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Sample records for inos mediate bacterial

  1. Increases in Calmodulin Abundance and Stabilization of Activated iNOS Mediate Bacterial Killing in RAW 264.7 Macrophages

    SciTech Connect

    Smallwood, Heather S.; Shi, Liang; Squier, Thomas C.

    2006-08-01

    The rapid activation of macrophages in response to bacterial antigens is central to the innate immune system that permits the recognition and killing of pathogens to limit infection. To understand regulatory mechanisms underlying macrophage activation, we have investigated changes in the abundance of calmodulin (CaM) and iNOS in response to the bacterial cell wall component lipopolysaccharide (LPS) using RAW 264.7 macrophages. Critical to these measurements was the ability to differentiate free iNOS from the CaM-bound (active) form of iNOS associated with nitric oxide generation. We observe a rapid two-fold increase in CaM abundance during the first 30 minutes that is blocked by inhibition of NF?B nuclear translocation or protein synthesis. A similar two-fold increase in the abundance of the complex between CaM and iNOS is observed with the same time dependence. In contrast, there are no detectable increases in the CaM-free (i.e., inactive) form of iNOS within the first hour; it remains at a very low abundance during the initial phase of macrophage activation. Increasing cellular CaM levels in stably transfected cells results in a corresponding increase in the abundance of the CaM/iNOS complex that promotes effective bacterial killing following challenge by Salmonella typhimurium. Thus, LPS-dependent increases in CaM abundance function in the stabilization and activation of iNOS on the rapid time-scale associated with macrophage activation and bacterial killing. These results explain how CaM and iNOS coordinately function to form a stable complex that is part of a rapid host-response that functions within the first 30 minutes following bacterial infection to up-regulate the innate immune system involving macrophage activation.

  2. Burkholderia pseudomallei rpoS mediates iNOS suppression in human hepatocyte (HC04) cells.

    PubMed

    Sanongkiet, Sucharat; Ponnikorn, Saranyoo; Udomsangpetch, Rachanee; Tungpradabkul, Sumalee

    2016-08-01

    Burkholderia pseudomallei is an intracellular Gram-negative bacterial pathogen and the causative agent of melioidosis, a widespread disease in Southeast Asia. Reactive nitrogen, in an intermediate form of nitric oxide (NO), is one of the first lines of defense used by host cells to eliminate intracellular pathogens, through the stimulation of inducible nitric oxide synthase (iNOS). Studies in phagocytotic cells have shown that the iNOS response is muted in B. pseudomallei infection, and implicated the rpoS sigma factor as a key regulatory factor mediating suppression. The liver is a main visceral organ affected by B. pseudomallei, and there is little knowledge about the interaction of liver cells and B. pseudomallei This study investigated the induction of iNOS, as well as autophagic flux and light-chain 3 (LC3) localization in human liver (HC04) cells in response to infection with B. pseudomallei and its rpoS deficient mutant. Results showed that the rpoS mutant was unable to suppress iNOS induction and that the mutant showed less induction of autophagy and lower co-localization with LC3, and this was coupled with a lower intracellular growth rate. Combining these results suggest that B. pseudomallei rpoS is an important factor in establishing infection in liver cells. PMID:27324398

  3. Burkholderia pseudomallei rpoS mediates iNOS suppression in human hepatocyte (HC04) cells

    PubMed Central

    Sanongkiet, Sucharat; Ponnikorn, Saranyoo; Udomsangpetch, Rachanee; Tungpradabkul, Sumalee

