nicola J; Smith, Leanne M; Anthony, Mark; May, Robin C

    2012-05-01

    Group B Streptococcus (GBS) is a leading cause of neonatal meningitis and septicemia. The ability of this organism to survive inside phagocytic cells is poorly understood but thought to be an important step for the establishment of disease in the host. Here, we demonstrate that GBS shows prolonged survival within J774 macrophages and that the capacity to survive is not significantly changed across a diverse range of strains representing different serotypes, multilocus sequence types (MLST), and sites of clinical isolation. Using staining for the lysosome-associated membrane protein (LAMP) and by pharmacological inhibition of phagosome acidification, we demonstrate that streptococci reside in a phagosome and that acidification of the phagosome is required for GBS to survive intracellularly. Moreover, we show that the GBS two-component system CovS/CovR, which is the major acid response regulator in this organism, is required for survival inside the phagosome. PMID:22331428

  5. The CovS/CovR Acid Response Regulator Is Required for Intracellular Survival of Group B Streptococcus in Macrophages

    PubMed Central

    Cumley, Nicola J.; Smith, Leanne M.; Anthony, Mark

    2012-01-01

    Group B Streptococcus (GBS) is a leading cause of neonatal meningitis and septicemia. The ability of this organism to survive inside phagocytic cells is poorly understood but thought to be an important step for the establishment of disease in the host. Here, we demonstrate that GBS shows prolonged survival within J774 macrophages and that the capacity to survive is not significantly changed across a diverse range of strains representing different serotypes, multilocus sequence types (MLST), and sites of clinical isolation. Using staining for the lysosome-associated membrane protein (LAMP) and by pharmacological inhibition of phagosome acidification, we demonstrate that streptococci reside in a phagosome and that acidification of the phagosome is required for GBS to survive intracellularly. Moreover, we show that the GBS two-component system CovS/CovR, which is the major acid response regulator in this organism, is required for survival inside the phagosome. PMID:22331428

  6. Role of Metal-Dependent Regulation of ESX-3 Secretion in Intracellular Survival of Mycobacterium tuberculosis.

    PubMed

    Tinaztepe, Emir; Wei, Jun-Rong; Raynowska, Jenelle; Portal-Celhay, Cynthia; Thompson, Victor; Philips, Jennifer A

    2016-08-01

    More people die every year from Mycobacterium tuberculosis infection than from infection by any other bacterial pathogen. Type VII secretion systems (T7SS) are used by both environmental and pathogenic mycobacteria to secrete proteins across their complex cell envelope. In the nonpathogen Mycobacterium smegmatis, the ESX-1 T7SS plays a role in conjugation, and the ESX-3 T7SS is involved in metal homeostasis. In M. tuberculosis, these secretion systems have taken on roles in virulence, and they also are targets of the host immune response. ESX-3 secretes a heterodimer composed of EsxG (TB9.8) and EsxH (TB10.4), which impairs phagosome maturation in macrophages and is essential for virulence in mice. Given the importance of EsxG and EsxH during infection, we examined their regulation. With M. tuberculosis, the secretion of EsxG and EsxH was regulated in response to iron and zinc, in accordance with the previously described transcriptional response of the esx-3 locus to these metals. While iron regulated the esx-3 expression in both M. tuberculosis and M. smegmatis, there is a significant difference in the dynamics of this regulation. In M. smegmatis, the esx-3 locus behaved like other iron-regulated genes such as mbtB In M. tuberculosis, both iron and zinc modestly repressed esx-3 expression. Diminished secretion of EsxG and EsxH in response to these metals altered the interaction of M. tuberculosis with macrophages, leading to impaired intracellular M. tuberculosis survival. Our findings detail the regulatory differences of esx-3 in M. tuberculosis and M. smegmatis and demonstrate the importance of metal-dependent regulation of ESX-3 for virulence in M. tuberculosis. PMID:27245412

  7. Dinosaur Peptides Suggest Mechanisms of Protein Survival

    SciTech Connect

    San Antonio, James D.; Schweitzer, Mary H.; Jensen, Shane T.; Kalluri, Raghu; Buckley, Michael; Orgel, Joseph P.R.O.

    2011-09-16

    Eleven collagen peptide sequences recovered from chemical extracts of dinosaur bones were mapped onto molecular models of the vertebrate collagen fibril derived from extant taxa. The dinosaur peptides localized to fibril regions protected by the close packing of collagen molecules, and contained few acidic amino acids. Four peptides mapped to collagen regions crucial for cell-collagen interactions and tissue development. Dinosaur peptides were not represented in more exposed parts of the collagen fibril or regions mediating intermolecular cross-linking. Thus functionally significant regions of collagen fibrils that are physically shielded within the fibril may be preferentially preserved in fossils. These results show empirically that structure-function relationships at the molecular level could contribute to selective preservation in fossilized vertebrate remains across geological time, suggest a 'preservation motif', and bolster current concepts linking collagen structure to biological function. This non-random distribution supports the hypothesis that the peptides are produced by the extinct organisms and suggests a chemical mechanism for survival.

  8. Dinosaur Peptides Suggest Mechanisms of Protein Survival

    PubMed Central

    San Antonio, James D.; Schweitzer, Mary H.; Jensen, Shane T.; Kalluri, Raghu; Buckley, Michael; Orgel, Joseph P. R. O.

    2011-01-01

    Eleven collagen peptide sequences recovered from chemical extracts of dinosaur bones were mapped onto molecular models of the vertebrate collagen fibril derived from extant taxa. The dinosaur peptides localized to fibril regions protected by the close packing of collagen molecules, and contained few acidic amino acids. Four peptides mapped to collagen regions crucial for cell-collagen interactions and tissue development. Dinosaur peptides were not represented in more exposed parts of the collagen fibril or regions mediating intermolecular cross-linking. Thus functionally significant regions of collagen fibrils that are physically shielded within the fibril may be preferentially preserved in fossils. These results show empirically that structure-function relationships at the molecular level could contribute to selective preservation in fossilized vertebrate remains across geological time, suggest a ‘preservation motif’, and bolster current concepts linking collagen structure to biological function. This non-random distribution supports the hypothesis that the peptides are produced by the extinct organisms and suggests a chemical mechanism for survival. PMID:21687667

  9. Intracellular transport driven by cytoskeletal motors: General mechanisms and defects

    NASA Astrophysics Data System (ADS)

    Appert-Rolland, C.; Ebbinghaus, M.; Santen, L.

    2015-09-01

    Cells are the elementary units of living organisms, which are able to carry out many vital functions. These functions rely on active processes on a microscopic scale. Therefore, they are strongly out-of-equilibrium systems, which are driven by continuous energy supply. The tasks that have to be performed in order to maintain the cell alive require transportation of various ingredients, some being small, others being large. Intracellular transport processes are able to induce concentration gradients and to carry objects to specific targets. These processes cannot be carried out only by diffusion, as cells may be crowded, and quite elongated on molecular scales. Therefore active transport has to be organized. The cytoskeleton, which is composed of three types of filaments (microtubules, actin and intermediate filaments), determines the shape of the cell, and plays a role in cell motion. It also serves as a road network for a special kind of vehicles, namely the cytoskeletal motors. These molecules can attach to a cytoskeletal filament, perform directed motion, possibly carrying along some cargo, and then detach. It is a central issue to understand how intracellular transport driven by molecular motors is regulated. The interest for this type of question was enhanced when it was discovered that intracellular transport breakdown is one of the signatures of some neuronal diseases like the Alzheimer. We give a survey of the current knowledge on microtubule based intracellular transport. Our review includes on the one hand an overview of biological facts, obtained from experiments, and on the other hand a presentation of some modeling attempts based on cellular automata. We present some background knowledge on the original and variants of the TASEP (Totally Asymmetric Simple Exclusion Process), before turning to more application oriented models. After addressing microtubule based transport in general, with a focus on in vitro experiments, and on cooperative effects in the

  10. Intracellular ice and cell survival in cryo-exposed embryonic axes of recalcitrant seeds of Acer saccharinum: an ultrastructural study of factors affecting cell and ice structures

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cryogenic technologies are required to preserve embryonic axes of recalcitrant seeds. Formation of potentially lethal intracellular ice limits successful cryopreservation; thus, it is important to understand the relationships among cryo-exposure techniques, water content and survival. In this pap...

  11. STING-Dependent 2'-5' Oligoadenylate Synthetase-Like Production Is Required for Intracellular Mycobacterium leprae Survival.

    PubMed

    de Toledo-Pinto, Thiago Gomes; Ferreira, Anna Beatriz Robottom; Ribeiro-Alves, Marcelo; Rodrigues, Luciana Silva; Batista-Silva, Leonardo Ribeiro; Silva, Bruno Jorge de Andrade; Lemes, Robertha Mariana Rodrigues; Martinez, Alejandra Nóbrega; Sandoval, Felipe Galvan; Alvarado-Arnez, Lucia Elena; Rosa, Patrícia Sammarco; Shannon, Edward Joseph; Pessolani, Maria Cristina Vidal; Pinheiro, Roberta Olmo; Antunes, Sérgio Luís Gomes; Sarno, Euzenir Nunes; Lara, Flávio Alves; Williams, Diana Lynn; Ozório Moraes, Milton

    2016-07-15

    Cytosolic detection of nucleic acids elicits a type I interferon (IFN) response and plays a critical role in host defense against intracellular pathogens. Herein, a global gene expression profile of Mycobacterium leprae-infected primary human Schwann cells identified the genes differentially expressed in the type I IFN pathway. Among them, the gene encoding 2'-5' oligoadenylate synthetase-like (OASL) underwent the greatest upregulation and was also shown to be upregulated in M. leprae-infected human macrophage cell lineages, primary monocytes, and skin lesion specimens from patients with a disseminated form of leprosy. OASL knock down was associated with decreased viability of M. leprae that was concomitant with upregulation of either antimicrobial peptide expression or autophagy levels. Downregulation of MCP-1/CCL2 release was also observed during OASL knock down. M. leprae-mediated OASL expression was dependent on cytosolic DNA sensing mediated by stimulator of IFN genes signaling. The addition of M. leprae DNA enhanced nonpathogenic Mycobacterium bovis bacillus Calmette-Guerin intracellular survival, downregulated antimicrobial peptide expression, and increased MCP-1/CCL2 secretion. Thus, our data uncover a promycobacterial role for OASL during M. leprae infection that directs the host immune response toward a niche that permits survival of the pathogen. PMID:27190175

  12. RipA, a cytoplasmic membrane protein conserved among Francisella species, is required for intracellular survival.

    PubMed

    Fuller, James R; Craven, Robin R; Hall, Joshua D; Kijek, Todd M; Taft-Benz, Sharon; Kawula, Thomas H

    2008-11-01

    Francisella tularensis is a highly virulent bacterial pathogen that invades and replicates within numerous host cell types, including macrophages and epithelial cells. In an effort to better understand this process, we screened a transposon insertion library of the F. tularensis live vaccine strain (LVS) for mutant strains that invaded but failed to replicate within alveolar epithelial cell lines. One such strain isolated from this screen contained an insertion in the gene FTL_1914, which is conserved among all sequenced Francisella species yet lacks significant homology to any gene with known function. A deletion strain lacking FTL_1914 was constructed. This strain did not replicate in either epithelial or macrophage-like cells, and intracellular replication was restored by the wild-type allele in trans. Based on the deletion mutant phenotype, FTL_1914 was termed ripA (required for intracellular proliferation, factor A). Following uptake by J774.A1 cells, F. tularensis LVS Delta ripA colocalized with LAMP-1 then escaped the phagosome at the same rate and frequency as wild-type LVS-infected cells. Electron micrographs of the F. tularensis LVS Delta ripA mutant demonstrated the reentry of the mutant bacteria into double membrane vacuoles characteristic of autophagosomes in a process that was not dependent on replication. The F. tularensis LVS Delta ripA mutant was significantly impaired in its ability to persist in the lung and in its capacity to disseminate and colonize the liver and spleen in a mouse model of pulmonary tularemia. The RipA protein was expressed during growth in laboratory media and localized to the cytoplasmic membrane. Thus, RipA is a cytoplasmic membrane protein conserved among Francisella species that is required for intracellular replication within the host cell cytoplasm as well as disease progression, dissemination, and virulence. PMID:18765722

  13. Immunological mechanisms contributing to the double burden of diabetes and intracellular bacterial infections

    PubMed Central

    Hodgson, Kelly; Morris, Jodie; Bridson, Tahnee; Govan, Brenda; Rush, Catherine; Ketheesan, Natkunam

    2015-01-01

    Diabetes has been recognized as an important risk factor for a variety of intracellular bacterial infections, but research into the dysregulated immune mechanisms contributing to the impaired host–pathogen interactions is in its infancy. Diabetes is characterized by a chronic state of low-grade inflammation due to activation of pro-inflammatory mediators and increased formation of advanced glycation end products. Increased oxidative stress also exacerbates the chronic inflammatory processes observed in diabetes. The reduced phagocytic and antibacterial activity of neutrophils and macrophages provides an intracellular niche for the pathogen to replicate. Phagocytic and antibacterial dysfunction may be mediated directly through altered glucose metabolism and oxidative stress. Furthermore, impaired activation of natural killer cells contributes to decreased levels of interferon-γ, required for promoting macrophage antibacterial mechanisms. Together with impaired dendritic cell function, this impedes timely activation of adaptive immune responses. Increased intracellular oxidation of antigen-presenting cells in individuals with diabetes alters the cytokine profile generated and the subsequent balance of T-cell immunity. The establishment of acute intracellular bacterial infections in the diabetic host is associated with impaired T-cell-mediated immune responses. Concomitant to the greater intracellular bacterial burden and potential cumulative effect of chronic inflammatory processes, late hyper-inflammatory cytokine responses are often observed in individuals with diabetes, contributing to systemic pathology. The convergence of intracellular bacterial infections and diabetes poses new challenges for immunologists, providing the impetus for multidisciplinary research. PMID:25262977

  14. Formation of extracellular and intracellular ice during warming of vitrified mouse morulae and its effect on embryo survival.

    PubMed

    Jin, Bo; Kusanagi, Kenji; Ueda, Makiko; Seki, Shinsuke; Valdez, Delgado M; Edashige, Keisuke; Kasai, Magosaburo

    2008-06-01

    In vitrified solutions, ice can form during warming if the concentration of the cryoprotectant is insufficient. For the cryopreservation of cells, ice is innocuous when it remains outside the cell, but intracellular ice (ICI) is lethal. We tried to estimate the conditions in which ICI forms in vitrified mouse morulae during warming. The solutions for the experiments (EFS10-EFS50) contained 10-50% ethylene glycol plus Ficoll plus sucrose. When vitrified EFS20, EFS30, and EFS40 were kept at -80 degrees C, they remained transparent after 3 min, but turned opaque after 60 min (EFS20, EFS30) or 24h (EFS40). Morulae were vitrified with EFS solutions after exposure for 30-120 s at 25 degrees C. They were warmed by various methods and survival was assessed in culture. After rapid warming (control), survival was high with EFS30 (79-93%) and EFS40 (96-99%). After slow warming, survival decreased with both EFS30 (48-62%) and EFS40 (44-64%). This must be from the formation of ICI. To examine the temperature at which ICI formed during slow warming, vitrified embryos were kept at various sub-zero temperatures during warming. Survival with EFS30 and EFS40 decreased on keeping samples for 3 min at -80 (25-75%), -60 (7-49%), -40 (0-41%), or -20 degrees C (26-60%). When samples were kept at -80 degrees C for 24h, the survival decreased to 0-14%. These results suggest that ICI forms at a wide range of temperatures including -80 and -20 degrees C, more likely between -60 and -40 degrees C, and the ice forms not only quickly but also slowly. PMID:18466891

  15. Neurodegeneration in Glaucoma: Progression and Calcium-Dependent Intracellular Mechanisms

    PubMed Central

    Crish, Samuel D.; Calkins, David J.

    2011-01-01

    Glaucoma is an age-related optic neuropathy involving sensitivity to ocular pressure. The disease is now seen increasingly as one of the central nervous system, as powerful new approaches highlight an increasing number of similarities with other age-related neurodegenerations such as Alzheimer’s and Parkinson’s. While the etiologies of these diseases are diverse, they involve many important common elements including compartmentalized programs of degeneration targeting axons, dendrites and finally cell bodies. Most age-related degenerations display early functional deficits that precede actual loss of neuronal substrate. These are linked to several specific neurochemical cascades that can be linked back to dysregulation of Ca2+-dependent processes. We are now in the midst of identifying similar cascades in glaucoma. Here we review recent evidence on the pathological progression of neurodegeneration in glaucoma and some of the Ca2+-dependent mechanisms that could underlie these changes. These mechanisms present clear implications for efforts to develop interventions targeting neuronal loss directly and make glaucoma an attractive model for both interrogating and informing other neurodegenerative diseases. PMID:21187126

  16. The Outer Membrane Protein A (OmpA) of Y. pestis promotes intracellular survival and virulence in mice

    PubMed Central

    Bartra, Sara Schesser; Gong, Xin; Lorica, Cherish D.; Jain, Chaitanya; Nair, Manoj K. M.; Schifferli, Dieter; Qian, Lianfen; Li, Zhongwei; Plano, Gregory V.; Schesser, Kurt

    2011-01-01

    The plague bacterium Yersinia pestis has a number of well-described strategies to protect itself from both host cells and soluble factors. In an effort to identify additional anti-host factors, we employed a transposon site hybridization (TraSH)-based approach to screen 105 Y. pestis mutants in an in vitro infection system. In addition to loci encoding various components of the well-characterized type III secretion system (T3SS), our screen unambiguously identified ompA as a pro-survival gene. We go on to show that an engineered Y. pestis ΔompA strain, as well as a ΔompA strain of the closely related pathogen Y. pseudotuberculosis, have fully functioning T3SSs but are specifically defective in surviving within macrophages. Additionally, the Y. pestis ΔompA strain was outcompeted by the wild-type strain in a mouse co-infection assay. Unlike in other bacterial pathogens in which OmpA can promote adherence, invasion, or serum resistance, the OmpA of Y. pestis is restricted to enhancing intracellular survival. Our data show that OmpA of the pathogenic Yersinia is a virulence factor on par with the T3SS. PMID:22023991

  17. Mycobacterium tuberculosis Rv1265 promotes mycobacterial intracellular survival and alters cytokine profile of the infected macrophage.

    PubMed

    Luo, Hongping; Zeng, Jie; Huang, Qinqin; Liu, Minqiang; Abdalla, Abualgasim Elgaili; Xie, Longxiang; Wang, Huan; Xie, Jianping

    2016-03-01

    Mycobacterium tuberculosis cAMP and underlying regulatory network are crucial for its survival and thrive in the presence of numerous stresses mounted by the host. Our studies mainly focus on the cAMP-induced M. tuberculosis gene Rv1265, which was shown to be up-regulated under hypoxia and during macrophage infection by addition of exogenous cAMP. To explore the role of Rv1265 in host-pathogen interactions, Rv1265 was expressed in a non-pathogenic Mycobacterium smegmatis. We found that Rv1265 was associated with cell envelope and can up-regulate some cell wall fatty acid components, especially the C26:0. The survival of the recombinant Ms_Rv1265 was enhanced within macrophages and under stress conditions such as low pH and SDS. Macrophages infected with Ms_Rv1265 increased transcription of pro-inflammatory cytokines IL-1β, IL-6, and IL-12 P40 and anti-inflammatory cytokine IL-10 possibly through activation of NF-κB and ERK1/2 pathway. Our findings indicate that Rv1265 can enhance mycobacterial survival within macrophages, and perturb the cytokine profile of macrophage. PMID:26156642

  18. Knocking down of the KCC2 in rat hippocampal neurons increases intracellular chloride concentration and compromises neuronal survival

    PubMed Central

    Pellegrino, Christophe; Gubkina, Olena; Schaefer, Michael; Becq, Hélène; Ludwig, Anastasia; Mukhtarov, Marat; Chudotvorova, Ilona; Corby, Severine; Salyha, Yuriy; Salozhin, Sergey; Bregestovski, Piotr; Medina, Igor

    2011-01-01

    Abstract KCC2 is a neuron-specific potassium–chloride co-transporter controlling intracellular chloride homeostasis in mature and developing neurons. It is implicated in the regulation of neuronal migration, dendrites outgrowth and formation of the excitatory and inhibitory synaptic connections. The function of KCC2 is suppressed under several pathological conditions including neuronal trauma, different types of epilepsies, axotomy of motoneurons, neuronal inflammations and ischaemic insults. However, it remains unclear how down-regulation of the KCC2 contributes to neuronal survival during and after toxic stress. Here we show that in primary hippocampal neuronal cultures the suppression of the KCC2 function using two different shRNAs, dominant-negative KCC2 mutant C568A or DIOA inhibitor, increased the intracellular chloride concentration [Cl−]i and enhanced the toxicity induced by lipofectamine-dependent oxidative stress or activation of the NMDA receptors. The rescuing of the KCC2 activity using over-expression of the active form of the KCC2, but not its non-active mutant Y1087D, effectively restored [Cl−]i and enhanced neuronal resistance to excitotoxicity. The reparative effects of KCC2 were mimicked by over-expression of the KCC3, a homologue transporter. These data suggest an important role of KCC2-dependent potassium/chloride homeostasis under neurototoxic conditions and reveal a novel role of endogenous KCC2 as a neuroprotective molecule. PMID:21486764

  19. Effect of warming rate on the survival of vitrified mouse oocytes and on the recrystallization of intracellular ice.

    PubMed

    Seki, Shinsuke; Mazur, Peter

    2008-10-01

    Successful cryopreservation demands there be little or no intracellular ice. One procedure is classical slow equilibrium freezing, and it has been successful in many cases. However, for some important cell types, including some mammalian oocytes, it has not. For the latter, there are increasing attempts to cryopreserve them by vitrification. However, even if intracellular ice formation (IIF) is prevented during cooling, it can still occur during the warming of a vitrified sample. Here, we examine two aspects of this occurrence in mouse oocytes. One took place in oocytes that were partly dehydrated by an initial hold for 12 min at -25 degrees C. They were then cooled rapidly to -70 degrees C and warmed slowly, or they were warmed rapidly to intermediate temperatures and held. These oocytes underwent no IIF during cooling but blackened from IIF during warming. The blackening rate increased about 5-fold for each five-degree rise in temperature. Upon thawing, they were dead. The second aspect involved oocytes that had been vitrified by cooling to -196 degrees C while suspended in a concentrated solution of cryoprotectants and warmed at rates ranging from 140 degrees C/min to 3300 degrees C/min. Survivals after warming at 140 degrees C/min and 250 degrees C/min were low (<30%). Survivals after warming at > or =2200 degrees C/min were high (80%). When warmed slowly, they were killed, apparently by the recrystallization of previously formed small internal ice crystals. The similarities and differences in the consequences of the two types of freezing are discussed. PMID:18562703

  20. Mechanisms of sensorineural cell damage, death and survival in the cochlea

    PubMed Central

    Wong, Ann C. Y.; Ryan, Allen F.

    2015-01-01

    The majority of acquired hearing loss, including presbycusis, is caused by irreversible damage to the sensorineural tissues of the cochlea. This article reviews the intracellular mechanisms that contribute to sensorineural damage in the cochlea, as well as the survival signaling pathways that can provide endogenous protection and tissue rescue. These data have primarily been generated in hearing loss not directly related to age. However, there is evidence that similar mechanisms operate in presbycusis. Moreover, accumulation of damage from other causes can contribute to age-related hearing loss (ARHL). Potential therapeutic interventions to balance opposing but interconnected cell damage and survival pathways, such as antioxidants, anti-apoptotics, and pro-inflammatory cytokine inhibitors, are also discussed. PMID:25954196

  1. Effects of Equex from different sources on post-thaw survival, longevity and intracellular Ca2+ concentration of dog spermatozoa.

    PubMed

    Peña, Ana I; Lugilde, Luz L; Barrio, Mónica; Herradón, Pedro G; Quintela, Luis A

    2003-04-15

    The aims of the present study were to compare the effects of two commercial preparations (Equex STM Paste or Equex Pasta), whose active ingredient is sodium dodecyl sulphate (SDS), added to a Tris-egg yolk-based extender, on post-thaw sperm survival and longevity, as well as on the intracellular Ca(2+) concentration of dog spermatozoa during incubation at 38 degrees C. One ejaculate was collected from each of eight dogs. Each ejaculate was centrifuged, the semen plasma discarded, and the sperm pellet rediluted with a Tris-glucose-egg yolk extender containing 3% glycerol (Ext-1) at a sperm concentration of 200 x 10(6) spermatozoa (spz)/ml. The diluted semen was divided in three aliquots of equal volume and allowed to equilibrate for 1h at 4 degrees C. After equilibration, the same volume of three different second extenders was added, respectively, to each of the three aliquots: (A) Ext-2A (same composition as Ext-1 except that it contained 7% glycerol and 1% Equex STM Paste), (B) Ext-2B (same composition as that of Ext-1 except that it contained 7% glycerol and 1% Equex Pasta), and (C) Ext-2 (CONTROL: same composition as that of Ext-1 except that it contained 7% glycerol). Semen samples were packed in 0.5 ml straws and frozen on a rack 4 cm above liquid nitrogen (LN(2)) in a styrofoam box. Thawing was at 70 degrees C for 8s. Sperm motility was evaluated after thawing and at 1 h intervals for 5h at 38 degrees C by subjective examination and by using a CASA system. Plasma membrane integrity and acrosomal status were evaluated at 1, 4 and 7h post-thaw using a triple staining procedure and flow cytometry. Intracellular Ca(2+) concentration of live spermatozoa was evaluated by flow cytometry at 1, 4 and 7h post-thaw after co-loading the sperm cells with the Ca(2+) indicators Fluo 3 AM and Fura Red AM, and with PI. Post-thaw sperm survival and longevity, as well as the quality of the sperm movement, were significantly better (P<0.005) when Ext-2A (containing Equex STM

  2. Two Novel Functions of Hyaluronidase from Streptococcus agalactiae Are Enhanced Intracellular Survival and Inhibition of Proinflammatory Cytokine Expression

    PubMed Central

    Wang, Zhaofei; Guo, Changming; Xu, Yannan; Liu, Guangjin; Lu, Chengping

    2014-01-01

    Streptococcus agalactiae is the causative agent of septicemia and meningitis in fish. Previous studies have shown that hyaluronidase (Hyl) is an important virulence factor in many Gram-positive bacteria. To investigate the role of S. agalactiae Hyl during interaction with macrophages, we inactivated the gene encoding extracellular hyaluronidase, hylB, in a clinical Hyl+ isolate. The isogenic hylb mutant (Δhylb) displayed reduced survival in macrophages compared to the wild type and stimulated a significantly higher release of proinflammatory cytokines, such as interleukin-1β (IL-1β), IL-6, and tumor necrosis factor alpha (TNF-α), than the wild type in macrophages as well as in mice. Furthermore, only Hyl+ strains could grow utilizing hyaluronic acid (HA) as the sole carbon source, suggesting that Hyl permits the organism to utilize host HA as an energy source. Fifty percent lethal dose (LD50) determinations in zebrafish demonstrated that the hylb mutant was highly attenuated relative to the wild-type strain. Experimental infection of BALB/c mice revealed that bacterial loads in the blood, spleen, and brain at 16 h postinfection were significantly reduced in the ΔhylB mutant compared to those in wild-type-infected mice. In conclusion, hyaluronidase has a strong influence on the intracellular survival of S. agalactiae and proinflammatory cytokine expression, suggesting that it plays a key role in S. agalactiae pathogenicity. PMID:24711564

  3. Biofilms: Survival Mechanisms of Clinically Relevant Microorganisms

    PubMed Central

    Donlan, Rodney M.; Costerton, J. William

    2002-01-01

    Though biofilms were first described by Antonie van Leeuwenhoek, the theory describing the biofilm process was not developed until 1978. We now understand that biofilms are universal, occurring in aquatic and industrial water systems as well as a large number of environments and medical devices relevant for public health. Using tools such as the scanning electron microscope and, more recently, the confocal laser scanning microscope, biofilm researchers now understand that biofilms are not unstructured, homogeneous deposits of cells and accumulated slime, but complex communities of surface-associated cells enclosed in a polymer matrix containing open water channels. Further studies have shown that the biofilm phenotype can be described in terms of the genes expressed by biofilm-associated cells. Microorganisms growing in a biofilm are highly resistant to antimicrobial agents by one or more mechanisms. Biofilm-associated microorganisms have been shown to be associated with several human diseases, such as native valve endocarditis and cystic fibrosis, and to colonize a wide variety of medical devices. Though epidemiologic evidence points to biofilms as a source of several infectious diseases, the exact mechanisms by which biofilm-associated microorganisms elicit disease are poorly understood. Detachment of cells or cell aggregates, production of endotoxin, increased resistance to the host immune system, and provision of a niche for the generation of resistant organisms are all biofilm processes which could initiate the disease process. Effective strategies to prevent or control biofilms on medical devices must take into consideration the unique and tenacious nature of biofilms. Current intervention strategies are designed to prevent initial device colonization, minimize microbial cell attachment to the device, penetrate the biofilm matrix and kill the associated cells, or remove the device from the patient. In the future, treatments may be based on inhibition of genes

  4. Swine TRIM21 restricts FMDV infection via an intracellular neutralization mechanism.

    PubMed

    Fan, Wenchun; Zhang, Dong; Qian, Ping; Qian, Suhong; Wu, Mengge; Chen, Huanchun; Li, Xiangmin

    2016-03-01

    The tripartite motif protein 21 (TRIM21) is a ubiquitously expressed E3 ubiquitin ligase and an intracellular antibody receptor. TRIM21 mediates antibody-dependent intracellular neutralization (ADIN) in cytosol and provides an intracellular immune response to protect host defense against pathogen infection. In this study, swine TRIM21 (sTRIM21) was cloned and its role in ADIN was investigated. The expression of sTRIM21 is induced by type I interferon in PK-15 cells. sTRIM21 restricts FMDV infection in the presence of FMDV specific antibodies. Furthermore, sTRIM21 interacts with Fc fragment of swine immunoglobulin G (sFc) fused VP1 of FMDV and thereby causing its degradation. Both the RING and SPRY domains are essential for sTRIM21 to degrade sFc-fused VP1. These results suggest that the intracellular neutralization features of FMDV contribute to the antiviral activity of sTRIM21. sTRIM21 provide another intracellular mechanism to inhibit FMDV infection in infected cells. PMID:26777733

  5. A role for interleukin-17A in modulating intracellular survival of Mycobacterium bovis bacillus Calmette-Guérin in murine macrophages.

    PubMed

    Ling, Wai Lim; Wang, Liang Jie; Pong, John C H; Lau, Allan S Y; Li, James C B

    2013-11-01

    Interleukin 17A IL-17A is a crucial immunomodulator in various chronic immunological diseases including rheumatoid arthritis and inflammatory bowel disease. The cytokine has also been demonstrated to control the pathogenesis of the Mycobacterium tuberculosis by dysregulating production of cytokines and chemokines and promoting granuloma formation. Whether IL-17A regulates innate defence mechanisms of macrophages in response to mycobacterial infection remains to be elucidated. In the current report, we investigated the effects of IL-17A on modulating the intracellular survival of Mycobacterium bovis bacillus Calmette-Guérin (BCG) in RAW264.7 murine macrophages. We observed that IL-17A pre-treatment for 24 hr was able to synergistically enhance BCG-induced nitric oxide (NO) production and inducible nitric oxide synthase expression in dose- and time-dependent manners. We further delineated the mechanisms involved in this synergistic reaction. IL-17A was found to specifically enhanced BCG-induced phosphorylation of Jun N-terminal kinase (JNK), but not of extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase. By using a specific JNK inhibitor (SP600125), we found that the production of NO in BCG-infected macrophages was significantly suppressed. Taken together, we confirmed the involvement of the JNK pathway in IL-17A-enhanced NO production in BCG-infected macrophages. We further demonstrated that IL-17A significantly enhanced the clearance of intracellular BCG by macrophages through an NO-dependent killing mechanism. In conclusion, our study revealed an anti-mycobacterial role of IL-17A through priming the macrophages to produce NO in response to mycobacterial infection. PMID:23808492

  6. Polycaprolactone/maltodextrin nanocarrier for intracellular drug delivery: formulation, uptake mechanism, internalization kinetics, and subcellular localization

    PubMed Central

    Korang-Yeboah, Maxwell; Gorantla, Yamini; Paulos, Simon A; Sharma, Pankaj; Chaudhary, Jaideep; Palaniappan, Ravi

    2015-01-01

    Prostate cancer (PCa) disease progression is associated with significant changes in intracellular and extracellular proteins, intracellular signaling mechanism, and cancer cell phenotype. These changes may have direct impact on the cellular interactions with nanocarriers; hence, there is the need for a much-detailed understanding, as nanocarrier cellular internalization and intracellular sorting mechanism correlate directly with bioavailability and clinical efficacy. In this study, we report the differences in the rate and mechanism of cellular internalization of a biocompatible polycaprolactone (PCL)/maltodextrin (MD) nanocarrier system for intracellular drug delivery in LNCaP, PC3, and DU145 PCa cell lines. PCL/MD nanocarriers were designed and characterized. PCL/MD nanocarriers significantly increased the intracellular concentration of coumarin-6 and fluorescein isothiocyanate-labeled bovine serum albumin, a model hydrophobic and large molecule, respectively. Fluorescence microscopy and flow cytometry analysis revealed rapid internalization of the nanocarrier. The extent of nanocarrier cellular internalization correlated directly with cell line aggressiveness. PCL/MD internalization was highest in PC3 followed by DU145 and LNCaP, respectively. Uptake in all PCa cell lines was metabolically dependent. Extraction of endogenous cholesterol by methyl-β-cyclodextrin reduced uptake by 75%±4.53% in PC3, 64%±6.01% in LNCaP, and 50%±4.50% in DU145, indicating the involvement of endogenous cholesterol in cellular internalization. Internalization of the nanocarrier in LNCaP was mediated mainly by macropinocytosis and clathrin-independent pathways, while internalization in PC3 and DU145 involved clathrin-mediated endocytosis, clathrin-independent pathways, and macropinocytosis. Fluorescence microscopy showed a very diffused and non-compartmentalized subcellular localization of the PCL/MD nanocarriers with possible intranuclear localization and minor colocalization in

  7. Noncontact three-dimensional mapping of intracellular hydro-mechanical properties by Brillouin microscopy

    PubMed Central

    Scarcelli, Giuliano; Polacheck, William J.; Nia, Hadi T.; Patel, Kripa; Grodzinsky, Alan J.; Kamm, Roger D.; Yun, Seok Hyun

    2015-01-01

    Current measurements of the biomechanical properties of cells require physical contact with cells or lack sub-cellular resolution. Here, we developed a label-free optical microscopy technique based on Brillouin light scattering capable of measuring intracellular longitudinal modulus with optical resolution. We obtained 3D Brillouin maps of cells in 2D and 3D microenvironments, which reveal mechanical changes due to cytoskeletal modulation and cell volume regulation. PMID:26436482

  8. A general mechanism for intracellular toxicity of metal-containing nanoparticles.

    PubMed

    Sabella, Stefania; Carney, Randy P; Brunetti, Virgilio; Malvindi, Maria Ada; Al-Juffali, Noura; Vecchio, Giuseppe; Janes, Sam M; Bakr, Osman M; Cingolani, Roberto; Stellacci, Francesco; Pompa, Pier Paolo

    2014-06-21

    The assessment of the risks exerted by nanoparticles is a key challenge for academic, industrial, and regulatory communities worldwide. Experimental evidence points towards significant toxicity for a range of nanoparticles both in vitro and in vivo. Worldwide efforts aim at uncovering the underlying mechanisms for this toxicity. Here, we show that the intracellular ion release elicited by the acidic conditions of the lysosomal cellular compartment--where particles are abundantly internalized--is responsible for the cascading events associated with nanoparticles-induced intracellular toxicity. We call this mechanism a "lysosome-enhanced Trojan horse effect" since, in the case of nanoparticles, the protective cellular machinery designed to degrade foreign objects is actually responsible for their toxicity. To test our hypothesis, we compare the toxicity of similar gold particles whose main difference is in the internalization pathways. We show that particles known to pass directly through cell membranes become more toxic when modified so as to be mostly internalized by endocytosis. Furthermore, using experiments with chelating and lysosomotropic agents, we found that the toxicity mechanism for different metal containing NPs (such as metallic, metal oxide, and semiconductor NPs) is mainly associated with the release of the corresponding toxic ions. Finally, we show that particles unable to release toxic ions (such as stably coated NPs, or diamond and silica NPs) are not harmful to intracellular environments. PMID:24842463

  9. Intracellular ice and cell survival in cryo-exposed embryonic axes of recalcitrant seeds of Acer saccharinum: an ultrastructural study of factors affecting cell and ice structures

    PubMed Central

    Wesley-Smith, James; Berjak, Patricia; Pammenter, N. W.; Walters, Christina

    2014-01-01

    Background and Aims Cryopreservation is the only long-term conservation strategy available for germplasm of recalcitrant-seeded species. Efforts to cryopreserve this form of germplasm are hampered by potentially lethal intracellular freezing events; thus, it is important to understand the relationships among cryo-exposure techniques, water content, structure and survival. Methods Undried embryonic axes of Acer saccharinum and those rapidly dried to two different water contents were cooled at three rates and re-warmed at two rates. Ultrastructural observations were carried out on radicle and shoot tips prepared by freeze-fracture and freeze-substitution to assess immediate (i.e. pre-thaw) responses to cooling treatments. Survival of axes was assessed in vitro. Key Results Intracellular ice formation was not necessarily lethal. Embryo cells survived when crystal diameter was between 0·2 and 0·4 µm and fewer than 20 crystals were distributed per μm2 in the cytoplasm. Ice was not uniformly distributed within the cells. In fully hydrated axes cooled at an intermediate rate, the interiors of many organelles were apparently ice-free; this may have prevented the disruption of vital intracellular machinery. Intracytoplasmic ice formation did not apparently impact the integrity of the plasmalemma. The maximum number of ice crystals was far greater in shoot apices, which were more sensitive than radicles to cryo-exposure. Conclusions The findings challenge the accepted paradigm that intracellular ice formation is always lethal, as the results show that cells can survive intracellular ice if crystals are small and localized in the cytoplasm. Further understanding of the interactions among water content, cooling rate, cell structure and ice structure is required to optimize cryopreservation treatments without undue reliance on empirical approaches. PMID:24368198

  10. The intracellular trafficking mechanism of Lipofectamine-based transfection reagents and its implication for gene delivery

    PubMed Central

    Cardarelli, Francesco; Digiacomo, Luca; Marchini, Cristina; Amici, Augusto; Salomone, Fabrizio; Fiume, Giuseppe; Rossetta, Alessandro; Gratton, Enrico; Pozzi, Daniela; Caracciolo, Giulio

    2016-01-01

    Lipofectamine reagents are widely accepted as “gold-standard” for the safe delivery of exogenous DNA or RNA into cells. Despite this, a satisfactory mechanism-based explanation of their superior efficacy has remained mostly elusive thus far. Here we apply a straightforward combination of live cell imaging, single-particle tracking microscopy, and quantitative transfection-efficiency assays on live cells to unveil the intracellular trafficking mechanism of Lipofectamine/DNA complexes. We find that Lipofectamine, contrary to alternative formulations, is able to efficiently avoid active intracellular transport along microtubules, and the subsequent entrapment and degradation of the payload within acidic/digestive lysosomal compartments. This result is achieved by random Brownian motion of Lipofectamine-containing vesicles within the cytoplasm. We demonstrate here that Brownian diffusion is an efficient route for Lipofectamine/DNA complexes to avoid metabolic degradation, thus leading to optimal transfection. By contrast, active transport along microtubules results in DNA degradation and subsequent poor transfection. Intracellular trafficking, endosomal escape and lysosomal degradation appear therefore as highly interdependent phenomena, in such a way that they should be viewed as a single barrier on the route for efficient transfection. As a matter of fact, they should be evaluated in their entirety for the development of optimized non-viral gene delivery vectors. PMID:27165510

  11. The intracellular trafficking mechanism of Lipofectamine-based transfection reagents and its implication for gene delivery.