    2016-01-01

    Burkholderia pseudomallei is an intracellular Gram-negative bacterial pathogen and the causative agent of melioidosis, a widespread disease in Southeast Asia. Reactive nitrogen, in an intermediate form of nitric oxide (NO), is one of the first lines of defense used by host cells to eliminate intracellular pathogens, through the stimulation of inducible nitric oxide synthase (iNOS). Studies in phagocytotic cells have shown that the iNOS response is muted in B. pseudomallei infection, and implicated the rpoS sigma factor as a key regulatory factor mediating suppression. The liver is a main visceral organ affected by B. pseudomallei, and there is little knowledge about the interaction of liver cells and B. pseudomallei. This study investigated the induction of iNOS, as well as autophagic flux and light-chain 3 (LC3) localization in human liver (HC04) cells in response to infection with B. pseudomallei and its rpoS deficient mutant. Results showed that the rpoS mutant was unable to suppress iNOS induction and that the mutant showed less induction of autophagy and lower co-localization with LC3, and this was coupled with a lower intracellular growth rate. Combining these results suggest that B. pseudomallei rpoS is an important factor in establishing infection in liver cells. PMID:27324398

  4. INO80 governs superenhancer-mediated oncogenic transcription and tumor growth in melanoma.

    PubMed

    Zhou, Bingying; Wang, Li; Zhang, Shu; Bennett, Brian D; He, Fan; Zhang, Yan; Xiong, Chengliang; Han, Leng; Diao, Lixia; Li, Pishun; Fargo, David C; Cox, Adrienne D; Hu, Guang

    2016-06-15

    Superenhancers (SEs) are large genomic regions with a high density of enhancer marks. In cancer, SEs are found near oncogenes and dictate cancer gene expression. However, how oncogenic SEs are regulated remains poorly understood. Here, we show that INO80, a chromatin remodeling complex, is required for SE-mediated oncogenic transcription and tumor growth in melanoma. The expression of Ino80, the SWI/SNF ATPase, is elevated in melanoma cells and patient melanomas compared with normal melanocytes and benign nevi. Furthermore, Ino80 silencing selectively inhibits melanoma cell proliferation, anchorage-independent growth, tumorigenesis, and tumor maintenance in mouse xenografts. Mechanistically, Ino80 occupies >90% of SEs, and its occupancy is dependent on transcription factors such as MITF and Sox9. Ino80 binding reduces nucleosome occupancy and facilitates Mediator recruitment, thus promoting oncogenic transcription. Consistently, genes co-occupied by Ino80 and Med1 are selectively expressed in melanomas compared with melanocytes. Together, our results reveal an essential role of INO80-dependent chromatin remodeling in SE function and suggest a novel strategy for disrupting SEs in cancer treatment. PMID:27340176

  5. Phylogenetic distinction of iNOS and IDO function in mesenchymal stem cell-mediated immunosuppression in mammalian species.

    PubMed

    Su, J; Chen, X; Huang, Y; Li, W; Li, J; Cao, K; Cao, G; Zhang, L; Li, F; Roberts, A I; Kang, H; Yu, P; Ren, G; Ji, W; Wang, Y; Shi, Y

    2014-03-01

    Mammalian mesenchymal stem cells (MSCs) have been shown to be strongly immunosuppressive in both animal disease models and human clinical trials. We have reported that the key molecule mediating immunosuppression by MSCs is species dependent: indoleamine 2,3-dioxygenase (IDO) in human and inducible nitric oxide synthase (iNOS) in mouse. In the present study, we isolated MSCs from several mammalian species, each of a different genus, and investigated the involvement of IDO and iNOS during MSC-mediated immunosuppression. The characterization of MSCs from different species was by adherence to tissue culture plastic, morphology, specific marker expression, and differentiation potential. On the basis of the inducibility of IDO and iNOS by inflammatory cytokines in MSCs, the tested mammalian species fall into two distinct groups: IDO utilizers and iNOS utilizers. MSCs from monkey, pig, and human employ IDO to suppress immune responses, whereas MSCs from mouse, rat, rabbit, and hamster utilize iNOS. Interestingly, based on the limited number of species tested, the iNOS-utilizing species all belong to the phylogenetic clade, Glires. Although the evolutionary significance of this divergence is not known, we believe that this study provides critical guidance for choosing appropriate animal models for preclinical studies of MSCs.