    PubMed

    Cardarelli, Francesco; Digiacomo, Luca; Marchini, Cristina; Amici, Augusto; Salomone, Fabrizio; Fiume, Giuseppe; Rossetta, Alessandro; Gratton, Enrico; Pozzi, Daniela; Caracciolo, Giulio

    2016-01-01

    Lipofectamine reagents are widely accepted as "gold-standard" for the safe delivery of exogenous DNA or RNA into cells. Despite this, a satisfactory mechanism-based explanation of their superior efficacy has remained mostly elusive thus far. Here we apply a straightforward combination of live cell imaging, single-particle tracking microscopy, and quantitative transfection-efficiency assays on live cells to unveil the intracellular trafficking mechanism of Lipofectamine/DNA complexes. We find that Lipofectamine, contrary to alternative formulations, is able to efficiently avoid active intracellular transport along microtubules, and the subsequent entrapment and degradation of the payload within acidic/digestive lysosomal compartments. This result is achieved by random Brownian motion of Lipofectamine-containing vesicles within the cytoplasm. We demonstrate here that Brownian diffusion is an efficient route for Lipofectamine/DNA complexes to avoid metabolic degradation, thus leading to optimal transfection. By contrast, active transport along microtubules results in DNA degradation and subsequent poor transfection. Intracellular trafficking, endosomal escape and lysosomal degradation appear therefore as highly interdependent phenomena, in such a way that they should be viewed as a single barrier on the route for efficient transfection. As a matter of fact, they should be evaluated in their entirety for the development of optimized non-viral gene delivery vectors. PMID:27165510

  12. A general mechanism for intracellular toxicity of metal-containing nanoparticles

    NASA Astrophysics Data System (ADS)

    Sabella, Stefania; Carney, Randy P.; Brunetti, Virgilio; Malvindi, Maria Ada; Al-Juffali, Noura; Vecchio, Giuseppe; Janes, Sam M.; Bakr, Osman M.; Cingolani, Roberto; Stellacci, Francesco; Pompa, Pier Paolo

    2014-05-01

    The assessment of the risks exerted by nanoparticles is a key challenge for academic, industrial, and regulatory communities worldwide. Experimental evidence points towards significant toxicity for a range of nanoparticles both in vitro and in vivo. Worldwide efforts aim at uncovering the underlying mechanisms for this toxicity. Here, we show that the intracellular ion release elicited by the acidic conditions of the lysosomal cellular compartment - where particles are abundantly internalized - is responsible for the cascading events associated with nanoparticles-induced intracellular toxicity. We call this mechanism a ``lysosome-enhanced Trojan horse effect'' since, in the case of nanoparticles, the protective cellular machinery designed to degrade foreign objects is actually responsible for their toxicity. To test our hypothesis, we compare the toxicity of similar gold particles whose main difference is in the internalization pathways. We show that particles known to pass directly through cell membranes become more toxic when modified so as to be mostly internalized by endocytosis. Furthermore, using experiments with chelating and lysosomotropic agents, we found that the toxicity mechanism for different metal containing NPs (such as metallic, metal oxide, and semiconductor NPs) is mainly associated with the release of the corresponding toxic ions. Finally, we show that particles unable to release toxic ions (such as stably coated NPs, or diamond and silica NPs) are not harmful to intracellular environments.The assessment of the risks exerted by nanoparticles is a key challenge for academic, industrial, and regulatory communities worldwide. Experimental evidence points towards significant toxicity for a range of nanoparticles both in vitro and in vivo. Worldwide efforts aim at uncovering the underlying mechanisms for this toxicity. Here, we show that the intracellular ion release elicited by the acidic conditions of the lysosomal cellular compartment - where

  13. Coupling mechanical forces to electrical signaling: molecular motors and the intracellular transport of ion channels.

    PubMed

    Barry, Joshua; Gu, Chen

    2013-04-01

    Proper localization of various ion channels is fundamental to neuronal functions, including postsynaptic potential plasticity, dendritic integration, action potential initiation and propagation, and neurotransmitter release. Microtubule-based forward transport mediated by kinesin motors plays a key role in placing ion channel proteins to correct subcellular compartments. PDZ- and coiled-coil-domain proteins function as adaptor proteins linking ionotropic glutamate and GABA receptors to various kinesin motors, respectively. Recent studies show that several voltage-gated ion channel/transporter proteins directly bind to kinesins during forward transport. Three major regulatory mechanisms underlying intracellular transport of ion channels are also revealed. These studies contribute to understanding how mechanical forces are coupled to electrical signaling and illuminating pathogenic mechanisms in neurodegenerative diseases. PMID:22910031

  14. Coupling Mechanical Forces to Electrical Signaling: Molecular Motors and the Intracellular Transport of Ion Channels

    PubMed Central

    Barry, Joshua; Gu, Chen

    2013-01-01

    Proper localization of various ion channels is fundamental to neuronal functions, including postsynaptic potential plasticity, dendritic integration, action potential initiation and propagation, and neurotransmitter release. Microtubule-based forward transport mediated by kinesin motors plays a key role in placing ion channel proteins to correct subcellular compartments. PDZ- and coiled-coil-domain proteins function as adaptor proteins linking ionotropic glutamate and GABA receptors to various kinesin motors, respectively. Recent studies show that several voltage-gated ion channel/transporter proteins directly bind to kinesins during forward transport. Three major regulatory mechanisms underlying intracellular transport of ion channels are also revealed. These studies contribute to understanding how mechanical forces are coupled to electrical signaling and illuminating pathogenic mechanisms in neurodegenerative diseases. PMID:22910031

  15. Kinetic insulation as an effective mechanism for achieving pathway specificity in intracellular signaling networks

    PubMed Central

    Behar, Marcelo; Dohlman, Henrik G.; Elston, Timothy C.

    2007-01-01

    Intracellular signaling pathways that share common components often elicit distinct physiological responses. In most cases, the biochemical mechanisms responsible for this signal specificity remain poorly understood. Protein scaffolds and cross-inhibition have been proposed as strategies to prevent unwanted cross-talk. Here, we report a mechanism for signal specificity termed “kinetic insulation.” In this approach signals are selectively transmitted through the appropriate pathway based on their temporal profile. In particular, we demonstrate how pathway architectures downstream of a common component can be designed to efficiently separate transient signals from signals that increase slowly over time. Furthermore, we demonstrate that upstream signaling proteins can generate the appropriate input to the common pathway component regardless of the temporal profile of the external stimulus. Our results suggest that multilevel signaling cascades may have evolved to modulate the temporal profile of pathway activity so that stimulus information can be efficiently encoded and transmitted while ensuring signal specificity. PMID:17913886

  16. Systematic analysis of intracellular mechanisms of propanol production in the engineered Thermobifida fusca B6 strain.

    PubMed

    Deng, Yu; Fisher, Adam B; Fong, Stephen S

    2015-10-01

    Thermobifida fusca is a moderately thermophilic actinobacterium naturally capable of utilizing lignocellulosic biomass. The B6 strain of T. fusca was previously engineered to produce 1-propanol directly on lignocellulosic biomass by expressing a bifunctional butyraldehyde/alcohol dehydrogenase (adhE2). To characterize the intracellular mechanisms related to the accumulation of 1-propanol, the engineered B6 and wild-type (WT) strains were systematically compared by analysis of the transcriptome and intracellular metabolome during exponential growth on glucose, cellobiose, and Avicel. Of the 18 known cellulases in T. fusca, 10 cellulase genes were transcriptionally expressed on all three substrates along with three hemicellulases. Transcriptomic analysis of cellodextrin and cellulose transport revealed that Tfu_0936 (multiple sugar transport system permease) was the key enzyme regulating the uptake of sugars in T. fusca. For both WT and B6 strains, it was found that growth in oxygen-limited conditions resulted in a blocked tricarboxylic acid (TCA) cycle caused by repressed expression of Tfu_1925 (aconitate hydratase). Further, the transcriptome suggested a pathway for synthesizing succinyl-CoA: oxaloacetate to malate (by malate dehydrogenase), malate to fumarate (by fumarate hydratase), and fumarate to succinate (by succinate dehydrogenase/fumarate reductase) which was ultimately converted to succinyl-CoA by succinyl-CoA synthetase. Both the transcriptome and the intracellular metabolome confirmed that 1-propanol was produced through succinyl-CoA, L-methylmalonyl-CoA, D-methylmalonyl-CoA, and propionyl-CoA in the B6 strain. PMID:26227414

  17. Neocortical GABA release at high intracellular sodium and low extracellular calcium: an anti-seizure mechanism.

    PubMed

    Rassner, Michael P; Moser, Andreas; Follo, Marie; Joseph, Kevin; van Velthoven-Wurster, Vera; Feuerstein, Thomas J

    2016-04-01

    In epilepsy, the GABA and glutamate balance may be disrupted and a transient decrease in extracellular calcium occurs before and during a seizure. Flow Cytometry based fluorescence activated particle sorting experiments quantified synaptosomes from human neocortical tissue, from both epileptic and non-epileptic patients (27.7% vs. 36.9% GABAergic synaptosomes, respectively). Transporter-mediated release of GABA in human and rat neocortical synaptosomes was measured using the superfusion technique for the measurement of endogenous GABA. GABA release was evoked by either a sodium channel activator or a sodium/potassium-ATPase inhibitor when exocytosis was possible or prevented, and when the sodium/calcium exchanger was active or inhibited. The transporter-mediated release of GABA is because of elevated intracellular sodium. A reduction in the extracellular calcium increased this release (in both non-epileptic and epileptic, except Rasmussen encephalitis, synaptosomes). The inverse was seen during calcium doubling. In humans, GABA release was not affected by exocytosis inhibition, that is, it was solely transporter-mediated. However, in rat synaptosomes, an increase in GABA release at zero calcium was only exhibited when the exocytosis was prevented. The absence of calcium amplified the sodium/calcium exchanger activity, leading to elevated intracellular sodium, which, together with the stimulation-evoked intracellular sodium increment, enhanced GABA transporter reversal. Sodium/calcium exchange inhibitors diminished GABA release. Thus, an important seizure-induced extracellular calcium reduction might trigger a transporter- and sodium/calcium exchanger-related anti-seizure mechanism by augmenting transporter-mediated GABA release, a mechanism absent in rats. Uniquely, the additional increase in GABA release because of calcium-withdrawal dwindled during the course of illness in Rasmussen encephalitis. Seizures cause high Na(+) influx through action potentials. A

  18. Origins of stochastic intracellular processes and consequences for cell-to-cell variability and cellular survival strategies.

    PubMed

    Schwabe, A; Dobrzyński, M; Rybakova, K; Verschure, P; Bruggeman, F J

    2011-01-01

    Quantitative analyses of the dynamics of single cells have become a powerful approach in current cell biology. They give us an unprecedented opportunity to study dynamics of molecular networks at a high level of accuracy in living single cells. Genetically identical cells, growing in the same environment and sharing the same growth history, can differ remarkably in their molecular makeup and physiological behaviors. The origins of this cell-to-cell variability have in many cases been traced to the inevitable stochasticity of molecular reactions. Those mechanisms can cause isogenic cells to have qualitatively different life histories. Many studies indicate that molecular noise can be exploited by cell populations to enhance survival prospects in uncertain environments. On the other hand, cells have evolved noise-suppression mechanisms to cope with the inevitable noise in their functioning so as to reduce the hazardous effects of noise. In this chapter, we discuss key experiments, theoretical results, and physiological consequences of molecular stochasticity to introduce this exciting field to a broader community of (systems) biologists. PMID:21943916

  19. Cisplatin Resistant Spheroids Model Clinically Relevant Survival Mechanisms in Ovarian Tumors

    PubMed Central

    Miller, Daniel H.; Medina, Jamie E.; Hamilton, Joshua W.; Messerli, Mark A.; Brodsky, Alexander S.

    2016-01-01

    The majority of ovarian tumors eventually recur in a drug resistant form. Using cisplatin sensitive and resistant cell lines assembled into 3D spheroids we profiled gene expression and identified candidate mechanisms and biological pathways associated with cisplatin resistance. OVCAR-8 human ovarian carcinoma cells were exposed to sub-lethal concentrations of cisplatin to create a matched cisplatin-resistant cell line, OVCAR-8R. Genome-wide gene expression profiling of sensitive and resistant ovarian cancer spheroids identified 3,331 significantly differentially expressed probesets coding for 3,139 distinct protein-coding genes (Fc >2, FDR < 0.05) (S2 Table). Despite significant expression changes in some transporters including MDR1, cisplatin resistance was not associated with differences in intracellular cisplatin concentration. Cisplatin resistant cells were significantly enriched for a mesenchymal gene expression signature. OVCAR-8R resistance derived gene sets were significantly more biased to patients with shorter survival. From the most differentially expressed genes, we derived a 17-gene expression signature that identifies ovarian cancer patients with shorter overall survival in three independent datasets. We propose that the use of cisplatin resistant cell lines in 3D spheroid models is a viable approach to gain insight into resistance mechanisms relevant to ovarian tumors in patients. Our data support the emerging concept that ovarian cancers can acquire drug resistance through an epithelial-to-mesenchymal transition. PMID:26986722

  20. Cisplatin Resistant Spheroids Model Clinically Relevant Survival Mechanisms in Ovarian Tumors.

    PubMed

    Chowanadisai, Winyoo; Messerli, Shanta M; Miller, Daniel H; Medina, Jamie E; Hamilton, Joshua W; Messerli, Mark A; Brodsky, Alexander S

    2016-01-01

    The majority of ovarian tumors eventually recur in a drug resistant form. Using cisplatin sensitive and resistant cell lines assembled into 3D spheroids we profiled gene expression and identified candidate mechanisms and biological pathways associated with cisplatin resistance. OVCAR-8 human ovarian carcinoma cells were exposed to sub-lethal concentrations of cisplatin to create a matched cisplatin-resistant cell line, OVCAR-8R. Genome-wide gene expression profiling of sensitive and resistant ovarian cancer spheroids identified 3,331 significantly differentially expressed probesets coding for 3,139 distinct protein-coding genes (Fc >2, FDR < 0.05) (S2 Table). Despite significant expression changes in some transporters including MDR1, cisplatin resistance was not associated with differences in intracellular cisplatin concentration. Cisplatin resistant cells were significantly enriched for a mesenchymal gene expression signature. OVCAR-8R resistance derived gene sets were significantly more biased to patients with shorter survival. From the most differentially expressed genes, we derived a 17-gene expression signature that identifies ovarian cancer patients with shorter overall survival in three independent datasets. We propose that the use of cisplatin resistant cell lines in 3D spheroid models is a viable approach to gain insight into resistance mechanisms relevant to ovarian tumors in patients. Our data support the emerging concept that ovarian cancers can acquire drug resistance through an epithelial-to-mesenchymal transition. PMID:26986722

  1. An integrative approach to understanding microbial diversity: from intracellular mechanisms to community structure

    PubMed Central

    Gudelj, Ivana; Weitz, Joshua S; Ferenci, Tom; Claire Horner-Devine, M; Marx, Christopher J; Meyer, Justin R; Forde, Samantha E

    2010-01-01

    Trade-offs have been put forward as essential to the generation and maintenance of diversity. However, variation in trade-offs is often determined at the molecular level, outside the scope of conventional ecological inquiry. In this study, we propose that understanding the intracellular basis for trade-offs in microbial systems can aid in predicting and interpreting patterns of diversity. First, we show how laboratory experiments and mathematical models have unveiled the hidden intracellular mechanisms underlying trade-offs key to microbial diversity: (i) metabolic and regulatory trade-offs in bacteria and yeast; (ii) life-history trade-offs in bacterial viruses. Next, we examine recent studies of marine microbes that have taken steps toward reconciling the molecular and the ecological views of trade-offs, despite the challenges in doing so in natural settings. Finally, we suggest avenues for research where mathematical modelling, experiments and studies of natural microbial communities provide a unique opportunity to integrate studies of diversity across multiple scales. PMID:20576029

  2. Political Mechanisms for Long-Range Survival and Development

    NASA Astrophysics Data System (ADS)

    Marshall, W.

    As the first species aware of extinction and capable of proactively ensuring our long-term survival and development, it is striking that we do not do so with the rigor, formality, and foresight it requires. Only from a reactive posture have we responded to the challenges of global warfare, human rights, environmental concerns, and sustainable development. Despite our awareness of the possibility for extinction and apocalyptic set-backs to our evolution, and despite the existence of long-range studies-which must still be dramatically increased-proactive global policy implementation regarding our long-term survival and development is arguably non-existent. This lack of long-term policy making can be attributed in part to the lack of formal political mechanisms to facilitate longer-range policy making that extends 30 years or more into the future. Political mechanisms for infusing long-range thinking, research, and strategic planning into the policy-making process can help correct this shortcoming and provide the motivation needed to adequately address long-term challenges with the political rigor required to effectively establish and implement long-term policies. There are some efforts that attempt to address longer-range issues, but those efforts often do not connect to the political process, do not extend 30 or more years into the future, are not well-funded, and are not sufficiently systemic. Political mechanisms for long-range survival and prosperity could correct these inadequacies by raising awareness, providing funding, and most importantly, leveraging political rigor to establish and enforce long-range strategic planning and policies. The feasibility of such mechanisms should first be rigorously studied and assessed in a feasibility study, which could then inform implementation. This paper will present the case for such a study and suggest some possible political mechanisms that should be investigated further in the proposed study. This work is being further

  3. Bacterial differentiation, development and disease: mechanisms for survival

    PubMed Central

    Justice, Sheryl S.; Harrison, Alistair; Becknell, Brian; Mason, Kevin M.

    2014-01-01

    Bacteria have the exquisite ability to maintain a precise diameter, cell length and shape. The dimensions of bacteria size and shape are a classical metric in the distinction of bacterial species. Much of what we know about the particular morphology of any given species is the result of investigations of planktonic cultures. As we explore deeper into the natural habitats of bacteria, it is increasingly clear that bacteria can alter their morphology in response to the environment in which they reside. Specific morphologies are also becoming recognized as advantageous for survival in hostile environments. This is of particular importance in the context of both colonization and infection in the host. There are multiple examples of bacterial pathogens that use morphological changes as a mechanism for evasion of host immune responses and continued persistence. This review will focus on two systems where specific morphological changes are essential for persistence in animal models of human disease. We will also offer insight into the mechanism underlying the morphological changes and how these morphotypes aid in persistence. Additional examples of morphological changes associated with survival will be presented. PMID:25228010

  4. Synergistic intracellular iron chelation combinations: mechanisms and conditions for optimizing iron mobilization.

    PubMed

    Vlachodimitropoulou Koumoutsea, Evangelia; Garbowski, Maciej; Porter, John

    2015-09-01

    Iron chelators are increasingly combined clinically but the optimal conditions for cellular iron mobilization and mechanisms of interaction are unclear. Speciation plots for iron(III) binding of paired combinations of the licensed iron chelators desferrioxamine (DFO), deferiprone (DFP) and deferasirox (DFX) suggest conditions under which chelators can combine as 'shuttle' and 'sink' molecules but this approach does not consider their relative access and interaction with cellular iron pools. To address this issue, a sensitive ferrozine-based detection system for intracellular iron removal from the human hepatocyte cell line (HuH-7) was developed. Antagonism, synergism or additivity with paired chelator combinations was distinguished using mathematical isobologram analysis over clinically relevant chelator concentrations. All combinations showed synergistic iron mobilization at 8 h with clinically achievable concentrations of sink and shuttle chelators. Greatest synergism was achieved by combining DFP with DFX, where about 60% of mobilized iron was attributable to synergistic interaction. These findings predict that the DFX dose required for a half-maximum effect can be reduced by 3·8-fold when only 1 μmol/l DFP is added. Mechanisms for the synergy are suggested by consideration of the iron-chelate speciation plots together with the size, charge and lipid solubilities for each chelator. Hydroxypyridinones with low lipid solubilities but otherwise similar properties to DFP were used to interrogate the mechanistic interactions of chelator pairs. These studies confirm that synergistic cellular iron mobilization requires one chelator to have the physicochemical properties to enter cells, chelate intracellular iron and subsequently donate iron to a second 'sink' chelator. PMID:26033030

  5. Identification of a type IV secretion substrate of Brucella abortus that participates in the early stages of intracellular survival.

    PubMed

    Döhmer, Peter H; Valguarnera, Ezequiel; Czibener, Cecilia; Ugalde, Juan E

    2014-03-01

    Brucella abortus, the aetiological agent of bovine brucellosis, is an intracellular pathogen whose virulence is completely dependent on a type IV secretion system. This secretion system translocates effector proteins into the host cell to modulate the intracellular fate of the bacterium in order to establish a secure niche were it actively replicates. Although much has been done in understanding how this secretion system participates in the virulence process, few effector proteins have been identified to date. We describe here the identification of a type IV secretion substrate (SepA) that is only present in Brucella spp. and has no detectable homology to known proteins. This protein is secreted in a virB-dependent manner in a two-step process involving a periplasmic intermediate and secretion is necessary for its function. The deletion mutant showed a defect in the early stages of intracellular replication in professional and non-professional phagocytes although it invades the cells more efficiently than the wild-type parental strain. Our results indicate that, even though the mutant was more invasive, it had a defect in excluding the lysosomal marker Lamp-1 and was inactivated more efficiently during the early phases of the intracellular life cycle. PMID:24119283

  6. Genome-Wide Transposon Mutagenesis Indicates that Mycobacterium marinum Customizes Its Virulence Mechanisms for Survival and Replication in Different Hosts

    PubMed Central

    Weerdenburg, Eveline M.; Abdallah, Abdallah M.; Rangkuti, Farania; Abd El Ghany, Moataz; Otto, Thomas D.; Adroub, Sabir A.; Molenaar, Douwe; Ummels, Roy; ter Veen, Kars; van Stempvoort, Gunny; van der Sar, Astrid M.; Ali, Shahjahan; Langridge, Gemma C.; Thomson, Nicholas R.

    2015-01-01

    The interaction of environmental bacteria with unicellular eukaryotes is generally considered a major driving force for the evolution of intracellular pathogens, allowing them to survive and replicate in phagocytic cells of vertebrate hosts. To test this hypothesis on a genome-wide level, we determined for the intracellular pathogen Mycobacterium marinum whether it uses conserved strategies to exploit host cells from both protozoan and vertebrate origin. Using transposon-directed insertion site sequencing (TraDIS), we determined differences in genetic requirements for survival and replication in phagocytic cells of organisms from different kingdoms. In line with the general hypothesis, we identified a number of general virulence mechanisms, including the type VII protein secretion system ESX-1, biosynthesis of polyketide lipids, and utilization of sterols. However, we were also able to show that M. marinum contains an even larger set of host-specific virulence determinants, including proteins involved in the modification of surface glycolipids and, surprisingly, the auxiliary proteins of the ESX-1 system. Several of these factors were in fact counterproductive in other hosts. Therefore, M. marinum contains different sets of virulence factors that are tailored for specific hosts. Our data imply that although amoebae could function as a training ground for intracellular pathogens, they do not fully prepare pathogens for crossing species barriers. PMID:25690095

  7. Adhesion, invasion, intracellular survival and cytotoxic activity of strains of Aeromonas spp. in HEp-2, Caco-2 and T-84 cell lines.

    PubMed

    dos Santos, Paula Azevedo; Pereira, Ana Claudia Machado; Ferreira, Andréa Fonseca; de Mattos Alves, Maria Angélica; Rosa, Ana Cláudia Paula; Freitas-Almeida, Angela Corrêa

    2015-05-01

    The genus Aeromonas contains important pathogen for both humans and other animals, being responsible for the etiology of intestinal and extraintestinal diseases. The pathology caused by these bacteria involves several virulence factors, such as the ability to produce toxins, adhesion and invasion. The properties conferred by these factors have been extensively studied in experiments of interaction between bacterial strains and cell culture. We evaluate the interaction of eight Aeromonas spp. strains, previously isolated from human faeces, food and water with HEp-2, Caco-2 and T-84 cell lines. Cytotoxic effects, the pattern of adhesion, invasive capacity and intracellular survival were analyzed. The results showed that Aeromonas strains were adherent to three cells lines in 6 h of incubation, displaying the aggregative adherence pattern. Among eight strains studied, 50% produced cytotoxic effects on HEp-2 cells, while none of the strains produced cytotoxic effects on Caco-2 and T-84 cells at 48 h. This study demonstrated that subsets of Aeromonas isolated from different sources were able to invade intestinal (T-84, Caco-2) and epithelial (HEp-2) cell lines cultivated in vitro surviving in intracellular environments up to 72 h. Finally, our results support the pathogenic potential of Aeromonas, especially those of food and clinical sources. PMID:25743539

  8. Existence of two groups of Staphylococcus aureus strains isolated from bovine mastitis based on biofilm formation, intracellular survival, capsular profile and agr-typing.

    PubMed

    Bardiau, Marjorie; Caplin, Jonathan; Detilleux, Johann; Graber, Hans; Moroni, Paolo; Taminiau, Bernard; Mainil, Jacques G

    2016-03-15

    Staphylococcus (S.) aureus is recognised worldwide as an important pathogen causing contagious acute and chronic bovine mastitis. Chronic mastitis account for a significant part of all bovine cases and represent an important economic problem for dairy producers. Several properties (biofilm formation, intracellular survival, capsular expression and group agr) are thought to be associated with this chronic status. In a previous study, we found the existence of two groups of strains based on the association of these features. The aim of the present work was to confirm on a large international and non-related collection of strains the existence of these clusters and to associate them with case history records. In addition, the genomes of eight strains were sequenced to study the genomic differences between strains of each cluster. The results confirmed the existence of both groups based on capsular typing, intracellular survival and agr-typing: strains cap8-positive, belonging to agr group II, showing a low invasion rate and strains cap5-positive, belonging to agr group I, showing a high invasion rate. None of the two clusters were associated with the chronic status of the cow. When comparing the genomes of strains belonging to both clusters, the genes specific to the group "cap5-agrI" would suggest that these strains are better adapted to live in hostile environment. The existence of these two groups is highly important as they may represent two clusters that are adapted differently to the host and/or the surrounding environment. PMID:26931384

  9. All sex steroids are made intracellularly in peripheral tissues by the mechanisms of intracrinology after menopause.

    PubMed

    Labrie, Fernand

    2015-01-01

    Following the arrest of estradiol secretion by the ovaries at menopause, all estrogens and all androgens in postmenopausal women are made locally in peripheral target tissues according to the physiological mechanisms of intracrinology. The locally made sex steroids exert their action and are inactivated intracellularly without biologically significant release of the active sex steroids in the circulation. The level of expression of the steroid-forming and steroid-inactivating enzymes is specific to each cell type in each tissue, thus permitting to each cell/tissue to synthesize a small amount of androgens and/or estrogens in order to meet the local physiological needs without affecting the other tissues of the organism. Achieved after 500 million years of evolution, combination of the arrest of ovarian estrogen secretion, the availability of high circulating levels of DHEA and the expression of the peripheral sex steroid-forming enzymes have permitted the appearance of menopause with a continuing access to intratissular sex steroids for the individual cells/tissues without systemic exposure to circulating estradiol. In fact, one essential condition of menopause is to maintain serum estradiol at biologically inactive (substhreshold) concentrations, thus avoiding stimulation of the endometrium and risk of endometrial cancer. Measurement of the low levels of serum estrogens and androgens in postmenopausal women absolutely requires the use of MS/MS-based technology in order to obtain reliable accurate, specific and precise assays. While the activity of the series of steroidogenic enzymes can vary, the serum levels of DHEA show large individual variations going from barely detectable to practically normal "premenopausal" values, thus explaining the absence of menopausal symptoms in about 25% of women. It should be added that the intracrine system has no feedback elements to adjust the serum levels of DHEA, thus meaning that women with low DHEA activity will not be

  10. Role of the 85-Kilobase Plasmid and Plasmid-Encoded Virulence-Associated Protein A in Intracellular Survival and Virulence of Rhodococcus equi

    PubMed Central

    Giguère, Steeve; Hondalus, Mary K.; Yager, Julie A.; Darrah, Patricia; Mosser, David M.; Prescott, John F.

    1999-01-01

    Rhodococcus equi is a facultative intracellular pathogen of macrophages and a cause of pneumonia in young horses (foals) and immunocompromised people. Isolates of R. equi from pneumonic foals typically contain large, 85- or 90-kb plasmids encoding a highly immunogenic virulence-associated protein (VapA). The objective of this study was to determine the role of the 85-kb plasmid and VapA in the intracellular survival and virulence of R. equi. Clinical isolates containing the plasmid and expressing VapA efficiently replicated within mouse macrophages in vitro, while plasmid-cured derivatives of these organisms did not multiply intracellularly. An isolate harboring the large plasmid also replicated in the tissues of experimentally infected mice, whereas its plasmid-cured derivative was rapidly cleared. All foals experimentally infected with a plasmid-containing clinical isolate developed severe bronchopneumonia, whereas the foals infected with its plasmid-cured derivative remained asymptomatic and free of visible lung lesions. By day 14 postinfection, lung bacterial burdens had increased considerably in foals challenged with the plasmid-containing clinical isolate. In contrast, bacteria could no longer be cultured from the lungs of foals challenged with the isogenic plasmid-cured derivative. A recombinant, plasmid-cured derivative expressing wild-type levels of VapA failed to replicate in macrophages and remained avirulent for both mice and foals. These results show that the 85-kb plasmid of R. equi is essential for intracellular replication within macrophages and for development of disease in the native host, the foal. However, expression of VapA alone is not sufficient to restore the virulence phenotype. PMID:10377138

  11. In situ intracellular calcium oscillations in osteocytes in intact mouse long bones under dynamic mechanical loading

    PubMed Central

    Jing, Da; Baik, Andrew D.; Lu, X. Lucas; Zhou, Bin; Lai, Xiaohan; Wang, Liyun; Luo, Erping; Guo, X. Edward

    2014-01-01

    Osteocytes have been hypothesized to be the major mechanosensors in bone. How in situ osteocytes respond to mechanical stimuli is still unclear because of technical difficulties. In vitro studies have shown that osteocytes exhibited unique calcium (Ca2+) oscillations to fluid shear. However, whether this mechanotransduction phenomenon holds for in situ osteocytes embedded within a mineralized bone matrix under dynamic loading remains unknown. Using a novel synchronized loading/imaging technique, we successfully visualized in real time and quantified Ca2+ responses in osteocytes and bone surface cells in situ under controlled dynamic loading on intact mouse tibia. The resultant fluid-induced shear stress on the osteocyte in the lacunocanalicular system (LCS) was also quantified. Osteocytes, but not surface cells, displayed repetitive Ca2+ spikes in response to dynamic loading, with spike frequency and magnitude dependent on load magnitude, tissue strain, and shear stress in the LCS. The Ca2+ oscillations were significantly reduced by endoplasmic reticulum (ER) depletion and P2 purinergic receptor (P2R)/phospholipase C (PLC) inhibition. This study provides direct evidence that osteocytes respond to in situ mechanical loading by Ca2+ oscillations, which are dependent on the P2R/PLC/inositol trisphosphate/ER pathway. This study develops a novel approach in skeletal mechanobiology and also advances our fundamental knowledge of bone mechanotransduction.—Jing, D., Baik, A. D., Lu, X. L., Zhou, B., Lai, X., Wang, L., Luo, E., Guo, X. E. In situ intracellular calcium oscillations in osteocytes in intact mouse long bones under dynamic mechanical loading. PMID:24347610

  12. Use of Agent-Based Modeling To Explore the Mechanisms of Intracellular Phosphorus Heterogeneity in Cultured Phytoplankton

    PubMed Central

    Fredrick, Neil D.; Berges, John A.; Twining, Benjamin S.; Nuñez-Milland, Daliangelis

    2013-01-01

    There can be significant intraspecific individual-level heterogeneity in the intracellular P of phytoplankton, which can affect the population-level growth rate. Several mechanisms can create this heterogeneity, including phenotypic variability in various physiological functions (e.g., nutrient uptake rate). Here, we use modeling to explore the contribution of various mechanisms to the heterogeneity in phytoplankton grown in a laboratory culture. An agent-based model simulates individual cells and their intracellular P. Heterogeneity is introduced by randomizing parameters (e.g., maximum uptake rate) of daughter cells at division. The model was calibrated to observations of the P quota of individual cells of the centric diatom Thalassiosira pseudonana, which were obtained using synchrotron X-ray fluorescence (SXRF). A number of simulations, with individual mechanisms of heterogeneity turned off, then were performed. Comparison of the coefficient of variation (CV) of these and the baseline simulation (i.e., all mechanisms turned on) provides an estimate of the relative contribution of these mechanisms. The results show that the mechanism with the largest contribution to variability is the parameter characterizing the maximum intracellular P, which, when removed, results in a CV of 0.21 compared to a CV of 0.37 with all mechanisms turned on. This suggests that nutrient/element storage capabilities/mechanisms are important determinants of intrapopulation heterogeneity. PMID:23666327

  13. Mechanisms of high-order photobleaching and its relationship to intracellular ablation

    PubMed Central

    Kalies, S.; Kuetemeyer, K.; Heisterkamp, A.

    2011-01-01

    In two-photon laser-scanning microscopy using femtosecond laser pulses, the dependence of the photobleaching rate on excitation power may have a quadratic, cubic or even biquadratic order. To date, there are still many open questions concerning this so-called high-order photobleaching. We studied the photobleaching kinetics of an intrinsic (enhanced Green Fluorescent Protein (eGFP)) and an extrinsic (Hoechst 33342) fluorophore in a cellular environment in two-photon microscopy. Furthermore, we examined the correlation between bleaching and the formation of reactive oxygen species. We observed bleaching-orders of three and four for eGFP and two and three for Hoechst increasing step-wise at a certain wavelength. An increase of reactive oxygen species correlating with the bleaching over time was recognized. Comparing our results to the mechanisms involved in intracellular ablation with respect to the amount of interacting photons and involved energetic states, we found that a low-density plasma is formed in both cases with a smooth transition in between. Photobleaching, however, is mediated by sequential-absorption and multiphoton-ionization, while ablation is dominated by the latter and cascade-ionization processes. PMID:21483605

  14. An Invertron-Like Linear Plasmid Mediates Intracellular Survival and Virulence in Bovine Isolates of Rhodococcus equi

    PubMed Central

    Valero-Rello, Ana; Hapeshi, Alexia; Anastasi, Elisa; Alvarez, Sonsiray; Scortti, Mariela; Meijer, Wim G.; MacArthur, Iain

    2015-01-01

    We report a novel host-associated virulence plasmid in Rhodococcus equi, pVAPN, carried by bovine isolates of this facultative intracellular pathogenic actinomycete. Surprisingly, pVAPN is a 120-kb invertron-like linear replicon unrelated to the circular virulence plasmids associated with equine (pVAPA) and porcine (pVAPB variant) R. equi isolates. pVAPN is similar to the linear plasmid pNSL1 from Rhodococcus sp. NS1 and harbors six new vap multigene family members (vapN to vapS) in a vap pathogenicity locus presumably acquired via en bloc mobilization from a direct predecessor of equine pVAPA. Loss of pVAPN rendered R. equi avirulent in macrophages and mice. Mating experiments using an in vivo transconjugant selection strategy demonstrated that pVAPN transfer is sufficient to confer virulence to a plasmid-cured R. equi recipient. Phylogenetic analyses assigned the vap multigene family complement from pVAPN, pVAPA, and pVAPB to seven monophyletic clades, each containing plasmid type-specific allelic variants of a precursor vap gene carried by the nearest vap island ancestor. Deletion of vapN, the predicted “bovine-type” allelic counterpart of vapA, essential for virulence in pVAPA, abrogated pVAPN-mediated intramacrophage proliferation and virulence in mice. Our findings support a model in which R. equi virulence is conferred by host-adapted plasmids. Their central role is mediating intracellular proliferation in macrophages, promoted by a key vap determinant present in the common ancestor of the plasmid-specific vap islands, with host tropism as a secondary trait selected during coevolution with specific animal species. PMID:25895973

  15. Mechanisms of intracellular calcium homeostasis in developing and mature bovine corpora lutea.

    PubMed

    Wright, Marietta F; Bowdridge, Elizabeth; McDermott, Erica L; Richardson, Samuel; Scheidler, James; Syed, Qaisar; Bush, Taylor; Inskeep, E Keith; Flores, Jorge A

    2014-03-01

    Although calcium (Ca(2+)) is accepted as an intracellular mediator of prostaglandin F2 alpha (PGF2alpha) actions on luteal cells, studies defining mechanisms of Ca(2+) homeostasis in bovine corpora lutea (CL) are lacking. The increase in intracellular Ca(2+) concentration ([Ca(2+)]i) induced by PGF2alpha in steroidogenic cells from mature CL is greater than in those isolated from developing CL. Our hypothesis is that differences in signal transduction associated with developing and mature CL contribute to the increased efficacy of PGF2alpha to induce a Ca(2+) signal capable of inducing regression in mature CL. To test this hypothesis, major genes participating in Ca(2+) homeostasis in the bovine CL were identified, and expression of mRNA, protein, or activity, in the case of phospholipase Cbeta (PLCbeta), in developing and mature bovine CL was compared. In addition, we examined the contribution of external and internal Ca(2+) to the PGF2alpha stimulated rise in [Ca(2+)]i in LLCs isolated from developing and mature bovine CL. Three differences were identified in mechanisms of calcium homeostasis between developing and mature CL, which could account for the lesser increase in [Ca(2+)]i in response to PGF2alpha in developing than in mature CL. First, there were lower concentrations of inositol 1,4,5-trisphosphate (IP3) after similar PGF2alpha challenge, indicating reduced phospholipase C beta (PLCbeta) activity, in developing than mature CL. Second, there was an increased expression of sorcin (SRI) in developing than in mature CL. This cytoplasmic Ca(2+) binding protein modulates the endoplasmic reticulum (ER) Ca(2+) release channel, ryanodine receptor (RyR), to be in the closed configuration. Third, there was greater expression of ATP2A2 or SERCA, which causes calcium reuptake into the ER, in developing than in mature CL. Developmental differences in expression detected in whole CL were confirmed by Western blots using protein samples from steroidogenic cells

  16. Intracellular Trafficking in Drosophila Visual System Development: A Basis for Pattern Formation Through Simple Mechanisms

    PubMed Central

    Chan, Chih-Chiang; Epstein, Daniel; Hiesinger, P. Robin

    2012-01-01

    Intracellular trafficking underlies cellular functions ranging from membrane remodeling to receptor activation. During multicellular organ development, these basic cell biological functions are required as both passive machinery and active signaling regulators. Exocytosis, endocytosis, and recycling of several key signaling receptors have long been known to actively regulate morphogenesis and pattern formation during Drosophila eye development. Hence, intracellular membrane trafficking not only sets the cell biological stage for receptor-mediated signaling but also actively controls signaling through spatiotemporally regulated receptor localization. In contrast to eye development, the role of intracellular trafficking for the establishment of the eye-to-brain connectivity map has only recently received more attention. It is still poorly understood how guidance receptors are spatiotemporally regulated to serve as meaningful synapse formation signals. Yet, the Drosophila visual system provides some of the most striking examples for the regulatory role of intracellular trafficking during multicellular organ development. In this review we will first highlight the experimental and conceptual advances that motivate the study of intracellular trafficking during Drosophila visual system development. We will then illuminate the development of the eye, the eye-to-brain connectivity map and the optic lobe from the perspective of cell biological dynamics. Finally, we provide a conceptual framework that seeks to explain how the interplay of simple genetically encoded intracellular trafficking events governs the seemingly complex cellular behaviors, which in turn determine the developmental product. PMID:21714102

  17. Intracellular mechanisms modulating gamma band activity in the pedunculopontine nucleus (PPN).

    PubMed

    Luster, Brennon R; Urbano, Francisco J; Garcia-Rill, Edgar

    2016-06-01

    The pedunculopontine nucleus is a part of the reticular activating system, and is active during waking and REM sleep. Previous results showed that all PPN cells tested fired maximally at gamma frequencies when depolarized. This intrinsic membrane property was shown to be mediated by high-threshold N- and P/Q-type Ca(2+) channels. Recent studies show that the PPN contains three independent populations of neurons which can generate gamma band oscillations through only N-type channels, only P/Q-type channels, or both N- and P/Q-type channels. This study investigated the intracellular mechanisms modulating gamma band activity in each population of neurons. We performed in vitro patch-clamp recordings of PPN neurons from Sprague-Dawley rat pups, and applied 1-sec ramps to induce intrinsic membrane oscillations. Our results show that there are two pathways modulating gamma band activity in PPN neurons. We describe populations of neurons mediating gamma band activity through only N-type channels and the cAMP/PKA pathway (presumed "REM-on" neurons), through only P/Q-type channels and the CaMKII pathway (presumed "Wake-on" neurons), and a third population which can mediate gamma activity through both N-type channels and cAMP/PK and P/Q-type channels and CaMKII (presumed "Wake/REM-on" neurons). These novel results suggest that PPN gamma oscillations are modulated by two independent pathways related to different Ca(2+) channel types. PMID:27354537

  18. Functional Genetic Diversity among Mycobacterium tuberculosis Complex Clinical Isolates: Delineation of Conserved Core and Lineage-Specific Transcriptomes during Intracellular Survival

    PubMed Central

    Homolka, Susanne; Niemann, Stefan; Russell, David G.; Rohde, Kyle H.

    2010-01-01

    Tuberculosis exerts a tremendous burden on global health, with ∼9 million new infections and ∼2 million deaths annually. The Mycobacterium tuberculosis complex (MTC) was initially regarded as a highly homogeneous population; however, recent data suggest the causative agents of tuberculosis are more genetically and functionally diverse than appreciated previously. The impact of this natural variation on the virulence and clinical manifestations of the pathogen remains largely unknown. This report examines the effect of genetic diversity among MTC clinical isolates on global gene expression and survival within macrophages. We discovered lineage-specific transcription patterns in vitro and distinct intracellular growth profiles associated with specific responses to host-derived environmental cues. Strain comparisons also facilitated delineation of a core intracellular transcriptome, including genes with highly conserved regulation across the global panel of clinical isolates. This study affords new insights into the genetic information that M. tuberculosis has conserved under selective pressure during its long-term interactions with its human host. PMID:20628579

  19. Three antagonistic cyclic di-GMP-catabolizing enzymes promote differential Dot/Icm effector delivery and intracellular survival at the early steps of Legionella pneumophila infection.

    PubMed

    Allombert, Julie; Lazzaroni, Jean-Claude; Baïlo, Nathalie; Gilbert, Christophe; Charpentier, Xavier; Doublet, Patricia; Vianney, Anne

    2014-03-01

    Legionella pneumophila is an intracellular pathogen which replicates within protozoan cells and can accidently infect alveolar macrophages, causing an acute pneumonia in humans. The second messenger cyclic di-GMP (c-di-GMP) has been shown to play key roles in the regulation of various bacterial processes, including virulence. While investigating the function of the 22 potential c-di-GMP-metabolizing enzymes of the L. pneumophila Lens strain, we found three that directly contribute to its ability to infect both protozoan and mammalian cells. These three enzymes display diguanylate cyclase (Lpl0780), phosphodiesterase (Lpl1118), and bifunctional diguanylate cyclase/phosphodiesterase (Lpl0922) activities, which are all required for the survival and intracellular replication of L. pneumophila. Mutants with deletions of the corresponding genes are efficiently taken up by phagocytic cells but are partially defective for the escape of the Legionella-containing vacuole (LCV) from the host degradative endocytic pathway and result in lower survival. In addition, Lpl1118 is required for efficient endoplasmic reticulum recruitment to the LCV. Trafficking and biogenesis of the LCV are dependent upon the orchestrated actions of several type 4 secretion system Dot/Icm effectors proteins, which exhibit differentially altered translocation in the three mutants. While translocation of some effectors remained unchanged, others appeared over- and undertranslocated. A general translocation offset of the large repertoire of Dot/Icm effectors may be responsible for the observed defects in the trafficking and biogenesis of the LCV. Our results suggest that L. pneumophila uses cyclic di-GMP signaling to fine-tune effector delivery and ensure effective evasion of the host degradative pathways and establishment of a replicative vacuole. PMID:24379287

  20. A eukaryotic-type signalling system of Pseudomonas aeruginosa contributes to oxidative stress resistance, intracellular survival and virulence

    PubMed Central

    2011-01-01

    Background The genome of Pseudomonas aeruginosa contains at least three genes encoding eukaryotic-type Ser/Thr protein kinases, one of which, ppkA, has been implicated in P. aeruginosa virulence. Together with the adjacent pppA phosphatase gene, they belong to the type VI secretion system (H1-T6SS) locus, which is important for bacterial pathogenesis. To determine the biological function of this protein pair, we prepared a pppA-ppkA double mutant and characterised its phenotype and transcriptomic profiles. Results Phenotypic studies revealed that the mutant grew slower than the wild-type strain in minimal media and exhibited reduced secretion of pyoverdine. In addition, the mutant had altered sensitivity to oxidative and hyperosmotic stress conditions. Consequently, mutant cells had an impaired ability to survive in murine macrophages and an attenuated virulence in the plant model of infection. Whole-genome transcriptome analysis revealed that pppA-ppkA deletion affects the expression of oxidative stress-responsive genes, stationary phase σ-factor RpoS-regulated genes, and quorum-sensing regulons. The transcriptome of the pppA-ppkA mutant was also analysed under conditions of oxidative stress and showed an impaired response to the stress, manifested by a weaker induction of stress adaptation genes as well as the genes of the SOS regulon. In addition, expression of either RpoS-regulated genes or quorum-sensing-dependent genes was also affected. Complementation analysis confirmed that the transcription levels of the differentially expressed genes were specifically restored when the pppA and ppkA genes were expressed ectopically. Conclusions Our results suggest that in addition to its crucial role in controlling the activity of P. aeruginosa H1-T6SS at the post-translational level, the PppA-PpkA pair also affects the transcription of stress-responsive genes. Based on these data, it is likely that the reduced virulence of the mutant strain results from an impaired

  1. Mechanisms of low Na+-induced increase in intracellular calcium in KCl-depolarized rat cardiomyocytes.

    PubMed

    Rathi, Satyajeet S; Saini, Harjot K; Xu, Yan-Jun; Dhalla, Naranjan S

    2004-08-01

    Although low Na+ is known to increase the intracellular Ca2+ concentration ([Ca2+]i) in cardiac muscle, the exact mechanisms of low Na+ -induced increases in [Ca2+]i are not completely defined. To gain information in this regard, we examined the effects of low Na+ (35 mM) on freshly isolated cardiomyocytes from rat heart in the absence and presence of different interventions. The [Ca2+]i in cardiomyocytes was measured fluorometrically with Fura-2 AM. Following a 10 min incubation, the low Na+ -induced increase in [Ca2+], was only observed in cardiomyocytes depolarized with 30 mM KCl, but not in quiescent cardiomyocytes. In contrast, low Na+ did not alter the ATP-induced increase in [Ca2+]i in the cardiomyocytes. This increase in [Ca2+]i due to low Na+ and elevated KCl was dependent on the extracellular concentration of Ca2+ (0.25-2.0 mM). The L-type Ca2+ -channel blockers, verapamil and diltiazem, at low concentrations (1 microM) depressed the low Na+, KCl-induced increase in [Ca2+]i without significantly affecting the response to low Na+ alone. The low Na+, high KCl-induced increase in [Ca2+]i was attenuated by treatments of cardiomyocytes with high concentrations of both verapamil (5 and 10 microM), and diltiazem (5 and 10 microM) as well as with amiloride (5-20 microM), nickel (1.25-5.0 mM), cyclopiazonic acid (25 and 50 microM) and thapsigargin (10 and 20 microM). On the other hand, this response was augmented by ouabain (1 and 2 mM) and unaltered by 5-(N-methyl-N-isobutyl) amiloride (5 and 10 microM). These data suggest that in addition to the sarcolemmal Na+ - Ca2+ exchanger, both sarcolemmal Na+ - K+ ATPase, as well as the sarcoplasmic reticulum Ca2+ -pump play prominent roles in the low Na+ -induced increase in [Ca2+]i. PMID:15524176

  2. Mechanisms of renal repair and survival following acute injury.

    PubMed

    Safirstein, R; DiMari, J; Megyesi, J; Price, P

    1998-09-01

    The reaction of the renal epithelium to injury is heterogenous. Some cells die, others survive apparently intact, while others commit to repair. The determinants of these responses appear to depend on signal transduction pathways and molecular responses that is segment specific and interactive. The kidney, as do cells in culture exposed to various noxious stimuli, react in a typical manner referred to as the stress response. The response is comprised of kinases and their molecular targets as well as cell cycle-specific factors that determine whether a cell survives the injury or not. We propose that this response can be modified by survival factors which upregulate those aspects of the response that are cytoprotective and which downregulate those that are cytoreductive. Preliminary data will be presented to demonstrate the feasibility of this approach. PMID:9754604

  3. Bud dormancy in perennial plants: a mechanism for survival. In:Dormancy of Cells and Organisms

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Many plants evolved the ability to reproduce asexually through vegetative buds as a survival mechanism. Identifying the genetic and physiological mechanisms regulating dormancy in these reproductive structures will allow manipulation of plant growth and development in both desirable and undesirable ...

  4. Depletion of autophagy-related genes ATG3 and ATG5 in Tenebrio molitor leads to decreased survivability against an intracellular pathogen, Listeria monocytogenes.

    PubMed

    Tindwa, Hamisi; Jo, Yong Hun; Patnaik, Bharat Bhusan; Noh, Mi Young; Kim, Dong Hyun; Kim, Iksoo; Han, Yeon Soo; Lee, Yong Seok; Lee, Bok Luel; Kim, Nam Jung

    2015-01-01

    Macroautophagy (autophagy) is an evolutionarily conserved catabolic process involved in physiological and developmental processes including cell survival, death, and innate immunity. Homologues of most of 36 originally discovered autophagy-related (ATG) genes in yeast have been characterized in higher eukaryotes including insects. In this study, the homologues of ATG3 (TmATG3) and ATG5 (TmATG5) were isolated from the coleopteran beetle, Tenebrio molitor by expressed sequence tag and RNAseq approaches. The cDNA of TmATG3 and TmATG5 comprise open-reading frame sizes of 963 and 792 bp encoding polypeptides of 320 and 263 amino acid residues, respectively. TmATG3 and TmATG5 mRNA are expressed in all developmental stages, and mainly in fat body and hemocytes of larvae. TmATG3 and TmATG5 showed an overall sequence identity of 58-95% to other insect Atg proteins. There exist clear one-to-one orthologs of TmATG3 and TmATG5 in Tribolium and that they clustered together in the gene tree. Depletion of TmATG3 and TmATG5 by RNA interference led to a significant reduction in survival ability of T. molitor larvae against an intracellular pathogen, Listeria monocytogenes. Six days post-Listeria challenge, the survival rate in the dsEGFP-injected (where EGFP is enhanced green fluorescent protein) control larvae was significantly higher (55%) compared to 4 and 3% for TmATG3 and TmATG5 double-stranded RNA injected larvae, respectively. These data suggested that TmATG3 and TmATG5 may play putative role in mediating autophagy-based clearance of Listeria in T. molitor model. PMID:25403020

  5. Homeostatic Systems--Mechanisms for Survival. Science IV.

    ERIC Educational Resources Information Center

    Pfeiffer, Carl H.

    The two student notebooks in this set provide the basic outline and assignments for the fourth and last year of a senior high school unified science program which builds on the technical third year course, Science IIIA (see SE 012 149). An introductory section considers the problems of survival inherent in living systems, matter-energy…

  6. Prolonged Oxaliplatin Exposure Alters Intracellular Calcium Signaling: A New Mechanism To Explain Oxaliplatin-Associated Peripheral Neuropathy

    PubMed Central

    Schulze, Christin; McGowan, Margit; Jordt, Sven; Ehrlich, Barbara E

    2012-01-01

    Oxaliplatin is a platinum based cytotoxic agent commonly used to treat colorectal cancers. Despite its effectiveness, oxaliplatin administration is associated with the development of cold-induced peripheral neuropathy. This potentially permanent side effect is provoked by cold exposure and can range from mild and self limited to severe and debilitating. Even with tumor shrinkage, these painful side effects can force dose-reduction or discontinuation of treatment. Neither the mechanism of action of oxaliplatin nor that of cold-induced neuropathy is understood. Paclitaxel, an entirely different chemotherapeutic agent used to treat a variety of malignancies, also is associated with the development of peripheral neuropathy. Unlike oxaliplatin, neurotoxicity arising from paclitaxel treatment is better understood and was found to have profound effects on intracellular calcium signaling (1,2). In this study we examined the effects of oxaliplatin on calcium signaling pathways and found that acute exposure of either a neuroblastoma cell line or primary neurons with therapeutic concentrations of oxaliplatin had no effect on intracellular calcium signaling. We also found that cellular temperature sensors (TRP channels) were also not activated by oxaliplatin. Interestingly, prolonged exposure of oxaliplatin sensitized cells to subsequent stimuli and enhanced the magnitude of intracellular calcium responses. Taken together, our results suggest that acute oxaliplatin exposure will not induce abnormal calcium signaling but oxaliplatin-primed cells do exhibit enhanced sensitivity. These findings provide new insight to the mechanism behind oxaliplatin-induced neuropathy. PMID:21859566

  7. Specific Monoclonal Antibody Overcomes the Salmonella enterica Serovar Typhimurium’s Adaptive Mechanisms of Intramacrophage Survival and Replication

    PubMed Central

    Aribam, Swarmistha Devi; Harada, Tomoyuki; Elsheimer-Matulova, Marta; Iwata, Taketoshi; Kanehira, Katsushi; Hikono, Hirokazu; Matsui, Hidenori; Ogawa, Yohsuke; Shimoji, Yoshihiro; Eguchi, Masahiro

    2016-01-01

    Salmonella-specific antibodies play an important role in host immunity; however, the mechanisms of Salmonella clearance by pathogen-specific antibodies remain to be completely elucidated since previous studies on antibody-mediated protection have yielded inconsistent results. These inconsistencies are at least partially attributable to the use of polyclonal antibodies against Salmonella antigens. Here, we developed a new monoclonal antibody (mAb)-449 and identified its related immunogen that protected BALB/c mice from infection with Salmonella enterica serovar Typhimurium. In addition, these data indicate that the mAb-449 immunogen is likely a major protective antigen. Using in vitro infection studies, we also analyzed the mechanism by which mAb-449 conferred host protection. Notably, macrophages infected with mAb-449-treated S. Typhimurium showed enhanced pathogen uptake compared to counterparts infected with control IgG-treated bacteria. Moreover, these macrophages produced elevated levels of pro-inflammatory cytokine TNFα and nitric oxide, indicating that mAb-449 enhanced macrophage activation. Finally, the number of intracellular bacteria in mAb-449-activated macrophages decreased considerably, while the opposite was found in IgG-treated controls. Based on these findings, we suggest that, although S. Typhimurium has the potential to survive and replicate within macrophages, host production of a specific antibody can effectively mediate macrophage activation for clearance of intracellular bacteria. PMID:26986057

  8. Effect of isoorientin on intracellular antioxidant defence mechanisms in hepatoma and liver cell lines.

    PubMed

    Yuan, Li; Wang, Jing; Wu, Wanqiang; Liu, Qian; Liu, Xuebo

    2016-07-01

    Isoorientin (ISO) is considered one of the most important flavonoid-like compounds responsible for health benefits, including the prevention of liver damage as well as antioxidant, anti-inflammatory, and anti-nociceptive activities. Our previous study showed that ISO inhibited the proliferation of hepatoma cells through increasing intracellular ROS levels. Interestingly, ISO protects rat liver cells against hydrogen peroxide-induced oxidation stress by decreasing intracellular ROS levels. Why are there different effects of ISO on ROS in different physiological and pathophysiological circumstances? The present study investigated the effect of ISO on mitochondrial respiratory chain complexes and phase II detoxifying enzyme activities in human hepatoblastoma cancer cells (HepG2), buffalo rat liver cells (BRL-3A) and human liver cancer cells (HL-7702). The results showed that intracellular ROS levels and the protein expression of the respiratory chain complexes was significantly (p<0.01) higher in the HepG2 cells than in the BRL-3A and HL-7702 cells. Additionally, ISO notably (p<0.01) increased ROS levels in the HepG2 cells, while no significance was found in the BRL-3A and HL-7702 cells. Furthermore, in the HepG2 cells, the protein expression of the respiratory chain complexes and the phase II detoxifying enzyme activities and GSH content were decreased by ISO (p<0.01), while ISO, in a certain range, enhanced the expression of the protein complexes and the phase II detoxifying enzyme activities and GSH content in BRL-3A and HL-7702 cells. All of these results demonstrated, for the first time, that ISO possesses a notable hepatoprotective effect, which might be mediated through the respiratory chain complexes and phase II detoxifying enzyme activities. PMID:27261613

  9. Anaerobic phosphate release from activated sludge with enhanced biological phosphorus removal. A possible mechanism of intracellular pH control

    SciTech Connect

    Bond, P.L.; Keller, J.; Blackall, L.L.

    1999-06-05

    The biochemical mechanisms of the wastewater treatment process known as enhanced biological phosphorus removal (EBPR) are presently described in a metabolic model. The authors investigated details of the EBPR model to determine the nature of the anaerobic phosphate release and how this may be metabolically associated with polyhydroxyalkanoate (PHA) formation. Iodoacetate, an inhibitor of glycolysis, was found to inhibit the anaerobic formation of PHA and phosphate release, supporting the pathways proposed in the EBPR metabolic model. In the metabolic model, it is proposed that polyphosphate degradation provides energy for the microorganisms in anaerobic regions of these treatment systems. Other investigations have shown that anaerobic phosphate release depends on the extracellular pH. The authors observed that when the intracellular pH of EBPR sludge was raised, substantial anaerobic phosphate release was caused without volatile fatty acid (VFA) uptake. Acidification of the sludge inhibited anaerobic phosphate release even in the presence of VFA. from these observations, the authors postulate that an additional possible role of anaerobic polyphosphate degradation in EBPR is for intracellular pH control. Intracellular pH control may be a metabolic feature of EBPR, not previously considered, that could have some use in the control and optimization of EBPR.

  10. Intracellular Protein Shuttling: A Mechanism Relevant for Myelin Repair in Multiple Sclerosis?

    PubMed

    Göttle, Peter; Küry, Patrick

    2015-01-01

    A prominent feature of demyelinating diseases such as multiple sclerosis (MS) is the degeneration and loss of previously established functional myelin sheaths, which results in impaired signal propagation and axonal damage. However, at least in early disease stages, partial replacement of lost oligodendrocytes and thus remyelination occur as a result of resident oligodendroglial precursor cell (OPC) activation. These cells represent a widespread cell population within the adult central nervous system (CNS) that can differentiate into functional myelinating glial cells to restore axonal functions. Nevertheless, the spontaneous remyelination capacity in the adult CNS is inefficient because OPCs often fail to generate new oligodendrocytes due to the lack of stimulatory cues and the presence of inhibitory factors. Recent studies have provided evidence that regulated intracellular protein shuttling is functionally involved in oligodendroglial differentiation and remyelination activities. In this review we shed light on the role of the subcellular localization of differentiation-associated factors within oligodendroglial cells and show that regulation of intracellular localization of regulatory factors represents a crucial process to modulate oligodendroglial maturation and myelin repair in the CNS. PMID:26151843

  11. Fever temperature enhances mechanisms of survival of Streptococcus agalactiae within human endothelial cells.

    PubMed

    Freitas Lione, Viviane Oliveira; Bittencourt Dos Santos, Michelle Hanthequeste; Ulisses Carvalho, Técia Maria; Hirata, Raphael; Mattos-Guaraldi, Ana Luiza; Arruda Mortara, Renato; Nagao, Prescilla Emy

    2010-10-01

    Group B streptococci (GBS) are the most common cause of pneumonia and sepsis during the neonatal period. However, the pathogenesis of invasive infection is poorly understood. We investigated the ability of GBS grown at 37 degrees C and 40 degrees C to adhere and invade human umbilical vein endothelial cells (HUVECs) at different periods of incubation (0, 0.5, 1, 2, 18 and 24 h). All strains tested, except strain 88641-vagina survived for 24 h in the intracellular environment at 40 degrees C. For serotype III grown at 40 degrees C, both strains (80340-vagina and 90356-liquor) showed increased adherence and intracellular survival when compared to bacteria grown at 37 degrees C (P<0.01). GBS serotype V strains (88641-vagina and 90186-blood) showed ability to survive inside HUVECs until 2 and 24 h post-infection at 40 degrees C and 37 degrees C, respectively (P<0.01). Influence of growth temperature in bacterial interaction with endothelial cells was partially dependent of serotypes and the clinical origin of strains. Serotypes III and V strains grown at both temperatures remained viable within acidic endothelial vacuoles which acquired Rab7 and LAMP-1 endosomal markers. The data emphasize the influence of temperature on cellular events of phagocytosis and pathogenesis of GBS diseases. PMID:20818490

  12. Decrease of intracellular pH as possible mechanism of embryotoxicity of glycol ether alkoxyacetic acid metabolites

    SciTech Connect

    Louisse, Jochem; Verwei, Miriam; Sandt, Johannes J.M. van de; Rietjens, Ivonne M.C.M.

    2010-06-01

    Embryotoxicity of glycol ethers is caused by their alkoxyacetic acid metabolites, but the mechanism underlying the embryotoxicity of these acid metabolites is so far not known. The present study investigates a possible mechanism underlying the embryotoxicity of glycol ether alkoxyacetic acid metabolites using the methoxyacetic acid (MAA) metabolite of ethylene glycol monomethyl ether as the model compound. The results obtained demonstrate an MAA-induced decrease of the intracellular pH (pH{sub i}) of embryonic BALB/c-3T3 cells as well as of embryonic stem (ES)-D3 cells, at concentrations that affect ES-D3 cell differentiation. These results suggest a mechanism for MAA-mediated embryotoxicity similar to the mechanism of embryotoxicity of the drugs valproic acid and acetazolamide (ACZ), known to decrease the pH{sub i}in vivo, and therefore used as positive controls. The embryotoxic alkoxyacetic acid metabolites ethoxyacetic acid, butoxyacetic acid and phenoxyacetic acid also caused an intracellular acidification of BALB/c-3T3 cells at concentrations that are known to inhibit ES-D3 cell differentiation. Two other embryotoxic compounds, all-trans-retinoic acid and 5-fluorouracil, did not decrease the pH{sub i} of embryonic cells at concentrations that affect ES-D3 cell differentiation, pointing at a different mechanism of embryotoxicity of these compounds. MAA and ACZ induced a concentration-dependent inhibition of ES-D3 cell differentiation, which was enhanced by amiloride, an inhibitor of the Na{sup +}/H{sup +}-antiporter, corroborating an important role of the pH{sub i} in the embryotoxic mechanism of both compounds. Together, the results presented indicate that a decrease of the pH{sub i} may be the mechanism of embryotoxicity of the alkoxyacetic acid metabolites of the glycol ethers.

  13. Systems biology approaches to understanding mycobacterial survival mechanisms

    PubMed Central

    Boshoff, Helena I.M.; Lun, Desmond S.

    2010-01-01

    The advent of high-throughput platforms for the interrogation of biological systems at the cellular and molecular level have allowed living cells to be observed and understood at a hitherto unprecedented level of detail and have enabled the construction of comprehensive, predictive in silico models. Here, we review the application of such high-throughput, systems-biological techniques to mycobacteria—specifically to the pernicious human pathogen Mycobacterium tuberculosis (MTb) and its ability to survive in human hosts. We discuss the development and application of transcriptomic, proteomic, regulomic, and metabolomic techniques for MTb as well as the development and application of genome-scale in silico models. Thus far, systems-biological approaches have largely focused on in vitro models of MTb growth; reliably extending these approaches to in vivo conditions relevant to infection is a significant challenge for the future that holds the ultimate promise of novel chemotherapeutic interventions. PMID:21072257

  14. High-density lipoprotein, mitochondrial dysfunction and cell survival mechanisms.

    PubMed

    White, C Roger; Giordano, Samantha; Anantharamaiah, G M

    2016-09-01

    Ischemic injury is associated with acute myocardial infarction, percutaneous coronary intervention, coronary artery bypass grafting and open heart surgery. The timely re-establishment of blood flow is critical in order to minimize cardiac complications. Reperfusion after a prolonged ischemic period, however, can induce severe cardiomyocyte dysfunction with mitochondria serving as a major target of ischemia/reperfusion (I/R) injury. An increase in the formation of reactive oxygen species (ROS) induces damage to mitochondrial respiratory complexes leading to uncoupling of oxidative phosphorylation. Mitochondrial membrane perturbations also contribute to calcium overload, opening of the mitochondrial permeability transition pore (mPTP) and the release of apoptotic mediators into the cytoplasm. Clinical and experimental studies show that ischemic preconditioning (ICPRE) and postconditioning (ICPOST) attenuate mitochondrial injury and improve cardiac function in the context of I/R injury. This is achieved by the activation of two principal cell survival cascades: 1) the Reperfusion Injury Salvage Kinase (RISK) pathway; and 2) the Survivor Activating Factor Enhancement (SAFE) pathway. Recent data suggest that high density lipoprotein (HDL) mimics the effects of conditioning protocols and attenuates myocardial I/R injury via activation of the RISK and SAFE signaling cascades. In this review, we discuss the roles of apolipoproteinA-I (apoA-I), the major protein constituent of HDL, and sphingosine 1-phosphate (S1P), a lysosphingolipid associated with small, dense HDL particles as mediators of cardiomyocyte survival. Both apoA-I and S1P exert an infarct-sparing effect by preventing ROS-dependent injury and inhibiting the opening of the mPTP. PMID:27150975

  15. Impact of mechanical shear on Listeria monocytogenes survival on surfaces

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Microbial inactivation using high temperatures is well known process and has contributed significantly toward food safety and shelf life extension for the food industry. Mechanical high pressure (hydrostatic) treatment is also gaining interest in food processing applications for achieving microbial...

  16. Mechanism of the Association between Na+ Binding and Conformations at the Intracellular Gate in Neurotransmitter:Sodium Symporters*

    PubMed Central

    Stolzenberg, Sebastian; Quick, Matthias; Zhao, Chunfeng; Gotfryd, Kamil; Khelashvili, George; Gether, Ulrik; Loland, Claus J.; Javitch, Jonathan A.; Noskov, Sergei; Weinstein, Harel; Shi, Lei

    2015-01-01

    Neurotransmitter:sodium symporters (NSSs) terminate neurotransmission by Na+-dependent reuptake of released neurotransmitters. Previous studies suggested that Na+-binding reconfigures dynamically coupled structural elements in an allosteric interaction network (AIN) responsible for function-related conformational changes, but the intramolecular pathway of this mechanism has remained uncharted. We describe a new approach for the modeling and analysis of intramolecular dynamics in the bacterial NSS homolog LeuT. From microsecond-scale molecular dynamics simulations and cognate experimental verifications in both LeuT and human dopamine transporter (hDAT), we apply the novel method to identify the composition and the dynamic properties of their conserved AIN. In LeuT, two different perturbations disrupting Na+ binding and transport (i.e. replacing Na+ with Li+ or the Y268A mutation at the intracellular gate) affect the AIN in strikingly similar ways. In contrast, other mutations that affect the intracellular gate (i.e. R5A and D369A) do not significantly impair Na+ cooperativity and transport. Our analysis shows these perturbations to have much lesser effects on the AIN, underscoring the sensitivity of this novel method to the mechanistic nature of the perturbation. Notably, this set of observations holds as well for hDAT, where the aligned Y335A, R60A, and D436A mutations also produce different impacts on Na+ dependence. Thus, the detailed AIN generated from our method is shown to connect Na+ binding with global conformational changes that are critical for the transport mechanism. That the AIN between the Na+ binding sites and the intracellular gate in bacterial LeuT resembles that in eukaryotic hDAT highlights the conservation of allosteric pathways underlying NSS function. PMID:25869126

  17. Mechanisms of cross-talk between G-protein-coupled receptors resulting in enhanced release of intracellular Ca2+.

    PubMed Central

    Werry, Tim D; Wilkinson, Graeme F; Willars, Gary B

    2003-01-01

    Alteration in [Ca(2+)](i) (the intracellular concentration of Ca(2+)) is a key regulator of many cellular processes. To allow precise regulation of [Ca(2+)](i) and a diversity of signalling by this ion, cells possess many mechanisms by which they are able to control [Ca(2+)](i) both globally and at the subcellular level. Among these are many members of the superfamily of GPCRs (G-protein-coupled receptors), which are characterized by the presence of seven transmembrane domains. Typically, those receptors able to activate PLC (phospholipase C) enzymes cause release of Ca(2+) from intracellular stores and influence Ca(2+) entry across the plasma membrane. It has been well documented that Ca(2+) signalling by one type of GPCR can be influenced by stimulation of a different type of GPCR. Indeed, many studies have demonstrated heterologous desensitization between two different PLC-coupled GPCRs. This is not surprising, given our current understanding of negative-feedback regulation and the likely shared components of the signalling pathway. However, there are also many documented examples of interactions between GPCRs, often coupling preferentially to different signalling pathways, which result in a potentiation of Ca(2+) signalling. Such interactions have important implications for both the control of cell function and the interpretation of in vitro cell-based assays. However, there is currently no single mechanism that adequately accounts for all examples of this type of cross-talk. Indeed, many studies either have not addressed this issue or have been unable to determine the mechanism(s) involved. This review seeks to explore a range of possible mechanisms to convey their potential diversity and to provide a basis for further experimental investigation. PMID:12790797

  18. Dietary antiaging phytochemicals and mechanisms associated with prolonged survival

    PubMed Central

    Si, Hongwei; Liu, Dongmin

    2014-01-01

    Aging is well-known an inevitable process that is influenced by genetic, lifestyle and environmental factors. However, the exact mechanisms underlying the aging process are not well understood. Increasing evidence shows that aging is highly associated with chronic increase in reactive oxygen species (ROS), accumulation of a low-grade proinflammatory phenotype and reduction in age-related autophagy, suggesting that these factors may play important roles in promoting aging. Indeed, reduction of ROS and low-grade inflammation and promotion of autophagy by calorie restriction or other dietary manipulation can extend lifespan in a wide spectrum of model organisms. Interestingly, recent studies show that some food-derived small molecules, also called phytochemicals, can extend lifespan in various animal species. In this paper, we review several recently identified potential antiaging phytochemicals that have been studied in cells, animals and humans and further highlight the cellular and molecular mechanisms underlying the antiaging actions by these molecules. PMID:24742470

  19. Merlin, a “Magic” Linker Between the Extracellular Cues and Intracellular Signaling Pathways that Regulate Cell Motility, Proliferation, and Survival

    PubMed Central

    Stamenkovic, Ivan; Yu, Qin

    2010-01-01

    Genetic alterations of neurofibromatosis type 2 (NF2) gene lead to the development of schwannomas, meningiomas, and ependymomas. Mutations of NF2 gene were also found in thyroid cancer, mesothelioma, and melanoma, suggesting that it functions as a tumor suppressor in a wide spectrum of cells. The product of NF2 gene is merlin (moesinezrin-radixin-like protein), a member of the Band 4.1 superfamily proteins. Merlin shares significant sequence homology with the ERM (Ezrin-Radixin-Moesin) family proteins and serves as a linker between transmembrane proteins and the actin-cytoskeleton. Merlin is a multifunctional protein and involved in integrating and regulating the extracellular cues and intracellular signaling pathways that control cell fate, shape, proliferation, survival, and motility. Recent studies showed that merlin regulates the cell-cell and cell-matrix adhesions and functions of the cell surface adhesion/extracellular matrix receptors including CD44 and that merlin and CD44 antagonize each other's function and work upstream of the mammalian Hippo signaling pathway. Furthermore, merlin plays important roles in stabilizing the contact inhibition of proliferation and in regulating activities of several receptor tyrosine kinases. Accumulating data also suggested an emerging role of merlin as a negative regulator of growth and progression of several non-NF2 associated cancer types. Together, these recent advances have improved our basic understanding about merlin function, its regulation, and the major signaling pathways regulated by merlin and provided the foundation for future translation of these findings into the clinic for patients bearing the cancers in which merlin function and/or its downstream signaling pathways are impaired or altered. PMID:20491622

  20. TcI Isolates of Trypanosoma cruzi Exploit the Antioxidant Network for Enhanced Intracellular Survival in Macrophages and Virulence in Mice.

    PubMed

    Zago, María Paola; Hosakote, Yashoda M; Koo, Sue-Jie; Dhiman, Monisha; Piñeyro, María Dolores; Parodi-Talice, Adriana; Basombrio, Miguel A; Robello, Carlos; Garg, Nisha J

    2016-06-01

    Trypanosoma cruzi species is categorized into six discrete typing units (TcI to TcVI) of which TcI is most abundantly noted in the sylvatic transmission cycle and considered the major cause of human disease. In our study, the TcI strains Colombiana (COL), SylvioX10/4 (SYL), and a cultured clone (TCC) exhibited different biological behavior in a murine model, ranging from high parasitemia and symptomatic cardiomyopathy (SYL), mild parasitemia and high tissue tropism (COL), to no pathogenicity (TCC). Proteomic profiling of the insect (epimastigote) and infective (trypomastigote) forms by two-dimensional gel electrophoresis/matrix-assisted laser desorption ionization-time of flight mass spectrometry, followed by functional annotation of the differential proteome data sets (≥2-fold change, P < 0.05), showed that several proteins involved in (i) cytoskeletal assembly and remodeling, essential for flagellar wave frequency and amplitude and forward motility of the parasite, and (ii) the parasite-specific antioxidant network were enhanced in COL and SYL (versus TCC) trypomastigotes. Western blotting confirmed the enhanced protein levels of cytosolic and mitochondrial tryparedoxin peroxidases and their substrate (tryparedoxin) and iron superoxide dismutase in COL and SYL (versus TCC) trypomastigotes. Further, COL and SYL (but not TCC) were resistant to exogenous treatment with stable oxidants (H2O2 and peroxynitrite [ONOO(-)]) and dampened the intracellular superoxide and nitric oxide response in macrophages, and thus these isolates escaped from macrophages. Our findings suggest that protein expression conducive to increase in motility and control of macrophage-derived free radicals provides survival and persistence benefits to TcI isolates of T. cruzi. PMID:27068090

  1. Autophagy and checkpoints for intracellular pathogen defense

    PubMed Central

    Paulus, Geraldine L.C.; Xavier, Ramnik J.

    2015-01-01

    Purpose of review Autophagy plays a crucial role in intracellular defense against various pathogens. Xenophagy is a form of selective autophagy that targets intracellular pathogens for degradation. In addition, several related yet distinct intracellular defense responses depend on autophagy-related (ATG) genes. This review gives an overview of these processes, pathogen strategies to subvert them, and their crosstalk with various cell death programs. Recent findings The recruitment of ATG proteins plays a key role in multiple intracellular defense programs, specifically xenophagy, LC3-associated phagocytosis (LAP), and the IFNγ-mediated elimination of pathogens such as Toxoplasma gondii and murine norovirus. Recent progress has revealed methods employed by pathogens to resist these intracellular defense mechanisms and/or persist in spite of them. The intracellular pathogen load can tip the balance between cell survival and cell death. Further, it was recently observed that LAP is indispensable for the efficient clearance of dying cells. Summary Autophagy-dependent and ATG gene-dependent pathways are essential in intracellular defense against a broad range of pathogens. PMID:25394238

  2. Construction, expression and characterization of chimaeric toxins containing the ribonucleolytic toxin restrictocin: intracellular mechanism of action.

    PubMed

    Rathore, D; Batra, J K

    1997-06-15

    Restrictocin is a ribonucleolytic toxin produced by the fungus Aspergillus restrictus. Two chimaeric toxins containing restrictocin directed at the human transferrin receptor have been constructed. Anti-TFR(scFv)-restrictocin is encoded by a gene produced by fusing the DNA encoding a single-chain antigen-combining region (scFv) of a monoclonal antibody, directed at the human transferrin receptor, at the 5' end of that encoding restrictocin. The other chimaeric toxin, restrictocin-anti-TFR(scFv), is encoded by a gene fusion containing the DNA encoding the single-chain antigen-combining region of antibody to human transferrin receptor at the 3' end of the DNA encoding restrictocin. These gene fusions were expressed in Escherichia coli, and fusion proteins purified from the inclusion bodies by simple chromatography techniques to near-homogeneity. The two chimaeric toxins were found to be equally active in inhibiting protein synthesis in a cell-free in vitro translation assay system. The chimaeric toxins were selectively toxic to the target cells in culture with potent cytotoxic activities. However, restrictocin-anti-TFR(scFv) was more active than anti-TFR(scFv)-restrictocin on all cell lines studied. By using protease and metabolic inhibitors, it can be shown that, to manifest their cytotoxic activity, the restrictocin-containing chimaeric toxins need to be proteolytically processed intracellularly and the free toxin or a fragment thereof thus generated is translocated to the target via a route involving the Golgi apparatus. PMID:9210405

  3. Identification of Two Intracellular Mechanisms Leading to Reduced Expression of Oncoretrovirus Envelope Glycoproteins at the Cell Surface

    PubMed Central

    Grange, Marie-Pierre; Blot, Vincent; Delamarre, Lelia; Bouchaert, Isabelle; Rocca, Anna; Dautry-Varsat, Alice; Dokhélar, Marie-Christine

    2000-01-01

    All retrovirus glycoproteins have a cytoplasmic domain that plays several roles in virus replication. We have determined whether and how the cytoplasmic domains of oncoretrovirus glycoproteins modulate their intracellular trafficking, by using chimeric proteins that combined the α-chain of the interleukin-2 receptor with the glycoprotein cytoplasmic domains of five oncoretroviruses: human T-cell leukemia virus type 1 (HTLV-1), Rous sarcoma virus (RSV), bovine leukemia virus (BLV), murine leukemia virus (MuLV), and Mason-Pfizer monkey virus (MPMV). All of these proteins were synthesized and matured in the same way as a control protein with no retrovirus cytoplasmic domain. However, the amounts of all chimeric proteins at the cell surface were smaller than that of the control protein. The protein appearing at and leaving the cell surface and endocytosis were measured in stable transfectants expressing the chimera. We identified two groups of proteins which followed distinct intracellular pathways. Group 1 included chimeric proteins that reached the cell surface normally but were rapidly endocytosed afterwards. This group included the chimeric proteins with HTLV-1, RSV, and BLV cytoplasmic domains. Group 2 included chimeric proteins that were not detected at the cell surface, despite normal intracellular concentrations, and were accumulated in the Golgi complex. This group included the chimeric proteins with MuLV and MPMV cytoplasmic domains. Finally, we verified that the MuLV envelope glycoproteins behaved in the same way as the corresponding chimeras. These results indicate that retroviruses have evolved two distinct mechanisms to ensure a similar biological feature: low concentrations of their glycoproteins at the cell surface. PMID:11090173

  4. Endocytosis of ABCG2 drug transporter caused by binding of 5D3 antibody: trafficking mechanisms and intracellular fate.

    PubMed

    Studzian, Maciej; Bartosz, Grzegorz; Pulaski, Lukasz

    2015-08-01

    ABCG2, a metabolite and xenobiotic transporter located at the plasma membrane (predominantly in barrier tissues and progenitor cells), undergoes a direct progressive endocytosis process from plasma membrane to intracellular compartments upon binding of 5D3 monoclonal antibody. This antibody is specific to an external epitope on the protein molecule and locks it in a discrete conformation within its activity cycle, presumably providing a structural trigger for the observed internalization phenomenon. Using routine and novel assays, we show that ABCG2 is endocytosed by a mixed mechanism: partially via a rapid, clathrin-dependent pathway and partially in a cholesterol-dependent, caveolin-independent manner. While the internalization process is entirely dynamin-dependent and converges initially at the early endosome, subsequent intracellular fate of ABCG2 is again twofold: endocytosis leads to only partial lysosomal degradation, while a significant fraction of the protein is retained in a post-endosomal compartment with the possibility of at least partial recycling back to the cell surface. This externally triggered, conformation-related trafficking pathway may serve as a general regulatory paradigm for membrane transporters, and its discovery was made possible thanks to consistent application of quantitative methods. PMID:25918011

  5. Engineered redox-responsive PEG detachment mechanism in PEGylated nano-graphene oxide for intracellular drug delivery.

    PubMed

    Wen, Huiyun; Dong, Chunyan; Dong, Haiqing; Shen, Aijun; Xia, Wenjuan; Cai, Xiaojun; Song, Yanyan; Li, Xuequan; Li, Yongyong; Shi, Donglu

    2012-03-12

    In biomedical applications, polyethylene glycol (PEG) functionalization has been a major approach to modify nanocarriers such as nano-graphene oxide for particular biological requirements. However, incorporation of a PEG shell poses a significant diffusion barrier that adversely affects the release of the loaded drugs. This study addresses this critical issue by employing a redox-responsive PEG detachment mechanism. A PEGylated nano-graphene oxide (NGO-SS-mPEG) with redox-responsive detachable PEG shell is developed that can rapidly release an encapsulated payload at tumor-relevant glutathione (GSH) levels. The PEG shell grafted onto NGO sheets gives the nanocomposite high physiological solubility and stability in circulation. It can selectively detach from NGO upon intracellular GSH stimulation. The surface-engineered structures are shown to accelerate the release of doxorubicin hydrochloride (DXR) from NGO-SS-mPEG 1.55 times faster than in the absence of GSH. Confocal microscopy shows clear evidence of NGO-SS-mPEG endocytosis in HeLa cells, mainly accumulated in cytoplasm. Furthermore, upon internalization of DXR-loaded NGO with a disulfide-linked PEG shell into HeLa cells, DXR is effectively released in the presence of an elevated GSH reducing environment, as observed in confocal microscopy and flow cytometric experiments. Importantly, inhibition of cell proliferation is directly correlated with increased intracellular GSH concentrations due to rapid DXR release. PMID:22228696

  6. Sulfur mustard-induced increase in intracellular calcium: A mechanism of mustard toxicity

    SciTech Connect

    Ray, R.; Majerus, B.J.; Munavalli, G.S.; Petrali, J.P.

    1993-05-13

    The effect of sulfur mustard SM, bis-(2-chloroethyl) sulfide on intracellular free Ca2+ concentration (Ca2+)i was studied in vitro using the clonal mouse neuroblastoma-rat glioma hybrid NG108-15 and primary normal human epidermal keratinocyte (NHEK) cell culture models. SM depletes cellular glutathione (GSH) and thus may inhibit GSH-dependent Ca2+-ATPase (Ca2+ pump), leading to a high (Ca2+) and consequent cellular toxicity. Following 0.3 mM SM exposure, GSH levels decreased 20-34% between 1-6 hr in NG108-15 cells. SM increased (Ca2+)i, measured using the Ca2+-specific fluorescent probe Fluo-3 AM, in both NG108-15 cells (1030% between 2-6 hr) and NHEK (23-30% between 0.5-3 hr) . Depletion of cellular GSH by buthionine sulfoximine (1 mM), a specific GSH biosynthesis inhibitor, also increased Ca2+, (88% at 1 hr) in NHEK, suggesting that GSH depletion may lead to increased (Ca2+)i. Calcium, localized cytochemically with antimony, accumulated in increased amounts around mitochondria and endoplasmic reticula, in the cytosol, and in particular in the euchromatin regions of the nucleus beginning at 6 hr after 0.3 mM SM exposure of NG108-15 cells. Cell membrane integrity examined with the fluorescent membrane probe calcein AM was unaffected through 6 hr following 1 mM SM exposure; and cell viability (NG108-15 cells) measured by trypan blue exclusion was >80% of control through 9 hr following 0.3 mM SM exposure.

  7. Sudden infant deaths: arousal as a survival mechanism.

    PubMed

    Kahn, Andre; Groswasser, Jose; Franco, Patricia; Scaillet, Sonia; Sawaguchi, Toshiko; Kelmanson, Igor; Bernanrd, Dan

    2002-12-01

    The mechanisms responsible for sudden infant death syndrome (SIDS) are still largely unknown. To explain what factors contribute to the deaths, we suggest a model: the '3 S model for SIDS' that includes 'sicknesses', 'stages of development' and 'surroundings': (1) 'sicknesses' refers to infectious diseases or other medical condition. (2) 'Stages of development' relates to the maturation of vital systems including respiratory, neurovegetative or sleep-wake behavioral controls. (3) 'Surroundings' refers to environmental conditions that enhance the deficiency of cardiorespiratory, vegetative and/or arousal controls. Such conditions were identified by epidemiological studies and include the following main risk factors: the prone body position during sleep, high environmental temperature, maternal smoking or sleep deprivation. An infant could be at higher risk for SIDS because of a deficiency in breathing and cardiac autonomic controls during sleep, inducing repeated episodes of hypoxia and hypoxemia. The risk is increased when the infant has a lower propensity to arouse from sleep and so, to autoresuscitate. The accident has a greater probability to occur when an infection, or an unfavorable environmental factor aggravates the immature cardiorespiratory and sleep/wake behaviors of the infant. The clinical findings could be related to the changes reported in the brainstems of SIDS victims. PMID:14592372

  8. Protection of Cells against Oxidative Stress by Nanomolar Levels of Hydroxyflavones Indicates a New Type of Intracellular Antioxidant Mechanism

    PubMed Central

    Hájek, Jan; Staňková, Veronika; Filipský, Tomáš; Balducci, Valentina; De Vito, Paolo; Leone, Stefano; Bavavea, Eugenia I.; Silvestri, Ilaria Proietti; Righi, Giuliana; Luly, Paolo; Saso, Luciano; Bovicelli, Paolo; Pedersen, Jens Z.; Incerpi, Sandra

    2013-01-01

    Natural polyphenol compounds are often good antioxidants, but they also cause damage to cells through more or less specific interactions with proteins. To distinguish antioxidant activity from cytotoxic effects we have tested four structurally related hydroxyflavones (baicalein, mosloflavone, negletein, and 5,6-dihydroxyflavone) at very low and physiologically relevant levels, using two different cell lines, L-6 myoblasts and THP-1 monocytes. Measurements using intracellular fluorescent probes and electron paramagnetic resonance spectroscopy in combination with cytotoxicity assays showed strong antioxidant activities for baicalein and 5,6-dihydroxyflavone at picomolar concentrations, while 10 nM partially protected monocytes against the strong oxidative stress induced by 200 µM cumene hydroperoxide. Wide range dose-dependence curves were introduced to characterize and distinguish the mechanism and targets of different flavone antioxidants, and identify cytotoxic effects which only became detectable at micromolar concentrations. Analysis of these dose-dependence curves made it possible to exclude a protein-mediated antioxidant response, as well as a mechanism based on the simple stoichiometric scavenging of radicals. The results demonstrate that these flavones do not act on the same radicals as the flavonol quercetin. Considering the normal concentrations of all the endogenous antioxidants in cells, the addition of picomolar or nanomolar levels of these flavones should not be expected to produce any detectable increase in the total cellular antioxidant capacity. The significant intracellular antioxidant activity observed with 1 pM baicalein means that it must be scavenging radicals that for some reason are not eliminated by the endogenous antioxidants. The strong antioxidant effects found suggest these flavones, as well as quercetin and similar polyphenolic antioxidants, at physiologically relevant concentrations act as redox mediators to enable endogenous

  9. Thioredoxin-1 promotes survival in cells exposed to S-nitrosoglutathione: Correlation with reduction of intracellular levels of nitrosothiols and up-regulation of the ERK1/2 MAP Kinases

    SciTech Connect

    Arai, Roberto J.; Debbas, Victor; Stern, Arnold; Monteiro, Hugo P.

    2008-12-01

    Accumulating evidence indicates that post-translational protein modifications by nitric oxide and its derived species are critical effectors of redox signaling in cells. These protein modifications are most likely controlled by intracellular reductants. Among them, the importance of the 12 kDa dithiol protein thioredoxin-1 (TRX-1) has been increasingly recognized. However, the effects of TRX-1 in cells exposed to exogenous nitrosothiols remain little understood. We investigated the levels of intracellular nitrosothiols and survival signaling in HeLa cells over-expressing TRX-1 and exposed to S-nitrosoglutahione (GSNO). A role for TRX-1 expression on GSNO catabolism and cell viability was demonstrated by the concentration-dependent effects of GSNO on decreasing TRX-1 expression, activation of caspase-3, and increasing cell death. The over-expression of TRX-1 in HeLa cells partially attenuated caspase-3 activation and enhanced cell viability upon GSNO treatment. This was correlated with reduction of intracellular levels of nitrosothiols and increasing levels of nitrite and nitrotyrosine. The involvement of ERK, p38 and JNK pathways were investigated in parental cells treated with GSNO. Activation of ERK1/2 MAP kinases was shown to be critical for survival signaling. In cells over-expressing TRX-1, basal phosphorylation levels of ERK1/2 MAP kinases were higher and further increased after GSNO treatment. These results indicate that the enhanced cell viability promoted by TRX-1 correlates with its capacity to regulate the levels of intracellular nitrosothiols and to up-regulate the survival signaling pathway mediated by the ERK1/2 MAP kinases.

  10. Increased extracellular and intracellular Ca{sup 2+} lead to adipocyte accumulation in bone marrow stromal cells by different mechanisms

    SciTech Connect

    Hashimoto, Ryota; Katoh, Youichi; Miyamoto, Yuki; Itoh, Seigo; Daida, Hiroyuki; Nakazato, Yuji; Okada, Takao

    2015-02-20

    Mesenchymal stem cells found in bone marrow stromal cells (BMSCs) are the common progenitors for both adipocyte and osteoblast. An increase in marrow adipogenesis is associated with age-related osteopenia and anemia. Both extracellular and intracellular Ca{sup 2+} ([Ca{sup 2+}]{sub o} and [Ca{sup 2+}]{sub i}) are versatile signaling molecules that are involved in the regulation of cell functions, including proliferation and differentiation. We have recently reported that upon treatment of BMSCs with insulin and dexamethasone, both high [Ca{sup 2+}]{sub o} and high [Ca{sup 2+}]{sub i} enhanced adipocyte accumulation, which suggested that increases in [Ca{sup 2+}]{sub o} caused by bone resorption may accelerate adipocyte accumulation in aging and diabetic patients. In this study, we used primary mouse BMSCs to investigate the mechanisms by which high [Ca{sup 2+}]{sub o} and high [Ca{sup 2+}]{sub i} may enhance adipocyte accumulation. In the process of adipocyte accumulation, two important keys are adipocyte differentiation and the proliferation of BMSCs, which have the potential to differentiate into adipocytes. Use of MTT assay and real-time RT-PCR revealed that high [Ca{sup 2+}]{sub i} (ionomycin)-dependent adipocyte accumulation is caused by enhanced proliferation of BMSCs but not enhanced differentiation into adipocytes. Using fura-2 fluorescence-based approaches, we showed that high [Ca{sup 2+}]{sub o} (addition of CaCl{sub 2}) leads to increases in [Ca{sup 2+}]{sub i}. Flow cytometric methods revealed that high [Ca{sup 2+}]{sub o} suppressed the phosphorylation of ERK independently of intracellular Ca{sup 2+}. The inhibition of ERK by U0126 and PD0325901 enhanced the differentiation of BMSCs into adipocytes. These data suggest that increased extracellular Ca{sup 2+} provides the differentiation of BMSCs into adipocytes by the suppression of ERK activity independently of increased intracellular Ca{sup 2+}, which results in BMSC proliferation. - Highlights:

  11. Arf6 controls retromer traffic and intracellular cholesterol distribution via a phosphoinositide-based mechanism

    PubMed Central

    Marquer, Catherine; Tian, Huasong; Yi, Julie; Bastien, Jayson; Dall'Armi, Claudia; Yang-Klingler, YoungJoo; Zhou, Bowen; Chan, Robin Barry; Di Paolo, Gilbert

    2016-01-01

    Small GTPases play a critical role in membrane traffic. Among them, Arf6 mediates transport to and from the plasma membrane, as well as phosphoinositide signalling and cholesterol homeostasis. Here we delineate the molecular basis for the link between Arf6 and cholesterol homeostasis using an inducible knockout (KO) model of mouse embryonic fibroblasts (MEFs). We find that accumulation of free cholesterol in the late endosomes/lysosomes of Arf6 KO MEFs results from mistrafficking of Niemann–Pick type C protein NPC2, a cargo of the cation-independent mannose-6-phosphate receptor (CI-M6PR). This is caused by a selective increase in an endosomal pool of phosphatidylinositol-4-phosphate (PI4P) and a perturbation of retromer, which controls the retrograde transport of CI-M6PR via sorting nexins, including the PI4P effector SNX6. Finally, reducing PI4P levels in KO MEFs through independent mechanisms rescues aberrant retromer tubulation and cholesterol mistrafficking. Our study highlights a phosphoinositide-based mechanism for control of cholesterol distribution via retromer. PMID:27336679

  12. Elevated intracellular calcium concentration increases secretory processing of the amyloid precursor protein by a tyrosine phosphorylation-dependent mechanism.

    PubMed Central

    Petryniak, M A; Wurtman, R J; Slack, B E

    1996-01-01

    Secretory cleavage of the amyloid precursor protein (APP), a process that releases soluble APP derivatives (APPs) into the extracellular space, is stimulated by the activation of muscarinic receptors coupled to phosphoinositide hydrolysis. The signalling pathways involved in the release process exhibit both protein kinase C- and protein tyrosine phosphorylation-dependent components [Slack, Breu, Petryniak, Srivastava and Wurtman (1995) J. Biol. Chem. 270, 8337-8344]. The possibility that elevations in intracellular Ca2+ concentration initiate the tyrosine phosphorylation-dependent release of APPs was examined in human embryonic kidney cells expressing muscarinic m3 receptors. Inhibition of protein kinase C with the bisindolylmaleimide GF 109203X decreased the carbachol-evoked release of APPs by approx. 30%, as shown previously. The residual response was further decreased, in an additive manner, by the Ca2+ chelator EGTA, or by the tyrosine kinase inhibitor tyrphostin A25. The Ca2+ ionophore, ionomycin, like carbachol, stimulated both the release of APPs and the tyrosine phosphorylation of several proteins, one of which was identified as paxillin, a component of focal adhesions. The effects of ionomycin on APPs release and on protein tyrosine phosphorylation were concentration-dependent, and occurred over similar concentration ranges; both effects were inhibited only partly by GF 109203X, but were abolished by EGTA or by tyrosine kinase inhibitors. The results demonstrate for the first time that ionophore-induced elevations in intracellular Ca2+ levels elicit APPs release via increased tyrosine phosphorylation. Part of the increase in APPs release evoked by muscarinic receptor activation might be attributable to a similar mechanism. PMID:9003386

  13. Mechanical models of the cellular cytoskeletal network for the analysis of intracellular mechanical properties and force distributions: a review.

    PubMed

    Chen, Ting-Jung; Wu, Chia-Ching; Su, Fong-Chin

    2012-12-01

    The cytoskeleton, which is the major mechanical component of cells, supports the cell body and regulates the cellular motility to assist the cell in performing its biological functions. Several cytoskeletal network models have been proposed to investigate the mechanical properties of cells. This review paper summarizes these models with a focus on the prestressed cable network, the semi-flexible chain network, the open-cell foam, the tensegrity, and the granular models. The components, material parameters, types of connection joints, tension conditions, and the advantages and disadvantages of each model are evaluated from a structural and biological point of view. The underlying mechanisms that are associated with the morphological changes of spreading cells are expected to be simulated using a cytoskeletal model; however, it is still paid less attention most likely due to the lack of a suitable cytoskeletal model that can accurately model the spreading process. In this review article, the established cytoskeletal models are hoped to provide useful information for the development of future cytoskeletal models with different degrees of cell attachment for the study of the mechanical mechanisms underlying the cellular behaviors in response to external stimulations. PMID:23062682

  14. Comparative transduction mechanisms of hair cells in the bullfrog utriculus. 1: Responses to intracellular current

    NASA Technical Reports Server (NTRS)

    Baird, Richard A.

    1994-01-01

    Hair cells in the bullfrog sacculus are specifically adapted to sense small-amplitude, high-frequency linear accelerations. These hair cells display many properties that are undesirable or inappropriate for hair cells that must provide static gravity sensitivity. This study resulted in part due to an interest in seeing how the transduction mechanisms of hair cells in a gravity-sensing otolith endorgan would differ from those in the bullfrog sacculus. The bullfrog utriculus is an appropriate model for these studies, because its structure is representative of higher vertebrates in general and its function as a sensor of static gravity and dynamic linear acceleration is well known. Hair cells in the bullfrog utriculus, classifiable as Type 2 by cell body and synapse morphology, differ markedly in hair bundle morphology from those in the bullfrog sacculus. Moreover, the hair bundle morphologies of utricular hair cells, unlike those in the sacculus, differ in different membrane regions.

  15. Mechanism of transport and intracellular binding of porfiromycin in HCT 116 human colon carcinoma cells.

    PubMed

    Pan, S S; Johnson, R; Gonzalez, H; Thohan, V

    1989-09-15

    The mechanism of uptake and efflux of porfiromycin (PFM) by HCT 116 human colon carcinoma cells or freshly obtained human RBC was investigated. The time course of uptake of radioactivity upon exposure of HCT 116 cells to [14C]PFM showed one fast and one slow phase of linear increase. The initial phase of PFM uptake was not saturable with external drug concentrations from 2 to 100 microM. PFM accumulation was temperature dependent with a temperature coefficient (Q10 24-37 degrees C) of 2.3 +/- 0.3. PFM uptake was not affected either by individual inhibitors such as 1 mM 2,4-dinitrophenol, sodium azide, iodoacetic acid, ouabain, 0.02 mM oligomycin, p-hydroxylmercuribenzoate, 0.2 mM N-ethylmaleimide, or by combinations of inhibitors. PFM uptake did not demonstrate competitive inhibition by unlabeled PFM and mitomycin C. Efflux of cellular radioactivity was not affected by the above mentioned inhibitors or by verapamil, diltiazem, or trifluoperazine. Only aliphatic alcohols accelerated the initial influx rate. The RBC, however, only exhibited the initial fast accumulation of [14C]PFM, and all the 14C accumulated by RBC was exchangeable. These data demonstrate that the uptake and the efflux of PFM in HCT 116 cells and RBC comprise a passive diffusion process. PMID:2766276

  16. Intracellular Uptake: A Possible Mechanism for Silver Engineered Nanoparticle Toxicity to a Freshwater Alga Ochromonas danica

    PubMed Central

    Miao, Ai-Jun; Luo, Zhiping; Chen, Chi-Shuo; Chin, Wei-Chun; Santschi, Peter H.; Quigg, Antonietta

    2010-01-01

    The behavior and toxicity of silver engineered nanoparticles (Ag-ENs) to the mixotrophic freshwater alga Ochromonas danica were examined in the present study to determine whether any other mechanisms are involved in their algal toxicity besides Ag+ liberation outside the cells. Despite their good dispersability, the Ag-ENs were found to continuously aggregate and dissolve rapidly. When the initial nanoparticle concentration was lower than 10 µM, the total dissolved Ag+ concentration ([Ag+]T) in the suspending media reached its maximum after 1 d and then decreased suggesting that Ag+ release might be limited by the nanoparticle surface area under these conditions. Furthermore, Ag-EN dissolution extent remarkably increased in the presence of glutathione. In the Ag-EN toxicity experiment, glutathione was also used to eliminate the indirect effects of Ag+ that was released. However, remarkable toxicity was still observed although the free Ag+ concentration in the media was orders of magnitude lower than the non-observed effect concentration of Ag+ itself. Such inhibitive effects were mitigated when more glutathione was added, but could never be completely eliminated. Most importantly, we demonstrate, for the first time, that Ag-ENs can be taken in and accumulated inside the algal cells, where they exerted their toxic effects. Therefore, nanoparticle internalization may be an alternative pathway through which algal growth can be influenced. PMID:21203552

  17. The Antimicrobial Domains of Wheat Puroindolines Are Cell-Penetrating Peptides with Possible Intracellular Mechanisms of Action

    PubMed Central

    Alfred, Rebecca L.; Palombo, Enzo A.; Panozzo, Joseph F.; Bhave, Mrinal

    2013-01-01

    The puroindoline proteins (PINA and PINB) of wheat display lipid-binding properties which affect the grain texture, a critical parameter for wheat quality. Interestingly, the same proteins also display antibacterial and antifungal properties, attributed mainly to their Tryptophan-rich domain (TRD). Synthetic peptides based on this domain also display selectivity towards bacterial and fungal cells and do not cause haemolysis of mammalian cells. However, the mechanisms of these activities are unclear, thus limiting our understanding of the in vivo roles of PINs and development of novel applications. This study investigated the mechanisms of antimicrobial activities of synthetic peptides based on the TRD of the PINA and PINB proteins. Calcein dye leakage tests and transmission electron microscopy showed that the peptides PuroA, Pina-M and Pina-W→F selectively permeabilised the large unilamellar vesicles (LUVs) made with negatively charged phospholipids mimicking bacterial membranes, but were ineffective against LUVs made with zwitterionic phospholipids mimicking eukaryotic membranes. Propidium iodide fluorescence tests of yeast (Saccharomyces cerevisiae) cells showed the peptides were able to cause loss of membrane integrity, PuroA and Pina-M being more efficient. Scanning electron micrographs of PINA-based peptide treated yeast cells showed the formation of pits or pores in cell membranes and release of cellular contents. Gel retardation assays indicated the peptides were able to bind to DNA in vitro, and the induction of filamental growth of E. coli cells indicated in vivo inhibition of DNA synthesis. Together, the results strongly suggest that the PIN-based peptides exert their antimicrobial effects by pore formation in the cell membrane, likely by a carpet-like mechanism, followed by intracellular mechanisms of activity. PMID:24098387

  18. Targets and Intracellular Signaling Mechanisms for Deoxynivalenol-Induced Ribosomal RNA Cleavage

    PubMed Central

    He, Kaiyu; Zhou, Hui-Ren; Pestka, James J.

    2012-01-01

    The trichothecene mycotoxin deoxynivalenol (DON), a known translational inhibitor, induces ribosomal RNA (rRNA) cleavage. Here, we characterized this process relative to (1) specific 18S and 28S ribosomal RNA cleavage sites and (2) identity of specific upstream signaling elements in this pathway. Capillary electrophoresis indicated that DON at concentrations as low as 200 ng/ml evoked selective rRNA cleavage after 6 h and that 1000 ng/ml caused cleavage within 2 h. Northern blot analysis revealed that DON exposure induced six rRNA cleavage fragments from 28S rRNA and five fragments from 18S rRNA. When selective kinase inhibitors were used to identify potential upstream signals, RNA-activated protein kinase (PKR), hematopoietic cell kinase (Hck), and p38 were found to be required for rRNA cleavage, whereas c-Jun N-terminal kinase and extracellular signal-regulated kinase were not. Furthermore, rRNA fragmentation was suppressed by the p53 inhibitors pifithrin-α and pifithrin-μ as well as the pan caspase inhibitor Z-VAD-FMK. Concurrent apoptosis was confirmed by acridine orange/ethidium bromide staining and flow cytometry. DON activated caspases 3, 8, and 9, thus suggesting the possible coinvolvement of both extrinsic and intrinsic apoptotic pathways in rRNA cleavage. Satratoxin G (SG), anisomycin, and ricin also induced specific rRNA cleavage profiles identical to those of DON, suggesting that ribotoxins might share a conserved rRNA cleavage mechanism. Taken together, DON-induced rRNA cleavage is likely to be closely linked to apoptosis activation and appears to involve the sequential activation of PKR/Hck →p38→p53→caspase 8/9→caspase 3. PMID:22491426

  19. Targets and intracellular signaling mechanisms for deoxynivalenol-induced ribosomal RNA cleavage.

    PubMed

    He, Kaiyu; Zhou, Hui-Ren; Pestka, James J

    2012-06-01

    The trichothecene mycotoxin deoxynivalenol (DON), a known translational inhibitor, induces ribosomal RNA (rRNA) cleavage. Here, we characterized this process relative to (1) specific 18S and 28S ribosomal RNA cleavage sites and (2) identity of specific upstream signaling elements in this pathway. Capillary electrophoresis indicated that DON at concentrations as low as 200 ng/ml evoked selective rRNA cleavage after 6 h and that 1000 ng/ml caused cleavage within 2 h. Northern blot analysis revealed that DON exposure induced six rRNA cleavage fragments from 28S rRNA and five fragments from 18S rRNA. When selective kinase inhibitors were used to identify potential upstream signals, RNA-activated protein kinase (PKR), hematopoietic cell kinase (Hck), and p38 were found to be required for rRNA cleavage, whereas c-Jun N-terminal kinase and extracellular signal-regulated kinase were not. Furthermore, rRNA fragmentation was suppressed by the p53 inhibitors pifithrin-α and pifithrin-μ as well as the pan caspase inhibitor Z-VAD-FMK. Concurrent apoptosis was confirmed by acridine orange/ethidium bromide staining and flow cytometry. DON activated caspases 3, 8, and 9, thus suggesting the possible coinvolvement of both extrinsic and intrinsic apoptotic pathways in rRNA cleavage. Satratoxin G (SG), anisomycin, and ricin also induced specific rRNA cleavage profiles identical to those of DON, suggesting that ribotoxins might share a conserved rRNA cleavage mechanism. Taken together, DON-induced rRNA cleavage is likely to be closely linked to apoptosis activation and appears to involve the sequential activation of PKR/Hck →p38→p53→caspase 8/9→caspase 3. PMID:22491426

  20. Retention mechanism of technetium-99m-HM-PAO: intracellular reaction with glutathione

    SciTech Connect

    Neirinckx, R.D.; Burke, J.F.; Harrison, R.C.; Forster, A.M.; Andersen, A.R.; Lassen, N.A.

    1988-12-01

    Preparations of d,l- and meso-hexamethylpropyleneamine oxime (HM-PAO) labeled with technetium-99m were added to rat brain homogenates diluted with phosphate buffer (1:10). The conversion of d,l-HM-PAO to hydrophilic forms took place with an initial rate constant of 0.12 min-1. Incubation of the brain homogenate with 2% diethyl maleate for 5 h decreased the homogenate's measured glutathione (GSH) concentration from 160 to 16 microM and decreased the conversion rate to 0.012 min-1. Buffered aqueous solutions of glutathione rapidly converted the HM-PAO tracers to hydrophilic forms having the same chromatographic characteristics as found in the brain homogenates. The rate constant for the conversion reaction of d,l-HM-PAO in GSH aqueous solution was 208 and 317 L/mol/min in two different assay systems and for meso-HM-PAO the values were 14.7 and 23.2 L/mol/min, respectively. Rat brain has a GSH concentration of about 2.3 mM and the conversion of the d,l-HM-PAO due to GSH alone should proceed with a rate constant of 0.48 to 0.73 min-1 and be correspondingly 14-fold slower for meso-HM-PAO. In human brain, the in vivo data of Lassen et al. show a conversion rate constant of 0.80 min-1. This correspondence of values supports the notion that GSH may be important for the in vivo conversion of 99mTc-labeled HM-PAO to hydrophilic forms and may be the mechanism of trapping in brain and other cells. A kinetic model for the trapping of d,l- and meso-HM-PAO in tissue is developed that is based on data of GSH concentration in various organs.

  1. A biphasic endothelial stress-survival mechanism regulates the cellular response to vascular endothelial growth factor A

    SciTech Connect

    Latham, Antony M.; Odell, Adam F.; Mughal, Nadeem A.; Issitt, Theo; Ulyatt, Clare; Walker, John H.; Homer-Vanniasinkam, Shervanthi; Ponnambalam, Sreenivasan

    2012-11-01

    Vascular endothelial growth factor A (VEGF-A) is an essential cytokine that regulates endothelial function and angiogenesis. VEGF-A binding to endothelial receptor tyrosine kinases such as VEGFR1 and VEGFR2 triggers cellular responses including survival, proliferation and new blood vessel sprouting. Increased levels of a soluble VEGFR1 splice variant (sFlt-1) correlate with endothelial dysfunction in pathologies such as pre-eclampsia; however the cellular mechanism(s) underlying the regulation and function of sFlt-1 are unclear. Here, we demonstrate the existence of a biphasic stress response in endothelial cells, using serum deprivation as a model of endothelial dysfunction. The early phase is characterized by a high VEGFR2:sFlt-1 ratio, which is reversed in the late phase. A functional consequence is a short-term increase in VEGF-A-stimulated intracellular signaling. In the late phase, sFlt-1 is secreted and deposited at the extracellular matrix. We hypothesized that under stress, increased endothelial sFlt-1 levels reduce VEGF-A bioavailability: VEGF-A treatment induces sFlt-1 expression at the cell surface and VEGF-A silencing inhibits sFlt-1 anchorage to the extracellular matrix. Treatment with recombinant sFlt-1 inhibits VEGF-A-stimulated in vitro angiogenesis and sFlt-1 silencing enhances this process. In this response, increased VEGFR2 levels are regulated by the phosphatidylinositol-3-kinase and PKB/Akt signaling pathways and increased sFlt-1 levels by the ERK1/2 signaling pathway. We conclude that during serum withdrawal, cellular sensing of environmental stress modulates sFlt-1 and VEGFR2 levels, regulating VEGF-A bioavailability and ensuring cell survival takes precedence over cell proliferation and migration. These findings may underpin an important mechanism contributing to endothelial dysfunction in pathological states. -- Highlights: Black-Right-Pointing-Pointer Endothelial cells mount a stress response under conditions of low serum. Black

  2. MECHANISMS OF THERMOPHILIC SURVIVAL, DEGRADATION BY THERMOPHILICS AND OPTIMIZATION OF THERMOPHILIC BACTERIA FOR BIODEGRADATION

    EPA Science Inventory

    We have developed a consortium of thermophilic methanotrophic bacteria from Yellowstone National Park that degrades TCE by the use of methyl monooxygenase. We are going to isolate the thermophiles in patent 5,858,763,determine their mechanisms of surviving extreme temperatures, d...

  3. Intracellular mechanisms of cocaine-memory reconsolidation in the basolateral amygdala and dorsal hippocampus

    NASA Astrophysics Data System (ADS)

    Wells, Audrey Marie

    The ability of cocaine-associated environmental contexts to promote relapse in abstinent humans and reinstatement of cocaine-seeking behavior in laboratory animals depends on the formation and maintenance of maladaptive context-response-cocaine associative memories, the latter of which can be disrupted by manipulations that interfere with memory reconsolidation. Memory reconsolidation refers to a protein synthesis-dependent phenomenon whereby memory traces are reincorporated back into long-term memory storage following their retrieval and subsequent destabilization. To elucidate the distinctive roles of the basolateral amygdala (BLA) and dorsal hippocampus (DH) in the reconsolidation of context-response-cocaine memories, Experiments 1-3 evaluated novel molecular mechanisms within each structure that control this phenomenon. Experiment 1 tested the hypothesis that activation of the extracellular signal-regulated kinase (ERK) in the BLA and nucleus accumbens core (NACc - a substrate for Pavlovian cocaine-memory reconsolidation) would critically control instrumental cocaine-memory reconsolidation. To determine this, rats were re-exposed to a context that had previously been used for cocaine self-administration (i.e., cocaine memory-reactivation) and immediately thereafter received bilateral intra-BLA or intra-NACc microinfusions of the ERK inhibitor U0126 or vehicle (VEH) and were subsequently tested for drug context-induced cocaine-seeking behavior (non-reinforced lever responding) ~72 h later. Re-exposure to the cocaine-paired context at test fully reinstated cocaine-seeking behavior, relative to responding in an alternate, extinction context, and post-reactivation U0126 treatment in the BLA, but not the NACc, impaired cocaine-seeking behavior, relative to VEH. This effect was associated with a temporary increase in ERK2, but not ERK1, phosphorylation in the BLA and required explicit reactivation of the target memory trace (i.e., did not similarly manifest when U

  4. Role of Sodium Bicarbonate Cotransporters in Intracellular pH Regulation and Their Regulatory Mechanisms in Human Submandibular Glands.

    PubMed

    Namkoong, Eun; Shin, Yong-Hwan; Bae, Jun-Seok; Choi, Seulki; Kim, Minkyoung; Kim, Nahyun; Hwang, Sung-Min; Park, Kyungpyo

    2015-01-01

    Sodium bicarbonate cotransporters (NBCs) are involved in the pH regulation of salivary glands. However, the roles and regulatory mechanisms among different NBC isotypes have not been rigorously evaluated. We investigated the roles of two different types of NBCs, electroneutral (NBCn1) and electrogenic NBC (NBCe1), with respect to pH regulation and regulatory mechanisms using human submandibular glands (hSMGs) and HSG cells. Intracellular pH (pHi) was measured and the pHi recovery rate from cell acidification induced by an NH4Cl pulse was recorded. Subcellular localization and protein phosphorylation were determined using immunohistochemistry and co-immunoprecipitation techniques. We determined that NBCn1 is expressed on the basolateral side of acinar cells and the apical side of duct cells, while NBCe1 is exclusively expressed on the apical membrane of duct cells. The pHi recovery rate in hSMG acinar cells, which only express NBCn1, was not affected by pre-incubation with 5 μM PP2, an Src tyrosine kinase inhibitor. However, in HSG cells, which express both NBCe1 and NBCn1, the pHi recovery rate was inhibited by PP2. The apparent difference in regulatory mechanisms for NBCn1 and NBCe1 was evaluated by artificial overexpression of NBCn1 or NBCe1 in HSG cells, which revealed that the pHi recovery rate was only inhibited by PP2 in cells overexpressing NBCe1. Furthermore, only NBCe1 was significantly phosphorylated and translocated by NH4Cl, which was inhibited by PP2. Our results suggest that both NBCn1 and NBCe1 play a role in pHi regulation in hSMG acinar cells, and also that Src kinase does not regulate the activity of NBCn1. PMID:26375462

  5. Role of Sodium Bicarbonate Cotransporters in Intracellular pH Regulation and Their Regulatory Mechanisms in Human Submandibular Glands

    PubMed Central

    Namkoong, Eun; Shin, Yong-Hwan; Bae, Jun-Seok; Choi, Seulki; Kim, Minkyoung; Kim, Nahyun; Hwang, Sung-Min; Park, Kyungpyo

    2015-01-01

    Sodium bicarbonate cotransporters (NBCs) are involved in the pH regulation of salivary glands. However, the roles and regulatory mechanisms among different NBC isotypes have not been rigorously evaluated. We investigated the roles of two different types of NBCs, electroneutral (NBCn1) and electrogenic NBC (NBCe1), with respect to pH regulation and regulatory mechanisms using human submandibular glands (hSMGs) and HSG cells. Intracellular pH (pHi) was measured and the pHi recovery rate from cell acidification induced by an NH4Cl pulse was recorded. Subcellular localization and protein phosphorylation were determined using immunohistochemistry and co-immunoprecipitation techniques. We determined that NBCn1 is expressed on the basolateral side of acinar cells and the apical side of duct cells, while NBCe1 is exclusively expressed on the apical membrane of duct cells. The pHi recovery rate in hSMG acinar cells, which only express NBCn1, was not affected by pre-incubation with 5 μM PP2, an Src tyrosine kinase inhibitor. However, in HSG cells, which express both NBCe1 and NBCn1, the pHi recovery rate was inhibited by PP2. The apparent difference in regulatory mechanisms for NBCn1 and NBCe1 was evaluated by artificial overexpression of NBCn1 or NBCe1 in HSG cells, which revealed that the pHi recovery rate was only inhibited by PP2 in cells overexpressing NBCe1. Furthermore, only NBCe1 was significantly phosphorylated and translocated by NH4Cl, which was inhibited by PP2. Our results suggest that both NBCn1 and NBCe1 play a role in pHi regulation in hSMG acinar cells, and also that Src kinase does not regulate the activity of NBCn1. PMID:26375462

  6. Survival in a recent cohort of mechanically ventilated pediatric allogeneic hematopoietic stem cell transplantation recipients.

    PubMed

    van Gestel, Josephus P J; Bollen, Casper W; Bierings, Marc B; Boelens, Jaap Jan; Wulffraat, Nico M; van Vught, Adrianus J

    2008-12-01

    There is ongoing discussion whether survival improved for children requiring mechanical ventilation after hematopoietic stem cell transplantation (HSCT). We reviewed the outcomes of 150 children who received an allogeneic HSCT between January 1999 and April 2007, in a pediatric university hospital in The Netherlands. Thirty-five of the 150 patients received mechanical ventilation on 38 occasions. None of the recorded risk factors was significantly associated with the requirement of mechanical ventilation. Sixteen admissions resulted in death in the intensive care unit (ICU), giving a case fatality rate of 42% (95% confidence interval 26%-58%). ICU mortality was associated with multiorgan failure on the second day of admission and with the use of high frequency oscillatory ventilation. Patients had higher pediatric risk of mortality scores than in previous studies, reflecting higher acuity of illness on admission to the ICU. Six-month survival in patients discharged from the ICU was 82%. Compared to previous studies, we found an improvement in ICU survival and survival 6 months after ICU discharge in a recent cohort of ventilated children after allogeneic HSCT, even though our patients were more severely ill. Our results are promising, but they need to be confirmed in larger, preferably multicenter, studies. PMID:19041061

  7. BDNF-stimulated intracellular signalling mechanisms underlie exercise-induced improvement in spatial memory in the male Wistar rat.

    PubMed

    Bechara, Ranya G; Lyne, Ronan; Kelly, Áine M

    2014-12-15

    Exercise-induced improvements in learning are associated with neurotrophic and neurogenic changes in the dentate gyrus, but the intracellular signalling mechanisms that may mediate these improvements remain unknown. In the current study we investigate the effects of one week of forced exercise on spatial memory and analyse in parallel BDNF-stimulated signalling pathways in cells of the dentate gyrus. Additionally, we test whether a single intracerebroventricular (i.c.v.) injection of BDNF can mimic the observed cognitive and signalling changes. Male Wistar rats were assigned to exercised and sedentary groups and tested in a spatial task post-exercise. Tissue from the dentate gyrus was assessed for expression and release of BDNF, and for changes in expression and activation of TrkB, ERK and synapsin-1. In a separate set of experiments, male Wistar rats received a single i.c.v. injection of BDNF and were then tested in the same spatial learning task. Exercised and BDNF-treated (but not control) rats could successfully complete an object displacement task that tests spatial learning. Exercised rats and BDNF-treated rats displayed increases BDNF expression and ERK1 activation, while exercised rats showed increases in cell division, stimulated BDNF release, TrkB activation, and synapsin-1 expression in the dentate gyrus. We conclude that exercise-induced increases in BDNF in the dentate gyrus are sufficient to cause improvements in spatial memory by activating signalling cascades that enhance synaptic transmission in the hippocampus. PMID:24269499

  8. Regulation of OGT by URI in Response to Glucose Confers c-MYC-Dependent Survival Mechanisms.

    PubMed

    Burén, Stefan; Gomes, Ana L; Teijeiro, Ana; Fawal, Mohamad-Ali; Yilmaz, Mahmut; Tummala, Krishna S; Perez, Manuel; Rodriguez-Justo, Manuel; Campos-Olivas, Ramón; Megías, Diego; Djouder, Nabil

    2016-08-01

    Cancer cells can adapt and survive under low nutrient conditions, but underlying mechanisms remain poorly explored. We demonstrate here that glucose maintains a functional complex between the co-chaperone URI, PP1γ, and OGT, the enzyme catalyzing O-GlcNAcylation. Glucose deprivation induces the activation of PKA, which phosphorylates URI at Ser-371, resulting in PP1γ release and URI-mediated OGT inhibition. Low OGT activity reduces O-GlcNAcylation and promotes c-MYC degradation to maintain cell survival. In the presence of glucose, PP1γ-bound URI increases OGT and c-MYC levels. Accordingly, mice expressing non-phosphorylatable URI (S371A) in hepatocytes exhibit high OGT activity and c-MYC stabilization, accelerating liver tumorigenesis in agreement with c-MYC oncogenic functions. Our work uncovers that URI-regulated OGT confers c-MYC-dependent survival functions in response to glucose fluctuations. PMID:27505673

  9. Longest Event-Free Survival without Anticoagulation in a Mechanical Aortic Valve Replacement

    PubMed Central

    Salmane, Chadi; Pandya, Bhavi; Lafferty, Kristen; Patel, Nileshkumar J; McCord, Donald

    2016-01-01

    Sixty percent of the patients going for valve replacement opt for mechanical valves and the remaining 40% choose bioprosthetics. Mechanical valves are known to have a higher risk of thrombosis; this risk further varies depending on the type of valve, its position, and certain individual factors. According to current guidelines, long-term anticoagulation is indicated in patients with metallic prosthetic valve disease. We report two unique cases of patients who survived 27 and 37 years event free, respectively, after mechanical aortic valve replacement (AVR) without being on any form of anticoagulation. The latter case described the longest survival in a human with a prosthetic aortic valve without anticoagulation. A review of literature demonstrated few cases of prosthetic valves with no anticoagulation in the long term without significant embolic events reported as case reports. These cases have been summarized in this article. Some cases of long-term survival (in the absence of anticoagulation) were attributed to good luck, and others as the result of genetic variations. New mechanical prosthetic valves can be promising, such as microporus-surfaced valves that may be used without full anticoagulation. The use of dual antiplatelet agents alone can be currently recommended only when a patient cannot take oral anticoagulation after AVR, and it should be followed with measuring and monitoring of platelet reactivity. PMID:27053922

  10. Longest Event-Free Survival without Anticoagulation in a Mechanical Aortic Valve Replacement.

    PubMed

    Salmane, Chadi; Pandya, Bhavi; Lafferty, Kristen; Patel, Nileshkumar J; McCord, Donald

    2016-01-01

    Sixty percent of the patients going for valve replacement opt for mechanical valves and the remaining 40% choose bioprosthetics. Mechanical valves are known to have a higher risk of thrombosis; this risk further varies depending on the type of valve, its position, and certain individual factors. According to current guidelines, long-term anticoagulation is indicated in patients with metallic prosthetic valve disease. We report two unique cases of patients who survived 27 and 37 years event free, respectively, after mechanical aortic valve replacement (AVR) without being on any form of anticoagulation. The latter case described the longest survival in a human with a prosthetic aortic valve without anticoagulation. A review of literature demonstrated few cases of prosthetic valves with no anticoagulation in the long term without significant embolic events reported as case reports. These cases have been summarized in this article. Some cases of long-term survival (in the absence of anticoagulation) were attributed to good luck, and others as the result of genetic variations. New mechanical prosthetic valves can be promising, such as microporus-surfaced valves that may be used without full anticoagulation. The use of dual antiplatelet agents alone can be currently recommended only when a patient cannot take oral anticoagulation after AVR, and it should be followed with measuring and monitoring of platelet reactivity. PMID:27053922

  11. Mathematical Modeling of Therapy-induced Cancer Drug Resistance: Connecting Cancer Mechanisms to Population Survival Rates.

    PubMed

    Sun, Xiaoqiang; Bao, Jiguang; Shao, Yongzhao

    2016-01-01

    Drug resistance significantly limits the long-term effectiveness of targeted therapeutics for cancer patients. Recent experimental studies have demonstrated that cancer cell heterogeneity and microenvironment adaptations to targeted therapy play important roles in promoting the rapid acquisition of drug resistance and in increasing cancer metastasis. The systematic development of effective therapeutics to overcome drug resistance mechanisms poses a major challenge. In this study, we used a modeling approach to connect cellular mechanisms underlying cancer drug resistance to population-level patient survival. To predict progression-free survival in cancer patients with metastatic melanoma, we developed a set of stochastic differential equations to describe the dynamics of heterogeneous cell populations while taking into account micro-environment adaptations. Clinical data on survival and circulating tumor cell DNA (ctDNA) concentrations were used to confirm the effectiveness of our model. Moreover, our model predicted distinct patterns of dose-dependent synergy when evaluating a combination of BRAF and MEK inhibitors versus a combination of BRAF and PI3K inhibitors. These predictions were consistent with the findings in previously reported studies. The impact of the drug metabolism rate on patient survival was also discussed. The proposed model might facilitate the quantitative evaluation and optimization of combination therapeutics and cancer clinical trial design. PMID:26928089

  12. Mathematical Modeling of Therapy-induced Cancer Drug Resistance: Connecting Cancer Mechanisms to Population Survival Rates

    PubMed Central

    Sun, Xiaoqiang; Bao, Jiguang; Shao, Yongzhao

    2016-01-01

    Drug resistance significantly limits the long-term effectiveness of targeted therapeutics for cancer patients. Recent experimental studies have demonstrated that cancer cell heterogeneity and microenvironment adaptations to targeted therapy play important roles in promoting the rapid acquisition of drug resistance and in increasing cancer metastasis. The systematic development of effective therapeutics to overcome drug resistance mechanisms poses a major challenge. In this study, we used a modeling approach to connect cellular mechanisms underlying cancer drug resistance to population-level patient survival. To predict progression-free survival in cancer patients with metastatic melanoma, we developed a set of stochastic differential equations to describe the dynamics of heterogeneous cell populations while taking into account micro-environment adaptations. Clinical data on survival and circulating tumor cell DNA (ctDNA) concentrations were used to confirm the effectiveness of our model. Moreover, our model predicted distinct patterns of dose-dependent synergy when evaluating a combination of BRAF and MEK inhibitors versus a combination of BRAF and PI3K inhibitors. These predictions were consistent with the findings in previously reported studies. The impact of the drug metabolism rate on patient survival was also discussed. The proposed model might facilitate the quantitative evaluation and optimization of combination therapeutics and cancer clinical trial design. PMID:26928089

  13. Dual actions of sphingosine-1-phosphate: extracellular through the Gi-coupled receptor Edg-1 and intracellular to regulate proliferation and survival.

    PubMed

    Van Brocklyn, J R; Lee, M J; Menzeleev, R; Olivera, A; Edsall, L; Cuvillier, O; Thomas, D M; Coopman, P J; Thangada, S; Liu, C H; Hla, T; Spiegel, S

    1998-07-13

    Sphingosine-1-phosphate (SPP), a bioactive lipid, acts both intracellularly and extracellularly to cause pleiotropic biological responses. Recently, we identified SPP as a ligand for the G protein-coupled receptor Edg-1 (Lee, M.-J., J.R. Van Brocklyn, S. Thangada, C.H. Liu, A.R. Hand, R. Menzeleev, S. Spiegel, and T. Hla. 1998. Science. 279:1552-1555). Edg-1 binds SPP with remarkable specificity as only sphinganine-1-phosphate displaced radiolabeled SPP, while other sphingolipids did not. Binding of SPP to Edg-1 resulted in inhibition of forskolin-stimulated cAMP accumulation, in a pertussis toxin-sensitive manner. In contrast, two well-characterized biological responses of SPP, mitogenesis and prevention of apoptosis, were clearly unrelated to binding to Edg-1 and correlated with intracellular uptake. SPP also stimulated signal transduction pathways, including calcium mobilization, activation of phospholipase D, and tyrosine phosphorylation of p125(FAK), independently of edg-1 expression. Moreover, DNA synthesis in Swiss 3T3 fibroblasts was significantly and specifically increased by microinjection of SPP. Finally, SPP suppresses apoptosis of HL-60 and pheochromocytoma PC12 cells, which do not have specific SPP binding or expression of Edg-1 mRNA. Conversely, sphinganine-1-phosphate, which binds to and signals via Edg-1, does not have any significant cytoprotective effect. Thus, SPP is a prototype for a novel class of lipid mediators that act both extracellularly as ligands for cell surface receptors and intracellularly as second messengers. PMID:9660876

  14. Intracellular proteoglycans.

    PubMed Central

    Kolset, Svein Olav; Prydz, Kristian; Pejler, Gunnar

    2004-01-01

    Proteoglycans (PGs) are proteins with glycosaminoglycan chains, are ubiquitously expressed and have a wide range of functions. PGs in the extracellular matrix and on the cell surface have been the subject of extensive structural and functional studies. Less attention has so far been given to PGs located in intracellular compartments, although several reports suggest that these have biological functions in storage granules, the nucleus and other intracellular organelles. The purpose of this review is, therefore, to present some of these studies and to discuss possible functions linked to PGs located in different intracellular compartments. Reference will be made to publications relevant for the topics we present. It is beyond the scope of this review to cover all publications on PGs in intracellular locations. PMID:14759226

  15. A novel mechanism for a survival advantage of vigilant individuals in groups.

    PubMed

    van der Post, Daniel J; de Weerd, Harmen; Verbrugge, Rineke; Hemelrijk, Charlotte K

    2013-11-01

    In many animal species, vigilance is crucial for avoiding predation. In groups, however, nonvigilant individuals could benefit from the vigilance of others without any of the associated costs. In an evolutionary sense, such exploitation may be compensated if vigilant individuals have a survival advantage. The novelty in our model is that the probability to detect a predator is "distance dependent." We show that even if nonvigilant individuals benefit fully from information produced by vigilant individuals, vigilant individuals nevertheless enjoy a survival advantage. This happens because detection of predators is more likely when vigilant individuals happen to be targets of predation. We expect this distance-dependent mechanism to be compatible with previously reported mechanisms. PMID:24107375

  16. A Functional Selectivity Mechanism at the Serotonin-2A GPCR Involves Ligand-Dependent Conformations of Intracellular Loop 2

    PubMed Central

    2014-01-01

    With recent progress in determination of G protein-coupled receptor (GPCR) structure with crystallography, a variety of other experimental approaches (e.g., NMR spectroscopy, fluorescent-based assays, mass spectrometry techniques) are also being used to characterize state-specific and ligand-specific conformational states. MD simulations offer a powerful complementary approach to elucidate the dynamic features associated with ligand-specific GPCR conformations. To shed light on the conformational elements and dynamics of the important aspect of GPCR functional selectivity, we carried out unbiased microsecond-length MD simulations of the human serotonin 2A receptor (5-HT2AR) in the absence of ligand and bound to four distinct serotonergic agonists. The 5-HT2AR is a suitable system to study the structural features involved in the ligand-dependent conformational heterogeneity of GPCRs because it is well-characterized experimentally and exhibits a strong agonist-specific phenotype in that some 5-HT2AR agonists induce LSD-like hallucinations, while others lack this psychoactive property entirely. Here we report evidence for structural and dynamic differences in 5-HT2AR interacting with such pharmacologically distinct ligands, hallucinogens, and nonhallucinogens obtained from all-atom MD simulations. Differential ligand binding contacts were identified for structurally similar hallucinogens and nonhallucinogens and found to correspond to different conformations in the intracellular loop 2 (ICL2). From the different ICL2 conformations, functional selective phenotypes are suggested through effects on dimerization and/or distinct direct interaction with effector proteins. The findings are presented in the context of currently proposed hallucinogenesis mechanisms, and ICL2 is proposed as a fine-tuning selective switch that can differentiates modes of 5-HT2AR activation. PMID:25314362

  17. The abcEDCBA-Encoded ABC Transporter and the virB Operon-Encoded Type IV Secretion System of Brucella ovis Are Critical for Intracellular Trafficking and Survival in Ovine Monocyte-Derived Macrophages

    PubMed Central

    Macedo, Auricelio A.; Silva, Ana P. C.; Mol, Juliana P. S.; Costa, Luciana F.; Garcia, Luize N. N.; Araújo, Marcio S.; Martins Filho, Olindo A.; Paixão, Tatiane A.; Santos, Renato L.

    2015-01-01

    Brucella ovis infection is associated with epididymitis, orchitis and infertility in rams. Most of the information available on B. ovis and host cell interaction has been generated using murine macrophages or epithelial cell lines, but the interaction between B. ovis and primary ovine macrophages has not been studied. The aim of this study was to evaluate the role of the B. ovis abcEDCBA-encoded ABC transporter and the virB operon-encoded Type IV Secretion System (T4SS) during intracellular survival of B. ovis in ovine peripheral blood monocyte-derived macrophages. ΔabcBA and ΔvirB2 mutant strains were unable to survive in the intracellular environment when compared to the WT B. ovis at 48 hours post infection (hpi). In addition, these mutant strains cannot exclude the lysosomal marker LAMP1 from its vacuolar membrane, and their vacuoles do not acquire the endoplasmic reticulum marker calreticulin, which takes place in the WT B. ovis containing vacuole. Higher levels of nitric oxide production were observed in macrophages infected with WT B. ovis at 48 hpi when compared to macrophages infected with the ΔabcBA or ΔvirB2 mutant strains. Conversely, higher levels of reactive oxygen species were detected in macrophages infected with the ΔabcBA or ΔvirB2 mutant strains at 48 hpi when compared to macrophages infected with the WT strain. Our results demonstrate that B. ovis is able to persist and multiply in ovine macrophages, while ΔabcBA and ΔvirB2 mutations prevent intracellular multiplication, favor phagolysosome fusion, and impair maturation of the B. ovis vacuole towards an endoplasmic reticulum-derived compartment. PMID:26366863

  18. Histoplasma capsulatum surmounts obstacles to intracellular pathogenesis.

    PubMed

    Garfoot, Andrew L; Rappleye, Chad A

    2016-02-01

    The fungal pathogen Histoplasma capsulatum causes respiratory and disseminated disease, even in immunocompetent hosts. In contrast to opportunistic pathogens, which are readily controlled by phagocytic cells, H. capsulatum yeasts are able to infect macrophages, survive antimicrobial defenses, and proliferate as an intracellular pathogen. In this review, we discuss some of the molecular mechanisms that enable H. capsulatum yeasts to overcome obstacles to intracellular pathogenesis. H. capsulatum yeasts gain refuge from extracellular obstacles such as antimicrobial lung surfactant proteins by engaging the β-integrin family of phagocytic receptors to promote entry into macrophages. In addition, H. capsulatum yeasts conceal immunostimulatory β-glucans to avoid triggering signaling receptors such as the β-glucan receptor Dectin-1. H. capsulatum yeasts counteract phagocyte-produced reactive oxygen species by expression of oxidative stress defense enzymes including an extracellular superoxide dismutase and an extracellular catalase. Within the phagosome, H. capsulatum yeasts block phagosome acidification, acquire essential metals such as iron and zinc, and utilize de novo biosynthesis pathways to overcome nutritional limitations. These mechanisms explain how H. capsulatum yeasts avoid and negate macrophage defense strategies and establish a hospitable intracellular niche, making H. capsulatum a successful intracellular pathogen of macrophages. PMID:26235362

  19. Modeling the effects of sodium chloride, acetic acid and intracellular pH on the survival of Escherichia coli O157:H7

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Microbiological safety has been a critical issue for acid and acidified foods since it became clear that acid-tolerant pathogens such as Escherichia coli O157:H7 can survive (even though they are unable to grow) in a pH range of 3 to 4, which is typical for these classes of food products. The primar...

  20. tPA promotes cortical neuron survival via mTOR-dependent mechanisms.

    PubMed

    Grummisch, Julia A; Jadavji, Nafisa M; Smith, Patrice D

    2016-07-01

    Tissue plasminogen activator (tPA) is a thrombolytic agent commonly used in the treatment of ischemic stroke. While the thrombolytic effects of tPA have been well established, the impact of this blood-brain barrier (BBB) crossing drug on neurons is not known. Given the widespread use of tPA in the clinical setting and the strict therapeutic window established for effective use of the drug, we examined the molecular mechanisms mediating the impact of tPA on postnatal cortical neurons isolated from the mouse brain. Dissociated postnatal primary cortical neurons were treated with tPA and the effects on neuron survival were evaluated. Pharmacological inhibitors of several signaling pathways previously implicated in neuroprotection (mTOR, JAK/STAT, MAPK and PKA-dependent mechanisms) were used to pinpoint the mechanistic effectors of tPA on neuron survival in vitro. We report here that tPA treatment results in a time-dependent neuroprotective effect on postnatal cortical neurons that relies predominantly on Janus kinase (JAK) and mammalian target of rapamycin (mTOR) signaling mechanisms. Taken together, these data suggest that tPA promotes neuroprotection in a temporally-regulated manner and that both JAK and mTOR signaling effectors are critical mediators of this neuroprotective effect. The results suggest the possibility of targeting these defined mechanisms to potentially expand the therapeutic window for tPA. PMID:26995507

  1. Hijacking and Use of Host Lipids by Intracellular Pathogens.

    PubMed

    Toledo, Alvaro; Benach, Jorge L

    2015-12-01

    Intracellular bacteria use a number of strategies to survive, grow, multiply, and disseminate within the host. One of the most striking adaptations that intracellular pathogens have developed is the ability to utilize host lipids and their metabolism. Bacteria such as Anaplasma, Chlamydia, or Mycobacterium can use host lipids for different purposes, such as a means of entry through lipid rafts, building blocks for bacteria membrane formation, energy sources, camouflage to avoid the fusion of phagosomes and lysosomes, and dissemination. One of the most extreme examples of lipid exploitation is Mycobacterium, which not only utilizes the host lipid as a carbon and energy source but is also able to reprogram the host lipid metabolism. Likewise, Chlamydia spp. have also developed numerous mechanisms to reprogram lipids onto their intracellular inclusions. Finally, while the ability to exploit host lipids is important in intracellular bacteria, it is not an exclusive trait. Extracellular pathogens, including Helicobacter, Mycoplasma, and Borrelia, can recruit and metabolize host lipids that are important for their growth and survival.Throughout this chapter we will review how intracellular and extracellular bacterial pathogens utilize host lipids to enter, survive, multiply, and disseminate in the host. PMID:27337282

  2. Therapeutic potential of analogues of amiloride: inhibition of the regulation of intracellular pH as a possible mechanism of tumour selective therapy.

    PubMed Central

    Maidorn, R. P.; Cragoe, E. J.; Tannock, I. F.

    1993-01-01

    The extracellular pH (pHe) in solid tumours is frequently lower than the pHe in normal tissues. Cells within an acidic environment depend on mechanisms which regulate intracellular pH (pHi) for their survival, including the Na+/H+ antiport which exports protons in exchange for Na+ ions. Amiloride and its analogues DMA (5-(N,N-dimethyl)amiloride), MIBA (5-(N-methyl-N-isobutyl)amiloride) and EIPA (5-(N-ethyl-N-isopropyl)amiloride) are known to inhibit the Na+/H+ antiport and therefore decrease the cells ability to regulate pHi. All three analogues were found to be potent inhibitors of the antiport in human MGH-U1 and murine EMT-6 cells, with DMA being approximately 20, MIBA 100 and EIPA 200-fold as potent as amiloride; EIPA also gave more complete suppression of the Na+/H+ antiport. These agents were not toxic to cells when used alone; however, in combination with nigericin, an agent which acidifies cells, all three analogues were toxic to cells at pHe < 7.0, and markedly enhanced the toxicity of nigericin alone. Cell killing was greatest for nigericin used with EIPA or MIBA. None of the agents were toxic to cells at pHe 7.0 or above. When used against variant cells lacking the Na+/H+ antiport (PS-120 cells) EIPA did not enhance the cytotoxicity of nigericin alone, suggesting that the observed effect was due to inhibition of Na+/H+ exchange, rather than due to non-specific effects. The combination of EIPA and nigericin gave similar cell killing in previously dissociated and intact MGH-U1 spheroids, suggesting that the agents have good penetration of solid tissue. Preliminary experiments using EMT-6 tumours in mice suggested that EIPA and nigericin were able to enhance the toxicity of radiation in vivo, presumably through selective effects against the hypoxic (and probably acidic) subpopulation of cells that is resistant to radiation. PMID:8381657

  3. Mechanisms of survival, responses and sources of Salmonella in low-moisture environments

    PubMed Central

    Finn, Sarah; Condell, Orla; McClure, Peter; Amézquita, Alejandro; Fanning, Séamus

    2013-01-01

    Some Enterobacteriaceae possess the ability to survive in low-moisture environments for extended periods of time. Many of the reported food-borne outbreaks associated with low-moisture foods involve Salmonella contamination. The control of Salmonella in low-moisture foods and their production environments represents a significant challenge for all food manufacturers. This review summarizes the current state of knowledge with respect to Salmonella survival in intermediate- and low-moisture food matrices and their production environments. The mechanisms utilized by this bacterium to ensure their survival in these dry conditions remain to be fully elucidated, however, in depth transcriptomic data is now beginning to emerge regarding this observation. Earlier research work described the effect(s) that low-moisture can exert on the long-term persistence and heat tolerance of Salmonella, however, data are also now available highlighting the potential cross-tolerance to other stressors including commonly used microbicidal agents. Sources and potential control measures to reduce the risk of contamination will be explored. By extending our understanding of these geno- and phenotypes, we may be able to exploit them to improve food safety and protect public health. PMID:24294212

  4. Mechanisms of survival, responses and sources of Salmonella in low-moisture environments.

    PubMed

    Finn, Sarah; Condell, Orla; McClure, Peter; Amézquita, Alejandro; Fanning, Séamus

    2013-01-01

    Some Enterobacteriaceae possess the ability to survive in low-moisture environments for extended periods of time. Many of the reported food-borne outbreaks associated with low-moisture foods involve Salmonella contamination. The control of Salmonella in low-moisture foods and their production environments represents a significant challenge for all food manufacturers. This review summarizes the current state of knowledge with respect to Salmonella survival in intermediate- and low-moisture food matrices and their production environments. The mechanisms utilized by this bacterium to ensure their survival in these dry conditions remain to be fully elucidated, however, in depth transcriptomic data is now beginning to emerge regarding this observation. Earlier research work described the effect(s) that low-moisture can exert on the long-term persistence and heat tolerance of Salmonella, however, data are also now available highlighting the potential cross-tolerance to other stressors including commonly used microbicidal agents. Sources and potential control measures to reduce the risk of contamination will be explored. By extending our understanding of these geno- and phenotypes, we may be able to exploit them to improve food safety and protect public health. PMID:24294212

  5. Intracellular signalling mechanism responsible for modulation of sarcolemmal ATP-sensitive potassium channels by nitric oxide in ventricular cardiomyocytes.

    PubMed

    Zhang, Dai-Min; Chai, Yongping; Erickson, Jeffrey R; Brown, Joan Heller; Bers, Donald M; Lin, Yu-Fung

    2014-03-01

    The ATP-sensitive potassium (KATP) channels are crucial for stress adaptation in the heart. It has previously been suggested that the function of KATP channels is modulated by nitric oxide (NO), a gaseous messenger known to be cytoprotective; however, the underlying mechanism remains poorly understood. Here we sought to delineate the intracellular signalling mechanism responsible for NO modulation of sarcolemmal KATP (sarcKATP) channels in ventricular cardiomyocytes. Cell-attached patch recordings were performed in transfected human embryonic kidney (HEK) 293 cells and ventricular cardiomyocytes freshly isolated from adult rabbits or genetically modified mice, in combination with pharmacological and biochemical approaches. Bath application of the NO donor NOC-18 increased the single-channel activity of Kir6.2/SUR2A (i.e., the principal ventricular-type KATP) channels in HEK293 cells, whereas the increase was abated by KT5823 [a selective cGMP-dependent protein kinase (PKG) inhibitor], mercaptopropionyl glycine [MPG; a reactive oxygen species (ROS) scavenger], catalase (an H2O2-degrading enzyme), myristoylated autocamtide-2 related inhibitory peptide (mAIP) selective for Ca2+ / calmodulin-dependent protein kinase II (CaMKII) and U0126 [an extracellular signal-regulated protein kinase 1/2 (ERK1/2) inhibitor], respectively. The NO donors NOC-18 and N-(2-deoxy-α,β-d-glucopyranose-2-)-N2-acetyl-S-nitroso-d,l-penicillaminamide (glycol-SNAP-2) were also capable of stimulating native sarcKATP channels preactivated by the channel opener pinacidil in rabbit ventricular myocytes, through reducing the occurrence and the dwelling time of the long closed states whilst increasing the frequency of channel opening; in contrast, all these changes were reversed in the presence of inhibitors selective for soluble guanylyl cyclase (sGC), PKG, calmodulin, CaMKII or ERK1/2. Mimicking the action of NO donors, exogenous H2O2 potentiated pinacidil-preactivated sarcKATP channel activity in

  6. Intracellular signalling mechanism responsible for modulation of sarcolemmal ATP-sensitive potassium channels by nitric oxide in ventricular cardiomyocytes

    PubMed Central

    Zhang, Dai-Min; Chai, Yongping; Erickson, Jeffrey R; Brown, Joan Heller; Bers, Donald M; Lin, Yu-Fung

    2014-01-01

    The ATP-sensitive potassium (KATP) channels are crucial for stress adaptation in the heart. It has previously been suggested that the function of KATP channels is modulated by nitric oxide (NO), a gaseous messenger known to be cytoprotective; however, the underlying mechanism remains poorly understood. Here we sought to delineate the intracellular signalling mechanism responsible for NO modulation of sarcolemmal KATP (sarcKATP) channels in ventricular cardiomyocytes. Cell-attached patch recordings were performed in transfected human embryonic kidney (HEK) 293 cells and ventricular cardiomyocytes freshly isolated from adult rabbits or genetically modified mice, in combination with pharmacological and biochemical approaches. Bath application of the NO donor NOC-18 increased the single-channel activity of Kir6.2/SUR2A (i.e. the principal ventricular-type KATP) channels in HEK293 cells, whereas the increase was abated by KT5823 [a selective cGMP-dependent protein kinase (PKG) inhibitor], mercaptopropionyl glycine [MPG; a reactive oxygen species (ROS) scavenger], catalase (an H2O2-degrading enzyme), myristoylated autocamtide-2 related inhibitory peptide (mAIP) selective for Ca2+/calmodulin-dependent protein kinase II (CaMKII) and U0126 [an extracellular signal-regulated protein kinase 1/2 (ERK1/2) inhibitor], respectively. The NO donors NOC-18 and N-(2-deoxy-α,β-d-glucopyranose-2-)-N2-acetyl-S-nitroso-d,l-penicillaminamide (glycol-SNAP-2) were also capable of stimulating native sarcKATP channels preactivated by the channel opener pinacidil in rabbit ventricular myocytes, through reducing the occurrence and the dwelling time of the long closed states whilst increasing the frequency of channel opening; in contrast, all these changes were reversed in the presence of inhibitors selective for soluble guanylyl cyclase (sGC), PKG, calmodulin, CaMKII or ERK1/2. Mimicking the action of NO donors, exogenous H2O2 potentiated pinacidil-preactivated sarcKATP channel activity in

  7. In-Situ Survival Mechanisms of U and Tc Reducing Bacteria in Contaminated Sediments

    SciTech Connect

    Krumholz, Lee R.

    2005-06-01

    Desulfovibrio desulfuricans G20 and Shewanella oneidensis MR-1 are model subsurface organisms for studying genes involving in situ radionuclide transformation and sediment survival. Our research objective for this project has been to develop a signature-tagged mutagenesis (STM) procedure and use it to identify mutants in genes of these subsurface bacteria involved in sediment survival and radionuclide reduction. The mutant genes identified in these studies allow us for the first time to describe at the genetic level microbial processes that are actually being used by environmental bacteria while growing in their natural ecosystems. Identification of these genes revealed facets of microbial physiology and ecology that are not accessible through laboratory studies. Ultimately, this information may be used to optimize bioremediation or other engineered microbial processes. Furthermore, the identification of a mutant in a gene conferring multidrug resistance in strain MR-1 shows that this widespread mechanism of antibiotic resistance, likely has its origins as a mechanism of bacterial defense against naturally occurring toxins. Studies with D. desulfuricans G20: The STM procedure first involved generating a library of 5760 G20 mutants and screening for potential non-survivors in subsurface sediment microcosms. After two rounds of screening, a total of 117 mutants were confirmed to be true non-survivors. 97 transposon insertion regions have been sequenced to date. Upon further analysis of these mutants, we classified the sediment survival genes into COG functional categories. STM mutant insertions were located in genes encoding proteins related to metabolism (33%), cellular processes (42%), and information storage and processing (17%). We also noted 8% of STM mutants identified had insertions in genes for hypothetical proteins or unknown functions. Interestingly, at least 64 of these genes encode cytoplasmic proteins, 46 encode inner membrane proteins, and only 7 encode

  8. Single-cell mass cytometry reveals intracellular survival/proliferative signaling in FLT3-ITD-mutated AML stem/progenitor cells.

    PubMed

    Han, Lina; Qiu, Peng; Zeng, Zhihong; Jorgensen, Jeffrey L; Mak, Duncan H; Burks, Jared K; Schober, Wendy; McQueen, Teresa J; Cortes, Jorge; Tanner, Scott D; Roboz, Gail J; Kantarjian, Hagop M; Kornblau, Steven M; Guzman, Monica L; Andreeff, Michael; Konopleva, Marina

    2015-04-01

    Understanding the unique phenotypes and complex signaling pathways of leukemia stem cells (LSCs) will provide insights and druggable targets that can be used to eradicate acute myeloid leukemia (AML). Current work on AML LSCs is limited by the number of parameters that conventional flow cytometry (FCM) can analyze because of cell autofluorescence and fluorescent dye spectral overlap. Single-cell mass cytometry (CyTOF) substitutes rare earth elements for fluorophores to label antibodies, which allows measurements of up to 120 parameters in single cells without correction for spectral overlap. The aim of this study was the evaluation of intracellular signaling in antigen-defined stem/progenitor cell subsets in primary AML. CyTOF and conventional FCM yielded comparable results on LSC phenotypes defined by CD45, CD34, CD38, CD123, and CD99. Intracellular phosphoprotein responses to ex vivo cell signaling inhibitors and cytokine stimulation were assessed in myeloid leukemia cell lines and one primary AML sample. CyTOF and conventional FCM results were confirmed by western blotting. In the primary AML sample, we investigated the cell responses to ex vivo stimulation with stem cell factor and BEZ235-induced inhibition of PI3K and identified activation patterns in multiple PI3K downstream signaling pathways including p-4EBP1, p-AKT, and p-S6, particularly in CD34(+) subsets. We evaluated multiple signaling pathways in antigen-defined subpopulations in primary AML cells with FLT3-ITD mutations. The data demonstrated the heterogeneity of cell phenotype distribution and distinct patterns of signaling activation across AML samples and between AML and normal samples. The mTOR targets p-4EBP1 and p-S6 were exclusively found in FLT3-ITD stem/progenitor cells, but not in their normal counterparts, suggesting both as novel targets in FLT3 mutated AML. Our data suggest that CyTOF can identify functional signaling pathways in antigen-defined subpopulations in primary AML, which may

  9. Single-cell mass cytometry reveals intracellular survival/proliferative signaling in FLT3-ITD-mutated AML stem/progenitor cells

    PubMed Central

    Han, Lina; Qiu, Peng; Zeng, Zhihong; Jorgensen, Jeffrey L; Mak, Duncan H; Burks, Jared K; Schober, Wendy; McQueen, Teresa J; Cortes, Jorge; Tanner, Scott D; Roboz, Gail J; Kantarjian, Hagop M; Kantarjian, Hagop M; Kornblau, Steven M; Guzman, Monica L; Andreeff, Michael; Konopleva, Marina

    2015-01-01

    Understanding the unique phenotypes and complex signaling pathways of leukemia stem cells (LSCs) will provide insights and druggable targets that can be used to eradicate acute myeloid leukemia (AML). Current work on AML LSCs is limited by the number of parameters that conventional flow cytometry (FCM) can analyze because of cell autofluorescence and fluorescent dye spectral overlap. Single-cell mass cytometry (CyTOF) substitutes rare earth elements for fluorophores to label antibodies, which allows measurements of up to 120 parameters in single cells without correction for spectral overlap. The aim of this study was the evaluation of intracellular signaling in antigen-defined stem/progenitor cell subsets in primary AML. CyTOF and conventional FCM yielded comparable results on LSC phenotypes defined by CD45, CD34, CD38, CD123, and CD99. Intracellular phosphoprotein responses to ex vivo cell signaling inhibitors and cytokine stimulation were assessed in myeloid leukemia cell lines and one primary AML sample. CyTOF and conventional FCM results were confirmed by western blotting. In the primary AML sample, we investigated the cell responses to ex vivo stimulation with stem cell factor (SCF) and BEZ235-induced inhibition of PI3K and identified activation patterns in multiple PI3K downstream signaling pathways including p-4EBP1, p-AKT, and p-S6, particularly in CD34+ subsets. We evaluated multiple signaling pathways in antigen-defined subpopulations in primary AML cells with FLT3-ITD mutations. The data demontrated the heterogeneity of cell phenotype distribution and distinct patterns of signaling activation across AML samples and between AML and normal samples. The mTOR targets p-4EBP1 and p-S6 were exclusively found in FLT3-ITD stem/progenitor cells, but not in their normal counterparts, suggesting both as novel targets in FLT3 mutated AML. Our data suggest that CyTOF can identify functional signaling pathways in antigen-defined subpopulations in primary AML, which

  10. Disruption of the intracellular Ca{sup 2+} homeostasis in the cardiac excitation-contraction coupling is a crucial mechanism of arrhythmic toxicity in aconitine-induced cardiomyocytes

    SciTech Connect

    Fu Min; Wu Meng; Wang Jifeng; Qiao Yanjiang; Wang Zhao . E-mail: zwang@tsinghua.edu.cn

    2007-03-23

    Aconitine is an effective ingredient in Aconite tuber, an important traditional Chinese medicine. Aconitine is also known to be a highly toxic diterpenoid alkaloid with arrhythmogenic effects. In the present study, we have characterized the properties of arrhythmic cytotoxicity and explored the possible mechanisms of aconitine-induced cardiomyocytes. Results show that aconitine induces significant abnormity in the spontaneous beating rate, amplitude of spontaneous oscillations and the relative intracellular Ca{sup 2+} concentration. Also, mRNA transcription levels and protein expressions of SR Ca{sup 2+} release channel RyR{sub 2} and sarcolemmal NCX were elevated in aconitine-induced cardiomyocytes. However, co-treatment with ruthenium red (RR), a RyR channel inhibitor, could reverse the aconitine-induced abnormity in intracellular Ca{sup 2+} signals. These results demonstrate that disruption of intracellular Ca{sup 2+} homeostasis in the cardiac excitation-contraction coupling (EC coupling) is a crucial mechanism of arrhythmic cytotoxicity in aconitine-induced cardiomyocytes. Moreover, certain inhibitors appear to play an important role in the detoxification of aconitine-induced Ca{sup 2+}-dependent arrhythmias.

  11. Autophagic clearance of bacterial pathogens: molecular recognition of intracellular microorganisms

    PubMed Central

    Mansilla Pareja, Maria Eugenia; Colombo, Maria I.

    2013-01-01

    Autophagy is involved in several physiological and pathological processes. One of the key roles of the autophagic pathway is to participate in the first line of defense against the invasion of pathogens, as part of the innate immune response. Targeting of intracellular bacteria by the autophagic machinery, either in the cytoplasm or within vacuolar compartments, helps to control bacterial proliferation in the host cell, controlling also the spreading of the infection. In this review we will describe the means used by diverse bacterial pathogens to survive intracellularly and how they are recognized by the autophagic molecular machinery, as well as the mechanisms used to avoid autophagic clearance. PMID:24137567

  12. Mobilization of Intracellular Copper by Gossypol and Apogossypolone Leads to Reactive Oxygen Species-Mediated Cell Death: Putative Anticancer Mechanism

    PubMed Central

    Zubair, Haseeb; Azim, Shafquat; Khan, Husain Yar; Ullah, Mohammad Fahad; Wu, Daocheng; Singh, Ajay Pratap; Hadi, Sheikh Mumtaz; Ahmad, Aamir

    2016-01-01

    There is compelling evidence that serum, tissue and intracellular levels of copper are elevated in all types of cancer. Copper has been suggested as an important co-factor for angiogenesis. It is also a major metal ion present inside the nucleus, bound to DNA bases, particularly guanine. We have earlier proposed that the interaction of phenolic-antioxidants with intracellular copper leads to the generation of reactive oxygen species (ROS) that ultimately serve as DNA cleaving agents. To further validate our hypothesis we show here that the antioxidant gossypol and its semi-synthetic derivative apogossypolone induce copper-mediated apoptosis in breast MDA-MB-231, prostate PC3 and pancreatic BxPC-3 cancer cells, through the generation of ROS. MCF10A breast epithelial cells refractory to the cytotoxic property of these compounds become sensitized to treatment against gossypol, as well as apogossypolone, when pre-incubated with copper. Our present results confirm our earlier findings and strengthen our hypothesis that plant-derived antioxidants mobilize intracellular copper instigating ROS-mediated cellular DNA breakage. As cancer cells exist under significant oxidative stress, this increase in ROS-stress to cytotoxic levels could be a successful anticancer approach. PMID:27331811

  13. Mobilization of Intracellular Copper by Gossypol and Apogossypolone Leads to Reactive Oxygen Species-Mediated Cell Death: Putative Anticancer Mechanism.

    PubMed

    Zubair, Haseeb; Azim, Shafquat; Khan, Husain Yar; Ullah, Mohammad Fahad; Wu, Daocheng; Singh, Ajay Pratap; Hadi, Sheikh Mumtaz; Ahmad, Aamir

    2016-01-01

    There is compelling evidence that serum, tissue and intracellular levels of copper are elevated in all types of cancer. Copper has been suggested as an important co-factor for angiogenesis. It is also a major metal ion present inside the nucleus, bound to DNA bases, particularly guanine. We have earlier proposed that the interaction of phenolic-antioxidants with intracellular copper leads to the generation of reactive oxygen species (ROS) that ultimately serve as DNA cleaving agents. To further validate our hypothesis we show here that the antioxidant gossypol and its semi-synthetic derivative apogossypolone induce copper-mediated apoptosis in breast MDA-MB-231, prostate PC3 and pancreatic BxPC-3 cancer cells, through the generation of ROS. MCF10A breast epithelial cells refractory to the cytotoxic property of these compounds become sensitized to treatment against gossypol, as well as apogossypolone, when pre-incubated with copper. Our present results confirm our earlier findings and strengthen our hypothesis that plant-derived antioxidants mobilize intracellular copper instigating ROS-mediated cellular DNA breakage. As cancer cells exist under significant oxidative stress, this increase in ROS-stress to cytotoxic levels could be a successful anticancer approach. PMID:27331811

  14. Endotoxin-stimulated Rat Hepatic Stellate Cells Induce Autophagy in Hepatocytes as a Survival Mechanism.

    PubMed

    Dangi, Anil; Huang, Chao; Tandon, Ashish; Stolz, Donna; Wu, Tong; Gandhi, Chandrashekhar R

    2016-01-01

    Bacterial lipopolysaccharide (LPS)-stimulated hepatic stellate cells (HSCs) produce many cytokines including IFNβ, TNFα, and IL6, strongly inhibit DNA synthesis, but induce apoptosis of a small number of hepatocytes. In vivo administration of LPS (up to 10 mg/mL) causes modest inflammation and weight loss in rats but not mortality. We determined whether LPS-stimulated HSCs instigate mechanisms of hepatocyte survival. Rats received 10 mg/kg LPS (i.p.) and determinations were made at 6 h. In vitro, HSCs were treated with 100 ng/mL LPS till 24 h. The medium was transferred to hepatocytes, and determinations were made at 0-12 h. Controls were HSC-conditioned medium or medium-containing LPS. LPS treatment of rats caused autophagy in hepatocytes, a physiological process for clearance of undesirable material including injured or damaged organelles. This was accompanied by activation of c-Jun NH2 terminal kinase (JNK) and apoptosis of ~4-5% of hepatocytes. In vitro, LPS-conditioned HSC medium (LPS/HSC) induced autophagy in hepatocytes but apoptosis of only ~10% of hepatocytes. While LPS/HSC stimulated activation of JNK (associated with cell death), it also activated NFkB and ERK1/2 (associated with cell survival). LPS-stimulated HSCs produced IFNβ, and LPS/HSC-induced autophagy in hepatocytes and their apoptosis were significantly inhibited by anti-IFNβ antibody. Blockade of autophagy, on the other hand, strongly augmented hepatocyte apoptosis. While LPS-stimulated HSCs cause apoptosis of a subpopulation of hepatocytes by producing IFNβ, they also induce cell survival mechanisms, which may be of critical importance in resistance to liver injury during endotoxemia. PMID:26031389

  15. Desiccation as a Long-Term Survival Mechanism for the Archaeon Methanosarcina barkeri

    PubMed Central

    Anderson, Kimberly L.; Apolinario, Ethel E.

    2012-01-01

    Viable methanogens have been detected in dry, aerobic environments such as dry reservoir sediment, dry rice paddies and aerobic desert soils, which suggests that methanogens have mechanisms for long-term survival in a desiccated state. In this study, we quantified the survival rates of the methanogenic archaeon Methanosarcina barkeri after desiccation under conditions equivalent to the driest environments on Earth and subsequent exposure to different stress factors. There was no significant loss of viability after desiccation for 28 days for cells grown with either hydrogen or the methylotrophic substrates, but recovery was affected by growth phase, with cells desiccated during the stationary phase of growth having a higher rate of recovery after desiccation. Synthesis of methanosarcinal extracellular polysaccharide (EPS) significantly increased the viability of desiccated cells under both anaerobic and aerobic conditions compared with that of non-EPS-synthesizing cells. Desiccated M. barkeri exposed to air at room temperature did not lose significant viability after 28 days, and exposure of M. barkeri to air after desiccation appeared to improve the recovery of viable cells compared with that of desiccated cells that were never exposed to air. Desiccated M. barkeri was more resistant to higher temperatures, and although resistance to oxidative conditions such as ozone and ionizing radiation was not as robust as in other desiccation-resistant microorganisms, the protection mechanisms are likely adequate to maintain cell viability during periodic exposure events. The results of this study demonstrate that after desiccation M. barkeri has the innate capability to survive extended periods of exposure to air and lethal temperatures. PMID:22194299

  16. Kinesin superfamily motor proteins and intracellular transport.

    PubMed

    Hirokawa, Nobutaka; Noda, Yasuko; Tanaka, Yosuke; Niwa, Shinsuke

    2009-10-01

    Intracellular transport is fundamental for cellular function, survival and morphogenesis. Kinesin superfamily proteins (also known as KIFs) are important molecular motors that directionally transport various cargos, including membranous organelles, protein complexes and mRNAs. The mechanisms by which different kinesins recognize and bind to specific cargos, as well as how kinesins unload cargo and determine the direction of transport, have now been identified. Furthermore, recent molecular genetic experiments have uncovered important and unexpected roles for kinesins in the regulation of such physiological processes as higher brain function, tumour suppression and developmental patterning. These findings open exciting new areas of kinesin research. PMID:19773780

  17. Intracellularly survived Staphylococcus aureus after phagocytosis are more virulent in inducing cytotoxicity in fresh murine peritoneal macrophages utilizing TLR-2 as a possible target.

    PubMed

    Nandi, Ajeya; Bishayi, Biswadev

    2016-08-01

    Staphylococcus aureus with high virulence potential is contributing to a current public health crisis in both hospital and community settings. TLR-2 and generation of reactive oxygen species (ROS) by phagocytic cells is thought to be an important component of the host's immunity against S. aureus infection. However, response of S. aureus against modulation of host-derived ROS in absence of TLR-2 during acute staphylococcal infection is still remains unclear. Peritoneal macrophages were pretreated with either inhibitors of superoxide dismutase (SOD) or catalase in presence or absence of anti TLR-2 antibody and were infected with S. aureus strain AG-789. Bacteria were recovered after time dependent phagocytosis; intracellular killing, level and expression of SOD and catalase were measured. Phagocytosed bacteria from respective groups were further used for infection to fresh peritoneal macrophages as well as for in vivo infection. Levels of ROS, cytokine, lysozyme, antioxidant enzymes activity and TLR-2 expression were measured. Results revealed that more bacteria were escaped killing in SOD and catalase inhibitor pretreated TLR-2 neutralized macrophages, found to express more catalase and are antibiotic resistant. Infection of fresh macrophages with S. aureus, recovered from SOD and catalase inhibited TLR-2 neutralized macrophages induced lower ROS, lysozyme and cytokine production and caused increased bacterial count. Furthermore, bacterial antioxidants by modulating host-derived ROS could regulate the cell surface TLR-2 expression in murine peritoneal macrophages. So, in the early phase of infection, TLR-2 participates in the innate immune response and targeting bacterial antioxidants might be useful in the alleviation of Staphylococcus aureus infection. PMID:27270212

  18. Mechanisms of PECAM-1-mediated cytoprotection and implications for cancer cell survival.

    PubMed

    Bergom, Carmen; Gao, Cunji; Newman, Peter J

    2005-10-01

    Defects in apoptotic pathways can promote cancer development and cause cancers to become resistant to chemotherapy. The cell adhesion and signaling molecule PECAM-1 has been shown to potently suppress apoptosis in a variety of cellular systems. PECAM-1 expression has been reported on a variety of human malignancies-especially hematopoietic and vascular cell cancers-but the significance of this expression has not been fully explored. The ability of PECAM-1 to inhibit apoptosis makes it an attractive candidate as a molecule that may promote cancer development and/or confer resistance to chemotherapeutic treatment. The exact mechanisms by which PECAM-1 mediates its cytoprotection have not been fully defined, but its anti-apoptotic effects have been shown to require both homophilic binding and intracellular signaling via its immunoreceptor tyrosine-based inhibitory motif (ITIM) domains. In this review, we will discuss the data regarding PECAM-1's anti-apoptotic effects and ways in which this cytoprotection may be clinically relevant to the development and/or treatment of hematologic malignancies that express this vascular cell-specific surface molecule. PMID:16194886

  19. Mitotic Intragenic Recombination: A Mechanism of Survival for Several Congenital Disorders of Glycosylation.

    PubMed

    Kane, Megan S; Davids, Mariska; Adams, Christopher; Wolfe, Lynne A; Cheung, Helen W; Gropman, Andrea; Huang, Yan; Ng, Bobby G; Freeze, Hudson H; Adams, David R; Gahl, William A; Boerkoel, Cornelius F

    2016-02-01

    Congenital disorders of glycosylation (CDGs) are disorders of abnormal protein glycosylation that affect multiple organ systems. Because most CDGs have been described in only a few individuals, our understanding of the associated phenotypes and the mechanisms of individual survival are limited. In the process of studying two siblings, aged 6 and 11 years, with MOGS-CDG and biallelic MOGS (mannosyl-oligosaccharide glucosidase) mutations (GenBank: NM_006302.2; c.[65C>A; 329G>A] p.[Ala22Glu; Arg110His]; c.[370C>T] p.[Gln124(∗)]), we noted that their survival was much longer than the previous report of MOGS-CDG, in a child who died at 74 days of age. Upon mutation analysis, we detected multiple MOGS genotypes including wild-type alleles in their cultured fibroblast and peripheral blood DNA. Further analysis of DNA from cultured fibroblasts of six individuals with compound heterozygous mutations of PMM2 (PMM2-CDG), MPI (MPI-CDG), ALG3 (ALG3-CDG), ALG12 (ALG12-CDG), DPAGT1 (DPAGT1-CDG), and ALG1 (ALG1-CDG) also identified multiple genotypes including wild-type alleles for each. Droplet digital PCR showed a ratio of nearly 1:1 wild-type to mutant alleles for most, but not all, mutations. This suggests that mitotic recombination contributes to the survival and the variable expressivity of individuals with compound heterozygous CDGs. This also provides an explanation for prior observations of a reduced frequency of homozygous mutations and might contribute to increased levels of residual enzyme activity in cultured fibroblasts of individuals with MPI- and PMM2-CDGs. PMID:26805780

  20. Survival and SOS response induction in ultraviolet B irradiated Escherichia coli cells with defective repair mechanisms.

    PubMed

    Prada Medina, Cesar Augusto; Aristizabal Tessmer, Elke Tatjana; Quintero Ruiz, Nathalia; Serment-Guerrero, Jorge; Fuentes, Jorge Luis

    2016-06-01

    Purpose In this paper, the contribution of different genes involved in DNA repair for both survival and SOS induction in Escherichia coli mutants exposed to ultraviolet B radiation (UVB, [wavelength range 280-315 nm]) was evaluated. Materials and methods E. coli strains defective in uvrA, oxyR, recO, recN, recJ, exoX, recB, recD or xonA genes were used to determine cell survival. All strains also had the genetic sulA::lacZ fusion, which allowed for the quantification of SOS induction through the SOS Chromotest. Results Five gene products were particularly important for survival, as follows: UvrA > RecB > RecO > RecJ > XonA. Strains defective in uvrA and recJ genes showed elevated SOS induction compared with the wild type, which remained stable for up to 240 min after UVB-irradiation. In addition, E. coli strains carrying the recO or recN mutation showed no SOS induction. Conclusions The nucleotide excision and DNA recombination pathways were equally used to repair UVB-induced DNA damage in E. coli cells. The sulA gene was not turned off in strains defective in UvrA and RecJ. RecO protein was essential for processing DNA damage prior to SOS induction. In this study, the roles of DNA repair proteins and their contributions to the mechanisms that induce SOS genes in E. coli are proposed. PMID:26967458

  1. Mitotic Intragenic Recombination: A Mechanism of Survival for Several Congenital Disorders of Glycosylation

    PubMed Central

    Kane, Megan S.; Davids, Mariska; Adams, Christopher; Wolfe, Lynne A.; Cheung, Helen W.; Gropman, Andrea; Huang, Yan; Ng, Bobby G.; Freeze, Hudson H.; Adams, David R.; Gahl, William A.; Boerkoel, Cornelius F.

    2016-01-01

    Congenital disorders of glycosylation (CDGs) are disorders of abnormal protein glycosylation that affect multiple organ systems. Because most CDGs have been described in only a few individuals, our understanding of the associated phenotypes and the mechanisms of individual survival are limited. In the process of studying two siblings, aged 6 and 11 years, with MOGS-CDG and biallelic MOGS (mannosyl-oligosaccharide glucosidase) mutations (GenBank: NM_006302.2; c.[65C>A; 329G>A] p.[Ala22Glu; Arg110His]; c.[370C>T] p.[Gln124∗]), we noted that their survival was much longer than the previous report of MOGS-CDG, in a child who died at 74 days of age. Upon mutation analysis, we detected multiple MOGS genotypes including wild-type alleles in their cultured fibroblast and peripheral blood DNA. Further analysis of DNA from cultured fibroblasts of six individuals with compound heterozygous mutations of PMM2 (PMM2-CDG), MPI (MPI-CDG), ALG3 (ALG3-CDG), ALG12 (ALG12-CDG), DPAGT1 (DPAGT1-CDG), and ALG1 (ALG1-CDG) also identified multiple genotypes including wild-type alleles for each. Droplet digital PCR showed a ratio of nearly 1:1 wild-type to mutant alleles for most, but not all, mutations. This suggests that mitotic recombination contributes to the survival and the variable expressivity of individuals with compound heterozygous CDGs. This also provides an explanation for prior observations of a reduced frequency of homozygous mutations and might contribute to increased levels of residual enzyme activity in cultured fibroblasts of individuals with MPI- and PMM2-CDGs. PMID:26805780

  2. Triiodothyronine enhances accumulation of intracellular lipids in adipocytes through thyroid hormone receptor α via direct and indirect mechanisms.

    PubMed

    Gambo, Yurina; Matsumura, Miki; Fujimori, Ko

    2016-08-15

    Triiodothyronine (T3) enhanced the expression of adipogenic and lipogenic genes with elevation of the intracellular lipids through thyroid hormone receptor (TR) α in mouse 3T3-L1 cells. However, the transcription of the SREBP-1c and HSL genes was decreased by T3. Such T3-mediated alterations were negated by TRα siRNA. Chromatin immunoprecipitation assay showed that the binding of TRα to the TR-responsive element (TRE) of the FAS promoter was elevated by T3. In contrast, the ability of TRα to bind to the TRE of the SREBP-1c promoter was decreased by T3. In addition, the binding of SREBP-1c to the SRE of the HSL promoter was lowered by T3. These results indicate that T3 increased the accumulation of intracellular lipids by enhancing the expression of the FAS gene through direct binding of TRα to the FAS promoter and simultaneously lowered the amount of lipolysis via reduced binding of T3-decreased SREBP-1c to the HSL promoter. PMID:27132806

  3. The cellular uptake mechanism, intracellular transportation, and exocytosis of polyamidoamine dendrimers in multidrug-resistant breast cancer cells

    PubMed Central

    Zhang, Jie; Liu, Dan; Zhang, Mengjun; Sun, Yuqi; Zhang, Xiaojun; Guan, Guannan; Zhao, Xiuli; Qiao, Mingxi; Chen, Dawei; Hu, Haiyang

    2016-01-01

    Polyamidoamine dendrimers, which can deliver drugs and genetic materials to resistant cells, are attracting increased research attention, but their transportation behavior in resistant cells remains unclear. In this paper, we performed a systematic analysis of the cellular uptake, intracellular transportation, and efflux of PAMAM-NH2 dendrimers in multidrug-resistant breast cancer cells (MCF-7/ADR cells) using sensitive breast cancer cells (MCF-7 cells) as the control. We found that the uptake rate of PAMAM-NH2 was much lower and exocytosis of PAMAM-NH2 was much greater in MCF-7/ADR cells than in MCF-7 cells due to the elimination of PAMAM-NH2 from P-glycoprotein and the multidrug resistance-associated protein in MCF-7/ADR cells. Macropinocytosis played a more important role in its uptake in MCF-7/ADR cells than in MCF-7 cells. PAMAM-NH2 aggregated and became more degraded in the lysosomal vesicles of the MCF-7/ADR cells than in those of the MCF-7 cells. The endoplasmic reticulum and Golgi complex were found to participate in the exocytosis rather than endocytosis process of PAMAM-NH2 in both types of cells. Our findings clearly showed the intracellular transportation process of PAMAM-NH2 in MCF-7/ADR cells and provided a guide of using PAMAM-NH2 as a drug and gene vector in resistant cells. PMID:27536106

  4. The cellular uptake mechanism, intracellular transportation, and exocytosis of polyamidoamine dendrimers in multidrug-resistant breast cancer cells.

    PubMed

    Zhang, Jie; Liu, Dan; Zhang, Mengjun; Sun, Yuqi; Zhang, Xiaojun; Guan, Guannan; Zhao, Xiuli; Qiao, Mingxi; Chen, Dawei; Hu, Haiyang

    2016-01-01

    Polyamidoamine dendrimers, which can deliver drugs and genetic materials to resistant cells, are attracting increased research attention, but their transportation behavior in resistant cells remains unclear. In this paper, we performed a systematic analysis of the cellular uptake, intracellular transportation, and efflux of PAMAM-NH2 dendrimers in multidrug-resistant breast cancer cells (MCF-7/ADR cells) using sensitive breast cancer cells (MCF-7 cells) as the control. We found that the uptake rate of PAMAM-NH2 was much lower and exocytosis of PAMAM-NH2 was much greater in MCF-7/ADR cells than in MCF-7 cells due to the elimination of PAMAM-NH2 from P-glycoprotein and the multidrug resistance-associated protein in MCF-7/ADR cells. Macropinocytosis played a more important role in its uptake in MCF-7/ADR cells than in MCF-7 cells. PAMAM-NH2 aggregated and became more degraded in the lysosomal vesicles of the MCF-7/ADR cells than in those of the MCF-7 cells. The endoplasmic reticulum and Golgi complex were found to participate in the exocytosis rather than endocytosis process of PAMAM-NH2 in both types of cells. Our findings clearly showed the intracellular transportation process of PAMAM-NH2 in MCF-7/ADR cells and provided a guide of using PAMAM-NH2 as a drug and gene vector in resistant cells. PMID:27536106

  5. Corynebacterium diphtheriae putative tellurite-resistance protein (CDCE8392_0813) contributes to the intracellular survival in human epithelial cells and lethality of Caenorhabditis elegans.

    PubMed

    Santos, Louisy Sanches Dos; Antunes, Camila Azevedo; Santos, Cintia Silva Dos; Pereira, José Augusto Adler; Sabbadini, Priscila Soares; Luna, Maria das Graças de; Azevedo, Vasco; Hirata Júnior, Raphael; Burkovski, Andreas; Asad, Lídia Maria Buarque de Oliveira; Mattos-Guaraldi, Ana Luíza

    2015-08-01

    Corynebacterium diphtheriae, the aetiologic agent of diphtheria, also represents a global medical challenge because of the existence of invasive strains as causative agents of systemic infections. Although tellurite (TeO32-) is toxic to most microorganisms, TeO32--resistant bacteria, including C. diphtheriae, exist in nature. The presence of TeO32--resistance (TeR) determinants in pathogenic bacteria might provide selective advantages in the natural environment. In the present study, we investigated the role of the putative TeR determinant (CDCE8392_813gene) in the virulence attributes of diphtheria bacilli. The disruption of CDCE8392_0813 gene expression in the LDCIC-L1 mutant increased susceptibility to TeO32- and reactive oxygen species (hydrogen peroxide), but not to other antimicrobial agents. The LDCIC-L1 mutant also showed a decrease in both the lethality of Caenorhabditis elegans and the survival inside of human epithelial cells compared to wild-type strain. Conversely, the haemagglutinating activity and adherence to and formation of biofilms on different abiotic surfaces were not regulated through the CDCE8392_0813 gene. In conclusion, the CDCE8392_813 gene contributes to the TeR and pathogenic potential of C. diphtheriae. PMID:26107188

  6. Corynebacterium diphtheriae putative tellurite-resistance protein (CDCE8392_0813) contributes to the intracellular survival in human epithelial cells and lethality of Caenorhabditis elegans

    PubMed Central

    dos Santos, Louisy Sanches; Antunes, Camila Azevedo; dos Santos, Cintia Silva; Pereira, José Augusto Adler; Sabbadini, Priscila Soares; de Luna, Maria das Graças; Azevedo, Vasco; Hirata, Raphael; Burkovski, Andreas; Asad, Lídia Maria Buarque de Oliveira; Mattos-Guaraldi, Ana Luíza

    2015-01-01

    Corynebacterium diphtheriae, the aetiologic agent of diphtheria, also represents a global medical challenge because of the existence of invasive strains as causative agents of systemic infections. Although tellurite (TeO32-) is toxic to most microorganisms, TeO32--resistant bacteria, including C. diphtheriae, exist in nature. The presence of TeO32--resistance (TeR) determinants in pathogenic bacteria might provide selective advantages in the natural environment. In the present study, we investigated the role of the putative TeR determinant (CDCE8392_813gene) in the virulence attributes of diphtheria bacilli. The disruption of CDCE8392_0813 gene expression in the LDCIC-L1 mutant increased susceptibility to TeO32- and reactive oxygen species (hydrogen peroxide), but not to other antimicrobial agents. The LDCIC-L1 mutant also showed a decrease in both the lethality of Caenorhabditis elegans and the survival inside of human epithelial cells compared to wild-type strain. Conversely, the haemagglutinating activity and adherence to and formation of biofilms on different abiotic surfaces were not regulated through the CDCE8392_0813 gene. In conclusion, the CDCE8392_813 gene contributes to the TeR and pathogenic potential of C. diphtheriae. PMID:26107188

  7. An inducible and secreted eukaryote-like serine/threonine kinase of Salmonella enterica serovar Typhi promotes intracellular survival and pathogenesis.

    PubMed

    Theeya, Nagaraja; Ta, Atri; Das, Sayan; Mandal, Rahul S; Chakrabarti, Oishee; Chakrabarti, Saikat; Ghosh, Amar N; Das, Santasabuj

    2015-02-01

    Eukaryote-like serine/threonine kinases (eSTKs) constitute an important family of bacterial virulence factors. Genome analysis had predicted putative eSTKs in Salmonella enterica serovar Typhi, although their functional characterization and the elucidation of their role in pathogenesis are still awaited. We show here that the primary sequence and secondary structure of the t4519 locus of Salmonella Typhi Ty2 have all the signatures of eukaryotic superfamily kinases. t4519 encodes a ∼39-kDa protein (T4519), which shows serine/threonine kinase activities in vitro. Recombinant T4519 (rT4519) is autophosphorylated and phosphorylates the universal substrate myelin basic protein. Infection of macrophages results in decreased viability of the mutant (Ty2Δt4519) strain, which is reversed by gene complementation. Moreover, reactive oxygen species produced by the macrophages signal to the bacteria to induce T4519, which is translocated to the host cell cytoplasm. That T4519 may target a host substrate(s) is further supported by the activation of host cellular signaling pathways and the induction of cytokines/chemokines. Finally, the role of T4519 in the pathogenesis of Salmonella Typhi is underscored by the significantly decreased mortality of mice infected with the Ty2Δt4519 strain and the fact that the competitive index of this strain for causing systemic infection is 0.25% that of the wild-type strain. This study characterizes the first eSTK of Salmonella Typhi and demonstrates its role in promoting phagosomal survival of the bacteria within macrophages, which is a key determinant of pathogenesis. This, to the best of our knowledge, is the first study to describe the essential role of eSTKs in the in vivo pathogenesis of Salmonella spp. PMID:25404028

  8. Compartmentation of malic acid in mesophyll cells of Kalanchoee daigremontiana: indications of a intracellular cytosolic vesicle transport mechanism

    SciTech Connect

    Balsamo, R.A.; Uribe, E.G.

    1987-04-01

    Leaf tissue was harvested over a 24hr period at one to three hour intervals. The malic acid levels in the tissue were assayed spectrophotometrically and the percent cell volume occupied by cytosolic vesicles in replicate samples was determined. The total volume of the cytosolic vesicles fluctuated throughout the photoperiod concommitantly with malic acid concentrations present in the tissue. An intact leaf tissue section (10.2cm/sup 2/) was radiolabeled with /sup 14/CO/sub 2/ seven hours into the dark period for thirty minutes. Two dimensional thin layer chromatography and electrophoresis of the tissue determined that 96% of the label was incorporated into malic acid. A freeze substitution procedure was initiated followed by microautoradiography (Fisher 1971) which allowed for the tracing of intracellular malic acid migration and compartmentation within the mesophyll cells. The results and interpretation of this experiment will be presented.

  9. Elevated Intracellular Calcium Increases Ferritin H Expression Through an NFAT-Independent Posttranscriptional Mechanism Involving mRNA Stabilization

    PubMed Central

    MacKenzie, Elizabeth L.; Tsuji, Yoshiaki

    2009-01-01

    An increase in intracellular Ca2+ is one of the initiating events in T cell activation. A calcium-mediated signaling cascade in T cells involves activation of calcineurin and the dephosphorylation and translocation of Nuclear Factor of Activated T-cells (NFAT), resulting in the transcriptional activation of target genes such as IL-2. In the present study, we found that increased intracellular calcium leads to induction of the antioxidant protein ferritin H. We previously reported that the ferritin H gene is transcriptionally activated under oxidative stress conditions through an antioxidant responsive element (ARE). The facts that the ferritin H ARE contains a composite AP1 site, and that NFAT collaborates with AP1 transcription factors, led us to test whether calcium-activated NFAT is involved in the ferritin H induction through the ARE. Treatment of Jurkat T cells with the calcium ionophore, ionomycin, increased ferritin H mRNA and protein expression. Though NFAT translocated to the nucleus and bound a consensus NFAT sequence located in the IL-2 promoter following ionomycin treatment, it did not activate ferritin H transcription despite the presence of a putative NFAT binding sequence in the ferritin H ARE. In addition, the calcineurin inhibitor cyclosporin A treatment blocked ionomycin-mediated NFAT nuclear translocation but failed to abrogate the increase in ferritin H mRNA. Analysis of mRNA stability following actinomycin D treatment revealed that ionomycin prolongs ferritin H mRNA half-life. Taken together, these results suggest that ionomycin-mediated induction of ferritin H may occur in an NFAT-independent manner but through posttranscriptional stabilization of the ferritin H mRNA. PMID:18076382

  10. Intracellular microlasers

    NASA Astrophysics Data System (ADS)

    Humar, Matjaž; Hyun Yun, Seok

    2015-09-01

    Optical microresonators, which confine light within a small cavity, are widely exploited for various applications ranging from the realization of lasers and nonlinear devices to biochemical and optomechanical sensing. Here we use microresonators and suitable optical gain materials inside biological cells to demonstrate various optical functions in vitro including lasing. We explore two distinct types of microresonator—soft and hard—that support whispering-gallery modes. Soft droplets formed by injecting oil or using natural lipid droplets support intracellular laser action. The laser spectra from oil-droplet microlasers can chart cytoplasmic internal stress (˜500 pN μm-2) and its dynamic fluctuations at a sensitivity of 20 pN μm-2 (20 Pa). In a second form, whispering-gallery modes within phagocytized polystyrene beads of different sizes enable individual tagging of thousands of cells easily and, in principle, a much larger number by multiplexing with different dyes.

  11. Molecular Mechanism of Flocculation Self-Recognition in Yeast and Its Role in Mating and Survival

    PubMed Central

    Goossens, Katty V. Y.; Ielasi, Francesco S.; Nookaew, Intawat; Stals, Ingeborg; Alonso-Sarduy, Livan; Daenen, Luk; Van Mulders, Sebastiaan E.; Stassen, Catherine; van Eijsden, Rudy G. E.; Siewers, Verena; Delvaux, Freddy R.; Kasas, Sandor; Nielsen, Jens; Devreese, Bart

    2015-01-01

    ABSTRACT We studied the flocculation mechanism at the molecular level by determining the atomic structures of N-Flo1p and N-Lg-Flo1p in complex with their ligands. We show that they have similar ligand binding mechanisms but distinct carbohydrate specificities and affinities, which are determined by the compactness of the binding site. We characterized the glycans of Flo1p and their role in this binding process and demonstrate that glycan-glycan interactions significantly contribute to the cell-cell adhesion mechanism. Therefore, the extended flocculation mechanism is based on the self-interaction of Flo proteins and this interaction is established in two stages, involving both glycan-glycan and protein-glycan interactions. The crucial role of calcium in both types of interaction was demonstrated: Ca2+ takes part in the binding of the carbohydrate to the protein, and the glycans aggregate only in the presence of Ca2+. These results unify the generally accepted lectin hypothesis with the historically first-proposed “Ca2+-bridge” hypothesis. Additionally, a new role of cell flocculation is demonstrated; i.e., flocculation is linked to cell conjugation and mating, and survival chances consequently increase significantly by spore formation and by introduction of genetic variability. The role of Flo1p in mating was demonstrated by showing that mating efficiency is increased when cells flocculate and by differential transcriptome analysis of flocculating versus nonflocculating cells in a low-shear environment (microgravity). The results show that a multicellular clump (floc) provides a uniquely organized multicellular ultrastructure that provides a suitable microenvironment to induce and perform cell conjugation and mating. PMID:25873380

  12. Intracellular distribution and mechanisms of actions of photosensitizer Zinc(II)-phthalocyanine solubilized in Cremophor EL against human hepatocellular carcinoma HepG2 cells.

    PubMed

    Shao, Jingwei; Dai, Yongchao; Zhao, Wenna; Xie, Jingjing; Xue, Jinping; Ye, Jianhui; Jia, Lee

    2013-03-01

    Zinc(II)-phthalocyanine (ZnPc) is a metal photosensitizer. In the present study, we formulated the poorly-soluble ZnPc in Cremophor EL solution to enhance its solubility and determined its intracellular distribution and mechanisms of action on human hepatocellular carcinoma HepG2 cells. ZnPc uptake by the cells reached a plateau by 8h. ZnPc primarily located in mitochondria, lysosome and endoplasmic reticulum. The concentration-growth inhibition curves of ZnPc on the cell lines were pharmacodynamically enhanced by 10-50 folds by irradiation. Once irradiated, ZnPc produced significant amount of reactive oxygen species (ROS), activated caspase-3 and caspase-9, arrested cell cycle mainly at G2/M stage, and decreased membrane potential (ΔΨm) of HepG2 cells. In conclusion, the present study first elucidated cellular and molecular mechanisms of ZnPc on HepG2 cells. PMID:23200672

  13. Isolation of a non-fermentative bacterium, Pseudomonas aeruginosa, using intracellular carbon for denitrification and phosphorus-accumulation and relevant metabolic mechanisms.

    PubMed

    Liu, Hui; Wang, Qin; Sun, Yanfu; Zhou, Kangqun; Liu, Wen; Lu, Qian; Ming, Caibing; Feng, Xidan; Du, Jianjun; Jia, Xiaoshan; Li, Jun

    2016-07-01

    A newly designed pilot-scale system was developed to enrich denitrifying phosphate-accumulating organisms (DNPAOs) for nitrogen and phosphorus nutrient removal synchronously. A strain of DNPAOs was isolated and its biochemical characteristics and metabolic mechanisms of this bacterial strain were analyzed. The results showed that compared with previously reported system, this newly designed system has higher removal rates of nutrients. Removal efficiencies of NH3-N, TN, TP, and COD in actual wastewater were 82.64%, 79.62%, 87.22%, and 90.41%, respectively. Metabolic activity of DNPAOs after anoxic stage in this study even reached 94.64%. Pseudomonas aeruginosa is a strain of non-fermentative DNPAOs with strong nitrogen and phosphorus removal abilities. Study on the metabolic mechanisms suggested that intracellular PHB of P. aeruginosa plays dual roles, supplying energy for phosphorus accumulation and serving as a major carbon source for denitrification. PMID:26995616

  14. Intracellular Zn(2+) Increase in Cardiomyocytes Induces both Electrical and Mechanical Dysfunction in Heart via Endogenous Generation of Reactive Nitrogen Species.

    PubMed

    Tuncay, Erkan; Turan, Belma

    2016-02-01

    Oxidants increase intracellular free Zn(2+) concentration ([Zn(2+)]i) in ventricular myocytes, which contributes to oxidant-induced alterations in excitation-contraction coupling (ECC). However, it is not clear whether increased [Zn(2+)]i in cardiomyocytes via increased reactive nitrogen species (RNS) has a role on heart function under pathological conditions, such as hyperglycemia. In this study, first we aimed to investigate the role of increased [Zn(2+)]i under in vitro condition in the development of both electrical and mechanical dysfunction of isolated papillary muscle strips from rat heart via exposed samples to a Zn(2+)-ionophore (Zn-pyrithione; 1 μM) for 20 min. Under simultaneous measurement of intracellular action potential and contractile activity in these preparations, Zn-pyrithione exposure caused marked prolongation in action potential repolarization phase and slowdown in both contraction and relaxation rates of twitch activity. Second, in order to demonstrate an association between increased [Zn(2+)]i and increased RNS, we monitored intracellular [Zn(2+)]i under an acute exposure of nitric oxide (NO) donor sodium nitroprusside, SNP, in freshly isolated quiescent cardiomyocytes loaded with FluoZin-3. Resting level of free Zn(2+) is significantly higher in cardiomyocytes under hyperglycemic condition compared to those of the controls, which seems to be associated with increased level of RNS production in hyperglycemic cardiomyocytes. Western blot analysis showed that Zn-pyrithione exposure induced a marked decrease in the activity of protein phosphatase 1 and 2A, member of macromolecular protein complex of cardiac ryanodine receptors, RyR2, besides significant increase in the phosphorylation level of extracellular signal-regulated kinase1/2 as a concentration-dependent manner. Overall, the present data demonstrated that there is a cross-relationship between increased RNS production and increased [Zn(2+)]i level in cardiomyocytes under pathological

  15. Managing intracellular transport

    PubMed Central

    Chua, John J.E.; Jahn, Reinhard; Klopfenstein, Dieter R.

    2013-01-01

    Formation and normal function of neuronal synapses are intimately dependent on the delivery to and removal of biological materials from synapses by the intracellular transport machinery. Indeed, defects in intracellular transport contribute to the development and aggravation of neurodegenerative disorders. Despite its importance, regulatory mechanisms underlying this machinery remain poorly defined. We recently uncovered a phosphorylation-regulated mechanism that controls FEZ1-mediated Kinesin-1-based delivery of Stx1 into neuronal axons. Using C. elegans as a model organism to investigate transport defects, we show that FEZ1 mutations resulted in abnormal Stx1 aggregation in neuronal cell bodies and axons. This phenomenon closely resembles transport defects observed in neurodegenerative disorders. Importantly, diminished transport due to mutations of FEZ1 and Kinesin-1 were concomitant with increased accumulation of autophagosomes. Here, we discuss the significance of our findings in a broader context in relation to regulation of Kinesin-mediated transport and neurodegenerative disorders. PMID:24058857

  16. Intracellular microlasers

    PubMed Central

    Humar, Matjaž; Yun, Seok Hyun

    2015-01-01

    Optical microresonators1 which confine light within a small cavity are widely exploited for various applications ranging from the realization of lasers2 and nonlinear devices3, 4, 5 to biochemical and optomechanical sensing6, 7, 8, 9, 10, 11. Here we employ microresonators and suitable optical gain materials inside biological cells to demonstrate various optical functions in vitro including lasing. We explored two distinct types of microresonators: soft and hard, that support whispering-gallery modes (WGM). Soft droplets formed by injecting oil or using natural lipid droplets support intracellular laser action. The laser spectra from oil-droplet microlasers can chart cytoplasmic internal stress (~500 pN/μm2) and its dynamic fluctuations at a sensitivity of 20 pN/μm2 (20 Pa). In a second form, WGMs within phagocytized polystyrene beads of different sizes enable individual tagging of thousands of cells easily and, in principle, a much larger number by multiplexing with different dyes. PMID:26417383

  17. Thrombolytic Therapy for Right-Sided Mechanical Pulmonic and Tricuspid Valves: The Largest Survival Analysis to Date

    PubMed Central

    Taherkhani, Maryam; Hashemi, Seyed Reza; Hekmat, Manouchehr; Safi, Morteza; Taherkhani, Adineh

    2015-01-01

    Data regarding thrombolytic treatment of right-sided mechanical valve thrombosis are almost nonexistent, and all current guidelines arise from very small case series. We retrospectively studied the in-hospital and long-term outcome data of a larger series of patients who had received, from September 2005 through June 2012, thrombolytic therapy for right-sided mechanical pulmonary valve or tricuspid valve thrombosis. We identified 16 patients aged 8–67 years who had undergone thrombolytic therapy for definite thrombotic mechanical valve obstruction in the tricuspid or pulmonary valve position (8 in each position). All study patients except one had subtherapeutic international normalized ratios. The 8 patients with pulmonary mechanical valve thrombosis had a 100% response rate to thrombolytic therapy, and their in-hospital survival rate was also 100%. The 8 patients with tricuspid mechanical valve thrombosis had a 75% response rate to thrombolytic therapy, with an in-hospital survival rate of 87.5%. The one-year survival rate for mechanical valve thrombosis treated with thrombolytic therapy (whether pulmonary or tricuspid) was 87.5%. On the basis of our data, we recommend that thrombolytic therapy remain the first-line therapy for right-sided mechanical valve thrombosis in adults or children—including children with complex congenital heart disease and patients with mechanical pulmonary valve thrombosis. Surgery should be reserved for patients in whom this treatment fails. PMID:26664307

  18. Tumor necrosis factor alpha and interleukin-12 contribute to resistance to the intracellular bacterium Brucella abortus by different mechanisms.

    PubMed Central

    Zhan, Y; Liu, Z; Cheers, C

    1996-01-01

    Both interleukin-12 (IL-12) and tumor necrosis factor alpha (TNF-alpha) are produced early in intracellular bacterial infection. Depletion of either IL-12 or TNF-alpha by a single injection of specific antibody 4 h before the injection of Brucella abortus 19 led to the exacerbation of infection 2 weeks later. Whereas the effect of IL-12 depletion on resistance was persistent and exacerbation was still significant 6 weeks later, the bacterial numbers in mice depleted of TNF-alpha were similar to the bacterial numbers in control infected mice by 6 weeks postinfection. Massive splenomegaly, which is often seen in 2-week Brucella-infected mice, was not observed in IL-12- or TNF-alpha-depleted mice. Both IL-12- and TNF-alpha-depleted mice showed reduced cell accumulation in the spleen compared with the massive cell accumulation in control infected mice. Granuloma formation in livers was much reduced in IL-12-depleted mice but not in TNF-alpha-depleted mice. Gamma interferon (IFN-gamma) production by cells from TNF-alpha-depleted mice was not significantly different from that of cells from control infected mice. In contrast, the production of IFN-gamma by both CD4+ and CD8+ T cells from IL-12-depleted mice was greatly reduced, compared with that from control infected mice. This effect was still observed when the antibody injection was delayed for up to 7 days postinfection, but injections of anti-IL-12 antibody into mice with established Brucella infection had no significant effect on IFN-gamma production by T cells. Taken together, these results suggested that IL-12 contributed to resistance mainly via an IFN-gamma-dependent pathway and had a profound effect on the induction of acquired cellular resistance. In contrast, TNF-alpha was involved in resistance possibly via direct action on effector cells and may not be essential for the induction of acquired cellular resistance. PMID:8698508

  19. The cancer glycocalyx mechanically primes integrin-mediated growth and survival

    PubMed Central

    Paszek, Matthew J.; DuFort, Christopher C.; Rossier, Olivier; Bainer, Russell; Mouw, Janna K.; Godula, Kamil; Hudak, Jason E.; Lakins, Jonathon N.; Wijekoon, Amanda C.; Cassereau, Luke; Rubashkin, Matthew G.; Magbanua, Mark J.; Thorn, Kurt S.; Davidson, Michael W.; Rugo, Hope S.; Park, John W.; Hammer, Daniel A.; Giannone, Grégory; Bertozzi, Carolyn R.; Weaver, Valerie M.

    2015-01-01

    Malignancy is associated with altered expression of glycans and glycoproteins that contribute to the cellular glycocalyx. We constructed a glycoprotein expression signature, which revealed that metastatic tumours upregulate expression of bulky glycoproteins. A computational model predicted that these glycoproteins would influence transmembrane receptor spatial organization and function. We tested this prediction by investigating whether bulky glycoproteins in the glycocalyx promote a tumour phenotype in human cells by increasing integrin adhesion and signalling. Our data revealed that a bulky glycocalyx facilitates integrin clustering by funnelling active integrins into adhesions and altering integrin state by applying tension to matrix-bound integrins, independent of actomyosin contractility. Expression of large tumour-associated glycoproteins in non-transformed mammary cells promoted focal adhesion assembly and facilitated integrin-dependent growth factor signalling to support cell growth and survival. Clinical studies revealed that large glycoproteins are abundantly expressed on circulating tumour cells from patients with advanced disease. Thus, a bulky glycocalyx is a feature of tumour cells that could foster metastasis by mechanically enhancing cell-surface receptor function. PMID:25030168

  20. Selection by differential molecular survival: a possible mechanism of early chemical evolution.

    PubMed Central

    de Duve, C

    1987-01-01

    A model is proposed to account for selective chemical evolution, progressing from a relatively simple initial set of abiotic synthetic phenomena up to the elaborately sophisticated processes that are almost certainly required to produce the complex molecules, such as replicatable RNA-like oligonucleotides, needed for a Darwinian form of selection to start operating. The model makes the following assumptions: (i) that a small number of micromolecular substances were present at high concentration; (ii) that a random assembly mechanism combined these molecules into a variety of multimeric compounds comprising a wide repertoire of rudimentary catalytic activities; and (iii) that a lytic system capable of breaking down the assembled products existed. The model assumes further that catalysts supplied with substrates were significantly protected against breakdown. It is shown that, by granting these assumptions, an increasingly complex network of metabolic pathways would progressively be established. At the same time, the catalysts concerned would accumulate selectively to become choice substrates for elongation and other modifications that could enhance their efficiency, as well as their survival. Chemical evolution would thus proceed by a dual process of metabolic extension and catalytic innovation. Such a process should be largely deterministic and predictable from initial conditions. PMID:3479788

  1. Metabolic mechanisms for anoxia tolerance and freezing survival in the intertidal gastropod, Littorina littorea.

    PubMed

    Storey, Kenneth B; Lant, Benjamin; Anozie, Obiajulu O; Storey, Janet M

    2013-08-01

    The gastropod mollusk, Littorina littorea L., is a common inhabitant of the intertidal zone along rocky coastlines of the north Atlantic. This species has well-developed anoxia tolerance and freeze tolerance and is extensively used as a model for exploring the biochemical adaptations that support these tolerances as well as for toxicological studies aimed at identifying effective biomarkers of aquatic pollution. This article highlights our current understanding of the molecular mechanisms involved in anaerobiosis and freezing survival of periwinkles, particularly with respect to anoxia-induced metabolic rate depression. Analysis of foot muscle and hepatopancreas metabolism includes anoxia-responsive changes in enzyme regulation, signal transduction, gene expression, post-transcriptional regulation of mRNA, control of translation, and cytoprotective strategies including chaperones and antioxidant defenses. New studies describe the regulation of glucose-6-phosphate dehydrogenase by reversible protein phosphorylation, the role of microRNAs in suppressing mRNA translation in the hypometabolic state, modulation of glutathione S-transferase isozyme patterns, and the regulation of the unfolded protein response. PMID:23507570

  2. Sulfide toxicity: Mechanical ventilation and hypotension determine survival rate and brain necrosis

    SciTech Connect

    Baldelli, R.J.; Green, F.H.Y.; Auer, R.N. )

    1993-09-01

    Occupational exposure to hydrogen sulfide is one of the leading causes of sudden death in the workplace, especially in the oil and gas industry. High-dose exposure causes immediate neurogenic apnea and death; lower doses cause [open quotes]knockdown[close quotes] (transient loss of consciousness, with apnea). Because permanent neurological sequelae have been reported, the authors sought to determine whether sulfide can directly kill central nervous system neurons. Ventilated and unventilated rats were studied to allow administration of higher doses of sulfide and to facilitate physiological monitoring. It was extremely difficult to produce cerebral necrosis with sulfide. Only one of eight surviving unventilated rats given high-dose sulfide (a dose that was lethal in [ge]50% of animals) showed cerebral necrosis. Mechanical ventilation shifted the dose that was lethal in 50% of the animals to 190 mg/kg from 94 mg/kg in the unventilated rats. Sulfide was found to potently depress blood pressure. Cerebral necrosis was absent in the ventilated rats (n = 11), except in one rat that showed profound and sustained hypotension to [le]35 Torr. Electroencephalogram activity ceased during exposure but recovered when the animals regained consciousness. The authors conclude that very-high-dose sulfide is incapable of producing cerebral necrosis by a direct histotoxic effect. 32 refs., 5 figs.

  3. A prognostic index for survival among mechanically ventilated hematopoietic cell transplant recipients.

    PubMed

    Solh, Melhem; Oommen, Sanjay; Vogel, Rachel Isaksson; Shanley, Ryan; Majhail, Navneet S; Burns, Linda J

    2012-09-01

    The prognosis of recipients of allogeneic hematopoietic cell transplantation (HCT) who require mechanical ventilation (MV) has historically been poor. Of 883 adults undergoing allogeneic HCT at the University of Minnesota between 1998 and 2009, 179 (20%) required MV before day 100 posttransplantation. We evaluated the outcomes of these patients to develop a prognostic index to predict the 100-day post-MV overall survival (OS) based on factors present at the time of MV. The 179 patients were divided at random into a training set (n = 119) and a validation set (n = 60). The 100-day postventilation OS was 17% for the total population. Multivariate Cox regression on the training set identified creatinine <2 mg/dL and platelet count >20 × 10(9)/L as significant predictors of better OS. Recursive partitioning classified patients with these good prognostic criteria into class A (n = 76); all other patients were classified as class B (n = 103). Among class A patients, 100-day OS was 29% in the training set and 30% in the validation set. Corresponding OS in class B patients was 5% and 15%, respectively. This prognostic index should help guide physicians in counseling HCT patients and their families regarding the use of MV and potential outcomes. PMID:22387348

  4. Mechanisms of spinal motoneurons survival in rats under simulated hypogravity on earth

    NASA Astrophysics Data System (ADS)

    Islamov, R. R.; Mishagina, E. A.; Tyapkina, O. V.; Shajmardanova, G. F.; Eremeev, A. A.; Kozlovskaya, I. B.; Nikolskij, E. E.; Grigorjev, A. I.

    2011-05-01

    It was previously shown that different cell types in vivo and in vitro may die via apoptosis under weightlessness conditions in space as well as in simulated hypogravity on the Earth. We assessed survivability of spinal motoneurons of rats after 35-day antiorthostatic hind limb suspension. Following weight bearing, unloading the total protein content in lumbar spinal cord is dropped by 21%. The electrophysiological studies of m. gastrocnemius revealed an elevated motoneurons' reflex excitability and conduction disturbances in the sciatic nerve axons. The number of myelinated fibers in the ventral root of experimental animals was insignificantly increased by 35-day of antiorthostatic hind limb suspension, although the retrograde axonal transport was significantly decreased during the first week of simulated hypogravity. The results of the immunohistochemical assay with antibodies against proapoptotic protein caspase 9 and cytotoxicity marker neuron specific nitric oxide synthase (nNOS) and the TUNEL staining did not reveal any signs of apoptosis in motoneurons of suspended and control animals. To examine the possible adaptation mechanisms activated in motoneurons in response to simulated hypogravity we investigated immunoexpression of Hsp25 and Hsp70 in lumbar spinal cord of the rats after 35-day antiorthostatic hind limb suspension. Comparative analysis of the immunohistochemical reaction with anti-Hsp25 antibodies revealed differential staining of motoneurons in intact and experimental animals. The density of immunoprecipitate with anti-Hsp25 antibodies was substantially higher in motoneurons of the 35-day suspended than control rats and the more intensive precipitate in this reaction was observed in motoneuron neuritis. Quantitative analysis of Hsp25 expression demonstrated an increase in the Hsp25 level by 95% in experimental rats compared to the control. The immunoexpression of Hsp70 found no qualitative and quantitative differences in control and experimental

  5. Inhibition of 6-phosphofructo-2-kinase (PFKFB3) induces autophagy as a survival mechanism

    PubMed Central

    2014-01-01

    Background Unlike glycolytic enzymes that directly catabolize glucose to pyruvate, the family of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFBs) control the conversion of fructose-6-phosphate to and from fructose-2,6-bisphosphate, a key regulator of the glycolytic enzyme phosphofructokinase-1 (PFK-1). One family member, PFKFB3, has been shown to be highly expressed and activated in human cancer cells, and derivatives of a PFKFB3 inhibitor, 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO), are currently being developed in clinical trials. However, the effectiveness of drugs such as 3PO that target energetic pathways is limited by survival pathways that can be activated by reduced ATP and nutrient uptake. One such pathway is the process of cellular self-catabolism termed autophagy. We hypothesized that the functional glucose starvation induced by inhibition of PFKFB3 in tumor cells would induce autophagy as a pro-survival mechanism and that inhibitors of autophagy could increase the anti-tumor effects of PFKFB3 inhibitors. Results We found that selective inhibition of PFKFB3 with either siRNA transfection or 3PO in HCT-116 colon adenocarcinoma cells caused a marked decrease in glucose uptake simultaneously with an increase in autophagy based on LC3-II and p62 protein expression, acridine orange fluorescence of acidic vacuoles and electron microscopic detection of autophagosomes. The induction of autophagy caused by PFKFB3 inhibition required an increase in reactive oxygen species since N-acetyl-cysteine blocked both the conversion of LC3-I to LC3-II and the increase in acridine orange fluorescence in acidic vesicles after exposure of HCT-116 cells to 3PO. We speculated that the induction of autophagy might protect cells from the pro-apoptotic effects of 3PO and found that agents that disrupt autophagy, including chloroquine, increased 3PO-induced apoptosis as measured by double staining with Annexin V and propidium iodide in both HCT-116 cells and

  6. In vitro Characterization of the Rapid Cytotoxicity of Anticancer Peptide HPRP-A2 through Membrane Destruction and Intracellular Mechanism against Gastric Cancer Cell Lines

    PubMed Central

    Zhao, Jing; Hao, Xueyu; Liu, Dong; Huang, Yibing; Chen, Yuxin

    2015-01-01

    In this study, HPRP-A2, a synthetic 15-mer cationic peptides with all D-amino acids, effectively inhibited the survival of gastric cell lines in a dose-dependent manner. Gastric tumor cells killing by HPRP-A2 involves a rapid collapse of the membrane integrity and intracellular pathways. Propidium iodide (PI) and lactate dehydrogenase (LDH) assays demonstrated that one-hour treatment with HPRP-A2 led to membrane permeability changes of BGC-823 cells in a dose-dependent manner. Moreover, HPRP-A2 induced apoptosis in BGC-823 cells involves a marked increase in generation of reactive oxygen species (ROS),caspase-3, -8 and -9 activation, a reduction of mitochondrial membrane potential (MMP), and cell cycle arrest in G1 phase. In addition to its inherent cytotoxicity, HPRP-A2 synergized strongly with doxorubicin (DOX) to enhance the efficacy of killing gastric tumor cells in vitro. We believe that HPRP-A2 with all D-amino acids could be a potent candidate of anticancer therapeutics, especially in combination therapy. PMID:26422386

  7. Combined modification of intracellular and extracellular loci on human gonadotropin-releasing hormone receptor provides a mechanism for enhanced expression.

    PubMed

    Maya-Núñez, G; Janovick, J A; Conn, P M

    2000-12-01

    The mammalian gonadotropin-releasing hormone (GnRH) receptor (GnRH-R) has been a therapeutic target for human and animal medicine. This receptor is a unique G-protein-coupled receptor that lacks the intracellular C-terminal domain commonly associated with this family. Development of highthrough put screens for agents active in humans has been hampered by low expression levels of the hGnRH-R in cellular models. Two sites have attracted the interest of laboratories studying regulation of expression. The chimeric addition of the C-terminal tail from catfish GnRH-R (cfGnRH-R) to the rat GnRH-R significantly augmented receptor expression in GH3 cells. In addition, rodent GnRH-R contains 327 amino acids, but cow, sheep, and human GnRH-R (hGnRH-R) contain 328 residues, the "additional" residue being a Lys 191. Deletion of Lys 191 (del 191) from the hGnRH-R resulted in increased receptor expression levels and decreased internalization rates in both COS-7 and HEK 293 cells. In this study, the combined effect of the addition of the C-tail from cfGnRH-R and deletion of the Lys 191 from the hGnRH-R was compared to expression of the wild-type (WT) or either alteration alone in a transient expression system using primate cells. The altered receptor (hGnRH-R[del 191]-C-tail) showed significantly increased receptor expression at the cell surface compared with the WT or either modification alone. The inositol phosphate response to stimulation was also significantly elevated in response to GnRH agonist. After treatment with a GnRH agonist, the altered receptors showed a slower internalization rate. The homologous steady-state regulation of the WT and the altered receptors was similar, although the response of the altered receptors was significantly decreased. These results suggest that the conformational change in the receptor as a result of the deletion of Lys 191 and the addition of the C-terminus tail substantially increased the steady-state receptor expression and decreased

  8. From mobility to crosstalk. A model of intracellular miRNAs motion may explain the RNAs interaction mechanism on the basis of target subcellular localization.

    PubMed

    Vasilescu, Catalin; Tanase, Mihai; Dragomir, Mihnea; Calin, George A

    2016-10-01

    MicroRNAs (miRNAs), 22 nucleotides long molecules with the function to reduce gene expression by inhibiting mRNA translation through partial complementary to one or more messenger RNA (mRNA) molecules. A single miRNA can reduce the expression levels of hundreds of genes and one mRNA can be a target for many miRNAs. Despite the study models used so far, miRNAs and mRNAs cannot be seen as acting in an isolated manner or even "in pairs". They most likely exert their complex actions through numerous overlapping interrelations. One of the models depicting interdependence of intracytoplasmic RNAs is the crosstalk model. It is based on a competition between several target mRNAs which are regulated by the same miRNA. In this paper, we will discuss the mobility mechanism of miRNAs, recently suggested by data from "single particle tracking" experiments. These data suggests that miRNA intracellular mobility may be of "intermittent active transport"(IAT) type. IAT is a mobility model composed by alternation of active transport (AT) and Brownian motion (BM). Based on a mathematical model, we concluded that, AT phase may explain the efficiency in reaching far targets and the BM phase may explain the competition. Furthermore, we suggest that the interaction between miRNAs and their targets depends on the concentration of the molecules, the affinity between the molecules and also on the intracellular localization of the molecules. Hence, the probability that a miRNA interacts with its target depends also on the distance to the target and the macromolecular crowding. Taken together, our data proposes an intracytoplasmic mobility mechanism for miRNA and shows that this model can partially explain the RNA crosstalk. PMID:27498347

  9. A Robust In Vivo-Like Persistent Firing Supported by a Hybrid of Intracellular and Synaptic Mechanisms

    PubMed Central

    Jochems, Arthur; Yoshida, Motoharu

    2015-01-01

    Persistent firing is believed to support short-term information retention in the brain. Established hypotheses make use of the recurrent synaptic connectivity to support persistent firing. However, this mechanism is known to suffer from a lack of robustness. On the other hand, persistent firing can be supported by an intrinsic cellular mechanism in multiple brain areas. However, the consequences of having both the intrinsic and the synaptic mechanisms (a hybrid model) on persistent firing remain largely unknown. The goal of this study is to investigate whether a hybrid neural network model with these two mechanisms has advantages over a conventional recurrent network based model. Our computer simulations were based on in vitro recordings obtained from hippocampal CA3 pyramidal cells under cholinergic receptor activation. Calcium activated non-specific cationic (CAN) current supported persistent firing in the Hodgkin-Huxley style cellular models. Our results suggest that the hybrid model supports persistent firing within a physiological frequency range over a wide range of different parameters, eliminating parameter sensitivity issues generally recognized in network based persistent firing. In addition, persistent firing in the hybrid model is substantially more robust against distracting inputs, can coexist with theta frequency oscillations, and supports pattern completion. PMID:25901969

  10. T-Type voltage-sensitive calcium channels mediate mechanically-induced intracellular calcium oscillations in osteocytes by regulating endoplasmic reticulum calcium dynamics.

    PubMed

    Brown, Genevieve N; Leong, Pui L; Guo, X Edward

    2016-07-01

    One of the earliest responses of bone cells to mechanical stimuli is a rise in intracellular calcium (Ca(2+)), and osteocytes in particular exhibit robust oscillations in Ca(2+) when subjected to loading. Previous studies implicate roles for both the endoplasmic reticulum (ER) and T-Type voltage-sensitive calcium channels (VSCC) in these responses, but their interactions or relative contributions have not been studied. By observing Ca(2+) dynamics in the cytosol (Ca(2+)cyt) and the ER (Ca(2+)ER), the focus of this study was to explore the role of the ER and T-Type channels in Ca(2+) signaling in bone cells. We demonstrate that inhibition of T-Type VSCC in osteocytes significantly reduces the number of Ca(2+)cyt responses and affects Ca(2+)ER depletion dynamics. Simultaneous observation of Ca(2+) exchange among these spaces revealed high synchrony between rises in Ca(2+)cyt and depressions in Ca(2+)ER, and this synchrony was significantly reduced by challenging T-Type VSCC. We further confirmed that this effect was mediated directly through the ER and not through store-operated Ca(2+) entry (SOCE) pathways. Taken together, our data suggests that T-Type VSCC facilitate the recovery of Ca(2+)ER in osteocytes to sustain mechanically-induced Ca(2+) oscillations, uncovering a new mechanism underlying the behavior of osteocytes as mechanosensors. PMID:27108342

  11. Adaptive Memory: Determining the Proximate Mechanisms Responsible for the Memorial Advantages of Survival Processing

    ERIC Educational Resources Information Center

    Burns, Daniel J.; Burns, Sarah A.; Hwang, Ana J.

    2011-01-01

    J. S. Nairne, S. R. Thompson, and J. N. S. Pandeirada (2007) suggested that our memory systems may have evolved to help us remember fitness-relevant information and showed that retention of words rated for their relevance to survival is superior to that of words encoded under other deep processing conditions. The authors present 4 experiments that…

  12. Exploring mechanisms of survival in rainbow trout selectively bred for increased resistance to Flavobacterium psychrophilum

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A challenge for selective breeding programs is to better understand how artificial selection alters host pathophysiologic and immunologic response following pathogen exposure. The National Center for Cool and Cold Water Aquaculture is exploring this in rainbow trout bred for increased survival (ARS...

  13. Nanotubes connect CD4+ T cells to airway smooth muscle cells: novel mechanism of T cell survival.

    PubMed

    Al Heialy, Saba; Zeroual, Melissa; Farahnak, Soroor; McGovern, Toby; Risse, Paul-André; Novali, Mauro; Lauzon, Anne-Marie; Roman, Horia N; Martin, James G

    2015-06-15

    Contact between airway smooth muscle (ASM) cells and activated CD4(+) T cells, a key interaction in diseases such as asthma, triggers ASM cell proliferation and enhances T cell survival. We hypothesized that direct contact between ASM and CD4(+) T cells facilitated the transfer of anti-apoptotic proteins via nanotubes, resulting in increased survival of activated CD4(+) T cells. CD4(+) T cells, isolated from PBMCs of healthy subjects, when activated and cocultured with ASM cells for 24 h, formed nanotubes that were visualized by immunofluorescence and atomic force microscopy. Cell-to-cell transfer of the fluorescent dye calcein-AM confirmed cytoplasmic communication via nanotubes. Immunoreactive B cell lymphoma 2 (Bcl-2) and induced myeloid leukemia cell differentiation protein (Mcl-1), two major anti-apoptotic proteins, were present within the nanotubes. Downregulation of Mcl-1 by small interfering RNA in ASM cells significantly increased T cell apoptosis, whereas downregulation of Bcl-2 had no effect. Transfer of GFP-tagged Mcl-1 from ASM cells to CD4(+) T cells via the nanotubes confirmed directionality of transfer. In conclusion, activated T cells communicate with ASM cells via nanotube formation. Direct transfer of Mcl-1 from ASM to CD(+) T cells via nanotubes is involved in T cell survival. This study provides a novel mechanism of survival of CD4(+) T cells that is dependent on interaction with a structural cell. PMID:25934863

  14. The amyloid precursor protein (APP) intracellular domain regulates translation of p44, a short isoform of p53, through an IRES-dependent mechanism.

    PubMed

    Li, Mi; Pehar, Mariana; Liu, Yan; Bhattacharyya, Anita; Zhang, Su-Chun; O'Riordan, Kenneth J; Burger, Corinna; D'Adamio, Luciano; Puglielli, Luigi

    2015-10-01

    p44 is a short isoform of the tumor suppressor protein p53 that is regulated in an age-dependent manner. When overexpressed in the mouse, it causes a progeroid phenotype that includes premature cognitive decline, synaptic defects, and hyperphosphorylation of tau. The hyperphosphorylation of tau has recently been linked to the ability of p44 to regulate transcription of relevant tau kinases. Here, we report that the amyloid precursor protein (APP) intracellular domain (AICD), which results from the processing of the APP, regulates translation of p44 through a cap-independent mechanism that requires direct binding to the second internal ribosome entry site (IRES) of the p53 mRNA. We also report that AICD associates with nucleolin, an already known IRES-specific trans-acting factor that binds with p53 IRES elements and regulates translation of p53 isoforms. The potential biological impact of our findings was assessed in a mouse model of Alzheimer's disease. In conclusion, our study reveals a novel aspect of AICD and p53/p44 biology and provides a possible molecular link between APP, p44, and tau. PMID:26174856

  15. Mechanism of antifungal activity of antimicrobial peptide APP, a cell-penetrating peptide derivative, against Candida albicans: intracellular DNA binding and cell cycle arrest.

    PubMed

    Li, Lirong; Sun, Jin; Xia, Shufang; Tian, Xu; Cheserek, Maureen Jepkorir; Le, Guowei

    2016-04-01

    We investigated the antifungal properties and anti-candidal mechanism of antimicrobial peptide APP. The minimum inhibitory concentration of APP was 8 μM against Candida albicans and Aspeogillus flavus, the concentration against Saccharomyces cerevisiae and Cryptococcus neoformans was 16 μM, while 32 μM inhibited Aspergilla niger and Trichopyton rubrum. APP caused slight depolarization (12.32 ± 0.87%) of the membrane potential of intact C. albicans cells when it exerted its anti-candidal activity and only caused 21.52 ± 0.48% C. albicans cell membrane damage. APP interacted with cell wall membrane, caused potassium efflux and nucleotide leakage. However, confocal fluorescence microscopy experiment and flow cytometry confirmed that FITC-labeled APP penetrated C. albicans cell membrane with 52.31 ± 1.88% cell-penetrating efficiency and accumulated in the cytoplasm. Then, APP interact with C. albicans genomic DNA and completely suppressed DNA migration above weight ratio (peptide/DNA) of 2, and significantly arrested cell cycles during the S-phase (S-phase cell population was 27.09 ± 0.73%, p < 0.05) after penetrating the cell membrane. Results indicated that APP kills C. albicans for efficient cell-penetrating efficiency, strong DNA-binding affinity and significant physiological changes inducing S-phase arrest in intracellular environment. PMID:26743655

  16. Molecular Mechanism for the Control of Eukaryotic Elongation Factor 2 Kinase by pH: Role in Cancer Cell Survival

    PubMed Central

    Xie, Jianling; Mikolajek, Halina; Pigott, Craig R.; Hooper, Kelly J.; Mellows, Toby; Moore, Claire E.; Mohammed, Hafeez; Werner, Jörn M.; Thomas, Gareth J.

    2015-01-01

    Acidification of the extracellular and/or intracellular environment is involved in many aspects of cell physiology and pathology. Eukaryotic elongation factor 2 kinase (eEF2K) is a Ca2+/calmodulin-dependent kinase that regulates translation elongation by phosphorylating and inhibiting eEF2. Here we show that extracellular acidosis elicits activation of eEF2K in vivo, leading to enhanced phosphorylation of eEF2. We identify five histidine residues in eEF2K that are crucial for the activation of eEF2K during acidosis. Three of them (H80, H87, and H94) are in its calmodulin-binding site, and their protonation appears to enhance the ability of calmodulin to activate eEF2K. The other two histidines (H227 and H230) lie in the catalytic domain of eEF2K. We also identify His108 in calmodulin as essential for activation of eEF2K. Acidification of cancer cell microenvironments is a hallmark of malignant solid tumors. Knocking down eEF2K in cancer cells attenuated the decrease in global protein synthesis when cells were cultured at acidic pH. Importantly, activation of eEF2K is linked to cancer cell survival under acidic conditions. Inhibition of eEF2K promotes cancer cell death under acidosis. PMID:25776553

  17. Early palliative care and metastatic non-small cell lung cancer: potential mechanisms of prolonged survival.

    PubMed

    Irwin, Kelly E; Greer, Joseph A; Khatib, Jude; Temel, Jennifer S; Pirl, William F

    2013-02-01

    Patients with advanced cancer experience a significant burden of physical symptoms and psychological distress at the end of life, and many elect to receive aggressive cancer-directed therapy. The goal of palliative care is to relieve suffering and promote quality of life (QOL) for patients and families. Traditionally, both the public and medical community have conceptualized the need for patients to make a choice between pursuing curative therapy or receiving palliative care. However, practice guidelines from the World Health Organization and leadership from the oncology and palliative care communities advocate a different model of palliative care that is introduced from the point of diagnosis of life-threatening illness. Early palliative care has been shown to provide benefits in QOL, mood, and health care utilization. Additionally, preliminary research has suggested that in contrast to fears about palliative care hastening death, referral to palliative care earlier in the course of illness may have the potential to lengthen survival, particularly in patients with advanced nonsmall-cell lung cancer. This review summarizes the literature on potential survival benefits of palliative care and presents a model of how early integrated palliative care could potentially influence survival in patients with advanced cancer. PMID:23355404

  18. Mitochondrial dysfunction and intracellular calcium dysregulation in ALS

    PubMed Central

    Kawamata, Hibiki; Manfredi, Giovanni

    2010-01-01

    Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder that affects the aging population. A progressive loss of motor neurons in the spinal cord and brain leads to muscle paralysis and death. As in other common neurodegenerative diseases, aging-related mitochondrial dysfunction is increasingly being considered among the pathogenic factors. Mitochondria are critical for cell survival: they provide energy to the cell, buffer intracellular calcium, and regulate apoptotic cell death. Whether mitochondrial abnormalities are a trigger or a consequence of the neurodegenerative process and the mechanisms whereby mitochondrial dysfunction contributes to disease are not clear yet. Calcium homeostasis is a major function of mitochondria in neurons, and there is ample evidence that intracellular calcium is dysregulated in ALS. The impact of mitochondrial dysfunction on intracellular calcium homeostasis and its role in motor neuron demise are intriguing issues that warrants in depth discussion. Clearly, unraveling the causal relationship between mitochondrial dysfunction, calcium dysregulation, and neuronal death is critical for the understanding of ALS pathogenesis. In this review, we will outline the current knowledge of various aspects of mitochondrial dysfunction in ALS, with a special emphasis on the role of these abnormalities on intracellular calcium handling. PMID:20493207

  19. Biochemical Mechanisms and Energy Strategies of Geobacter sulfurreducens for Long- Term Survival

    NASA Astrophysics Data System (ADS)

    Helmus, R. A.; Liermann, L. J.; Brantley, S. L.; Tien, M.

    2008-12-01

    Numerous species of bacteria have been observed to exhibit a growth advantage in stationary phase (GASP) phenotype, indicating that microorganisms starved of an energy source may adapt to allow for long-term survival. Understanding how Geobacter sulfurreducens persists using various metal forms as energy sources and whether a GASP phenotype develops during long-term growth are important for efficient application of this bacterium to sites requiring engineered bioremediation of soluble metals. Thus, we investigated the growth kinetics and survival of G. sulfurreducens. The growth rate of G. sulfurreducens was highest when cultured with soluble iron and generally higher on iron oxide than manganese oxide, suggesting that soluble metal forms are more readily utilized as energy sources by G. sulfurreducens. By monitoring the abundance of G. sulfurreducens in batch cultures for >6 months, distinct growth, stationary, and prolonged starvation phases were observed and a cell density of 105- 106 cells/mL persisted under long-term starvation conditions. The outgrowth of an aged G. sulfurreducens strain co-cultured with a young strain was monitored as a measure of the existence of the GASP phenotype. As the strains aged, the rpoS gene was cloned and sequenced at different stages of growth to identify mutations corresponding to a growth advantage. The results of these studies provide insight into the use of various metal forms for growth by G. sulfurreducens and its ability to persist when starved of energy sources.

  20. Improved Vascular Survival and Growth in the Mouse Model of Hindlimb Ischemia by a Remote Signaling Mechanism

    PubMed Central

    Takeda, Kotaro; Duan, Li-Juan; Takeda, Hiromi; Fong, Guo-Hua

    2015-01-01

    Deficiencies in prolyl hydroxylase domain proteins (PHDs) may lead to the accumulation of hypoxia-inducible factor-α proteins, the latter of which activate local angiogenic responses by paracrine mechanisms. Here, we investigate whether a keratinocyte-specific PHD deficiency may promote vascular survival and growth in a distantly located ischemic tissue by a remote signaling mechanism. We generated mice that carry a keratinocyte-specific Phd2 knockout (kPhd2KO) and performed femoral artery ligation. Relative to wild-type controls, kPhd2KO mice displayed improved vascular survival and arteriogenesis in ischemic hind limbs, leading to the accelerated recovery of hindlimb perfusion and superior muscle regeneration. Similar protective effects were also seen in type 1 and type 2 diabetic mice. Molecularly, both abundance of hypoxia-inducible factor-1α protein and expression of vascular endothelial growth factor-A were increased in epidermal tissues of kPhd2KO mice, accompanied by increased plasma concentration of vascular endothelial growth factor-A. Contrary to kPhd2KO mice, which are PHD2 deficient in all skin tissues, localized kPhd2KO in hindlimb skin tissues did not have similar effects, excluding paracrine signaling as a major mechanism. Confirming the existence of remote effects, hepatocyte-specific Phd2 knockout also protected hind limbs from ischemia injury. These data indicate that vascular survival and growth in ischemia-injured tissue may be stimulated by suppressing PHD2 in a remotely located tissue and may provide highly effective angiogenesis therapies without the need for directly accessing target tissues. PMID:24440788

  1. Activation of Akt pathway by transcription-independent mechanisms of retinoic acid promotes survival and invasion in lung cancer cells

    PubMed Central

    2013-01-01

    Background All-trans retinoic acid (ATRA) is currently being used in clinical trials for cancer treatment. The use of ATRA is limited because some cancers, such as lung cancer, show resistance to treatment. However, little is known about the molecular mechanisms that regulate resistance to ATRA treatment. Akt is a kinase that plays a key role in cell survival and cell invasion. Akt is often activated in lung cancer, suggesting its participation in resistance to chemotherapy. In this study, we explored the hypothesis that activation of the Akt pathway promotes resistance to ATRA treatment at the inhibition of cell survival and invasion in lung cancer. We aimed to provide guidelines for the proper use of ATRA in clinical trials and to elucidate basic biological mechanisms of resistance. Results We performed experiments using the A549 human lung adenocarcinoma cell line. We found that ATRA treatment promotes PI3k-Akt pathway activation through transcription-independent mechanisms. Interestingly, ATRA treatment induces the translocation of RARα to the plasma membrane, where it colocalizes with Akt. Immunoprecipitation assays showed that ATRA promotes Akt activation mediated by RARα-Akt interaction. Activation of the PI3k-Akt pathway by ATRA promotes invasion through Rac-GTPase, whereas pretreatment with 15e (PI3k inhibitor) or over-expression of the inactive form of Akt blocks ATRA-induced invasion. We also found that treatment with ATRA induces cell survival, which is inhibited by 15e or over-expression of an inactive form of Akt, through a subsequent increase in the levels of the active form of caspase-3. Finally, we showed that over-expression of the active form of Akt significantly decreases expression levels of the tumor suppressors RARβ2 and p53. In contrast, over-expression of the inactive form of Akt restores RARβ2 expression in cells treated with ATRA, indicating that activation of the PI3k-Akt pathway inhibits the expression of ATRA target genes

  2. Interactions of Oryza sativa OsCONTINUOUS VASCULAR RING-LIKE 1 (OsCOLE1) and OsCOLE1-INTERACTING PROTEIN reveal a novel intracellular auxin transport mechanism.

    PubMed

    Liu, Fei; Zhang, Lan; Luo, Yanzhong; Xu, Miaoyun; Fan, Yunliu; Wang, Lei

    2016-10-01

    Little is known about the transport mechanism of intracellular auxin. Here, we report two vacuole-localized proteins, Oryza sativa OsCONTINUOUS VASCULAR RING-LIKE 1 (OsCOLE1) and OsCOLE1-INTERACTING PROTEIN (OsCLIP), that regulate intracellular auxin transport and homoeostasis. Overexpression of OsCOLE1 markedly increased the internode length and auxin content of the stem base, whereas these parameters were decreased in RNA interference (RNAi) plants. OsCOLE1 was localized on the tonoplast and preferentially expressed in mature tissues. We further identified its interacting protein OsCLIP, which was co-localized on the tonoplast. Protein-protein binding assays demonstrated that the N-terminus of OsCOLE1 directly interacted with OsCLIP in yeast cells and the rice protoplast. Furthermore, (3) H-indole-3-acetic acid ((3) H-IAA) transport assays revealed that OsCLIP transported IAA into yeast cells, which was promoted by OsCOLE1. The results indicate that OsCOLE1 affects rice development by regulating intracellular auxin transport through interaction with OsCLIP, which provides a new insight into the regulatory mechanism of intracellular transport of auxin and the roles of vacuoles in plant development. PMID:27265035

  3. Indoprofen Upregulates the Survival Motor Neuron Protein through a Cyclooxygenase-Independent Mechanism

    PubMed Central

    Lunn, Mitchell R.; Root, David E.; Martino, Allison M.; Flaherty, Stephen P.; Kelley, Brian P.; Coovert, Daniel D.; Burghes, Arthur H.; Man, Nguyen thi; Morris, Glenn E.; Zhou, Jianhua; Androphy, Elliot J.; Sumner, Charlotte J.; Stockwell, Brent R.

    2011-01-01

    Most patients with the pediatric neurodegenerative disease spinal muscular atrophy have a homozygous deletion of the survival motor neuron 1 (SMN1) gene, but retain one or more copies of the closely related SMN2gene. TheSMN2gene encodes the same protein (SMN) but produces it at a low efficiency compared with the SMN1 gene. We performed a high-throughput screen of ~47,000 compounds to identify those that increase production of an SMN2-luciferase reporter protein, but not an SMN1-luciferase reporter protein. Indoprofen, a nonsteroidal anti-inflammatory drug (NSAID) and cyclooxygenase (COX) inhibitor, selectively increased SMN2-luciferase reporter protein and endogenous SMN protein and caused a 5-fold increase in the number of nuclear gems in fibroblasts from SMA patients. No other NSAIDs or COX inhibitors tested exhibited this activity. PMID:15555999

  4. p62/SQSTM1 upregulation constitutes a survival mechanism that occurs during granulocytic differentiation of acute myeloid leukemia cells

    PubMed Central

    Trocoli, A; Bensadoun, P; Richard, E; Labrunie, G; Merhi, F; Schläfli, A M; Brigger, D; Souquere, S; Pierron, G; Pasquet, J-M; Soubeyran, P; Reiffers, J; Ségal-Bendirdjian, E; Tschan, M P; Djavaheri-Mergny, M

    2014-01-01

    The p62/SQSTM1 adapter protein has an important role in the regulation of several key signaling pathways and helps transport ubiquitinated proteins to the autophagosomes and proteasome for degradation. Here, we investigate the regulation and roles of p62/SQSTM1 during acute myeloid leukemia (AML) cell maturation into granulocytes. Levels of p62/SQSTM1 mRNA and protein were both significantly increased during all-trans retinoic acid (ATRA)-induced differentiation of AML cells through a mechanism that depends on NF-κB activation. We show that this response constitutes a survival mechanism that prolongs the life span of mature AML cells and mitigates the effects of accumulation of aggregated proteins that occurs during granulocytic differentiation. Interestingly, ATRA-induced p62/SQSTM1 upregulation was impaired in maturation-resistant AML cells but was reactivated when differentiation was restored in these cells. Primary blast cells of AML patients and CD34+ progenitors exhibited significantly lower p62/SQSTM1 mRNA levels than did mature granulocytes from healthy donors. Our results demonstrate that p62/SQSTM1 expression is upregulated in mature compared with immature myeloid cells and reveal a pro-survival function of the NF-κB/SQSTM1 signaling axis during granulocytic differentiation of AML cells. These findings may help our understanding of neutrophil/granulocyte development and will guide the development of novel therapeutic strategies for refractory and relapsed AML patients with previous exposure to ATRA. PMID:25034783

  5. Intracellular Accumulation of Glycine in Polyphosphate-Accumulating Organisms in Activated Sludge, a Novel Storage Mechanism under Dynamic Anaerobic-Aerobic Conditions

    PubMed Central

    Nguyen, Hien Thi Thu; Kristiansen, Rikke; Vestergaard, Mette; Wimmer, Reinhard

    2015-01-01

    Dynamic anaerobic-aerobic feast-famine conditions are applied to wastewater treatment plants to select polyphosphate-accumulating organisms to carry out enhanced biological phosphorus removal. Acetate is a well-known substrate to stimulate this process, and here we show that different amino acids also are suitable substrates, with glycine as the most promising. 13C-labeled glycine and nuclear magnetic resonance (NMR) were applied to investigate uptake and potential storage products when activated sludge was fed with glycine under anaerobic conditions. Glycine was consumed by the biomass, and the majority was stored intracellularly as free glycine and fermentation products. Subsequently, in the aerobic phase without addition of external substrate, the stored glycine was consumed. The uptake of glycine and oxidation of intracellular metabolites took place along with a release and uptake of orthophosphate, respectively. Fluorescence in situ hybridization combined with microautoradiography using 3H-labeled glycine revealed uncultured actinobacterial Tetrasphaera as a dominant glycine consumer. Experiments with Tetrasphaera elongata as representative of uncultured Tetrasphaera showed that under anaerobic conditions it was able to take up labeled glycine and accumulate this and other labeled metabolites to an intracellular concentration of approximately 4 mM. All components were consumed under subsequent aerobic conditions. Intracellular accumulation of amino acids seems to be a novel storage strategy for polyphosphate-accumulating bacteria under dynamic anaerobic-aerobic feast-famine conditions. PMID:25956769

  6. Intracellular Accumulation of Glycine in Polyphosphate-Accumulating Organisms in Activated Sludge, a Novel Storage Mechanism under Dynamic Anaerobic-Aerobic Conditions.

    PubMed

    Nguyen, Hien Thi Thu; Kristiansen, Rikke; Vestergaard, Mette; Wimmer, Reinhard; Nielsen, Per Halkjær

    2015-07-01

    Dynamic anaerobic-aerobic feast-famine conditions are applied to wastewater treatment plants to select polyphosphate-accumulating organisms to carry out enhanced biological phosphorus removal. Acetate is a well-known substrate to stimulate this process, and here we show that different amino acids also are suitable substrates, with glycine as the most promising. (13)C-labeled glycine and nuclear magnetic resonance (NMR) were applied to investigate uptake and potential storage products when activated sludge was fed with glycine under anaerobic conditions. Glycine was consumed by the biomass, and the majority was stored intracellularly as free glycine and fermentation products. Subsequently, in the aerobic phase without addition of external substrate, the stored glycine was consumed. The uptake of glycine and oxidation of intracellular metabolites took place along with a release and uptake of orthophosphate, respectively. Fluorescence in situ hybridization combined with microautoradiography using (3)H-labeled glycine revealed uncultured actinobacterial Tetrasphaera as a dominant glycine consumer. Experiments with Tetrasphaera elongata as representative of uncultured Tetrasphaera showed that under anaerobic conditions it was able to take up labeled glycine and accumulate this and other labeled metabolites to an intracellular concentration of approximately 4 mM. All components were consumed under subsequent aerobic conditions. Intracellular accumulation of amino acids seems to be a novel storage strategy for polyphosphate-accumulating bacteria under dynamic anaerobic-aerobic feast-famine conditions. PMID:25956769

  7. A general mechanism for intracellular toxicity of metal-containing nanoparticles† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr01234h Click here for additional data file.

    PubMed Central

    Sabella, Stefania; Carney, Randy P.; Brunetti, Virgilio; Malvindi, Maria Ada; Al-Juffali, Noura; Vecchio, Giuseppe; Janes, Sam M.; Bakr, Osman M.; Cingolani, Roberto

    2014-01-01

    The assessment of the risks exerted by nanoparticles is a key challenge for academic, industrial, and regulatory communities worldwide. Experimental evidence points towards significant toxicity for a range of nanoparticles both in vitro and in vivo. Worldwide efforts aim at uncovering the underlying mechanisms for this toxicity. Here, we show that the intracellular ion release elicited by the acidic conditions of the lysosomal cellular compartment – where particles are abundantly internalized – is responsible for the cascading events associated with nanoparticles-induced intracellular toxicity. We call this mechanism a “lysosome-enhanced Trojan horse effect” since, in the case of nanoparticles, the protective cellular machinery designed to degrade foreign objects is actually responsible for their toxicity. To test our hypothesis, we compare the toxicity of similar gold particles whose main difference is in the internalization pathways. We show that particles known to pass directly through cell membranes become more toxic when modified so as to be mostly internalized by endocytosis. Furthermore, using experiments with chelating and lysosomotropic agents, we found that the toxicity mechanism for different metal containing NPs (such as metallic, metal oxide, and semiconductor NPs) is mainly associated with the release of the corresponding toxic ions. Finally, we show that particles unable to release toxic ions (such as stably coated NPs, or diamond and silica NPs) are not harmful to intracellular environments. PMID:24842463

  8. Bacterial biofilm mechanical properties persist upon antibiotic treatment and survive cell death

    NASA Astrophysics Data System (ADS)

    Zrelli, K.; Galy, O.; Latour-Lambert, P.; Kirwan, L.; Ghigo, J. M.; Beloin, C.; Henry, N.

    2013-12-01

    Bacteria living on surfaces form heterogeneous three-dimensional consortia known as biofilms, where they exhibit many specific properties one of which is an increased tolerance to antibiotics. Biofilms are maintained by a polymeric network and display physical properties similar to that of complex fluids. In this work, we address the question of the impact of antibiotic treatment on the physical properties of biofilms based on recently developed tools enabling the in situ mapping of biofilm local mechanical properties at the micron scale. This approach takes into account the material heterogeneity and reveals the spatial distribution of all the small changes that may occur in the structure. With an Escherichia coli biofilm, we demonstrate using in situ fluorescent labeling that the two antibiotics ofloxacin and ticarcillin—targeting DNA replication and membrane assembly, respectively—induced no detectable alteration of the biofilm mechanical properties while they killed the vast majority of the cells. In parallel, we show that a proteolytic enzyme that cleaves extracellular proteins into short peptides, but does not alter bacterial viability in the biofilm, clearly affects the mechanical properties of the biofilm structure, inducing a significant increase of the material compliance. We conclude that conventional biofilm control strategy relying on the use of biocides targeting cells is missing a key target since biofilm structural integrity is preserved. This is expected to efficiently promote biofilm resilience, especially in the presence of persister cells. In contrast, the targeting of polymer network cross-links—among which extracellular proteins emerge as major players—offers a promising route for the development of rational multi-target strategies to fight against biofilms.

  9. Delivery of host cell-directed therapeutics for intracellular pathogen clearance

    PubMed Central

    Collier, Michael A.; Gallovic, Matthew D.; Peine, Kevin J.; Duong, Anthony D.; Bachelder, Eric M.; Gunn, John S.; Schlesinger, Larry S.; Ainslie, Kristy M.

    2014-01-01

    Intracellular pathogens present a major health risk because of their innate ability to evade clearance. Their location within host cells and ability to react to the host environment by mutation or transcriptional changes often enables survival mechanisms to resist standard therapies. Host-directed drugs do not target the pathogen, minimizing the potential development of drug resistance; however, they can be difficult to deliver efficiently to intracellular sites. Vehicle delivery of host-mediated response drugs not only improves drug distribution and toxicity profiles, but can reduce the total amount of drug necessary to clear infection. In this article, we will review some host-directed drugs and current drug delivery techniques that can be used to efficiently clear intracellular infections. PMID:24134600

  10. Bacillus and other spore-forming genera: variations in responses and mechanisms for survival.

    PubMed

    Checinska, Aleksandra; Paszczynski, Andrzej; Burbank, Malcolm

    2015-01-01

    The ubiquity of Bacilli endospores in soils facilitates their easy transfer routes to other environments, including cleanrooms and low-biomass sites required by many industries such as food production and processing. A bacterial endospore is a metabolically dormant form of life that is much more resistant to heat, desiccation, lack of nutrients, exposure to UV and gamma radiation, organic chemicals, and oxidizing agents than is a vegetative cell. For example, the heat tolerance of endospores depends on multiple factors such as sporulation temperature, core dehydration, and the presence of minerals and small, acid-soluble proteins (SASPs) in the core. This review describes our current understanding of the persistence mechanisms related to sporeformers' biochemical properties and discusses in detail spores' heat, radiation, and reactive chemical resistance. In addition, it discusses the impact of contamination with spores on many areas of human activity, spore adhesive properties, and biofilm contribution to resistance. PMID:25705935