  6. Glycogen synthase kinase 3 regulates IL-1β mediated iNOS expression in hepatocytes by down-regulating c-Jun.

    PubMed

    Lakshmanan, Jaganathan; Zhang, Baochun; Nweze, Ikenna C; Du, Yibo; Harbrecht, Brian G

    2015-01-01

    Excessive nitric oxide from the inducible nitric oxide synthase (iNOS) increases shock-induced hepatic injury, hepatic dysfunction, inflammation, and mortality in animal models. Cytokines increase the expression of iNOS in hepatocytes, but the signaling mechanisms involved are not completely understood. We have previously demonstrated that Akt mediates the inhibitory effect of cAMP and insulin on cytokine-induced hepatocyte iNOS expression. We hypothesized that glycogen synthase kinase 3 (GSK3), a target of Akt phosphorylation, would regulate hepatocyte iNOS expression. In cultured rat hepatocytes, GSK3 inhibitors decreased IL-1β mediated nitric oxide (NO) production and iNOS protein expression, while the phosphatidylinositol 3-kinase (PI3K)/Akt pathway inhibitor LY294002 increased the cytokine-mediated NO production and iNOS expression. Over-expression of the constitutively active form of GSK3β enhanced IL-1β-mediated iNOS expression. GSK3 catalyzes the phosphorylation of c-Jun at the c-terminal Thr239 that facilitates c-Jun degradation. Inhibition of GSK3 with SB216763 and lithium chloride significantly reduced, whereas blocking PI3K/Akt increased phosphorylation of c-Jun at Thr239. The levels of total-c-Jun and c-Jun phosphorylated at Ser63 inversely correlated with c-Jun phosphorylated at Thr239, GSK3 activation and iNOS expression. Over-expression of a dominant negative c-Jun not only caused an increase in IL-1β-mediated iNOS promoter activity and iNOS protein expression but was also able to reverse the SB216763-mediated suppression of iNOS. These results demonstrate that GSK3, a downstream target of Akt, regulates IL-1β-stimulated iNOS expression in hepatocytes by directly phosphorylating c-Jun in an inhibitory manner. PMID:25160751

  7. CPEB1 modulates lipopolysaccharide-mediated iNOS induction in rat primary astrocytes

    SciTech Connect

    Kim, Ki Chan; Hyun Joo, So; Shin, Chan Young

    2011-06-17

    Highlights: {yields} Expression and phosphorylation of CPEB1 is increased by LPS stimulation in rat primary astrocytes. {yields} JNK regulates expression and phosphorylation of CPEB1 in reactive astrocytes. {yields} Down-regulation of CPEB1 using siRNA inhibits oxidative stress and iNOS induction by LPS stimulation. {yields} CPEB1 may play an important role in regulating inflammatory responses in reactive astrocytes induced by LPS. -- Abstract: Upon CNS damage, astrocytes undergo a series of biological changes including increased proliferation, production of inflammatory mediators and morphological changes, in a response collectively called reactive gliosis. This process is an essential part of the brains response to injury, yet much is unknown about the molecular mechanism(s) that induce these changes. In this study, we investigated the role of cytoplasmic polyadenylation element binding protein 1 (CPEB1) in the regulation of inflammatory responses in a model of reactive gliosis, lipopolysaccharide-stimulated astrocytes. CPEB1 is an mRNA-binding protein recently shown to be expressed in astrocytes that may play a role in astrocytes migration. After LPS stimulation, the expression and phosphorylation of CPEB1 was increased in rat primary astrocytes in a JNK-dependent process. siRNA-induced knockdown of CPEB1 expression inhibited the LPS-induced up-regulation of iNOS as well as NO and ROS production, a hallmark of immunological activation of astrocytes. The results from the study suggest that CPEB1 is actively involved in the regulation of inflammatory responses in astrocytes, which might provide new insights into the regulatory mechanism after brain injury.

  8. Bacterially mediated mineralization of vaterite

    NASA Astrophysics Data System (ADS)

    Rodriguez-Navarro, Carlos; Jimenez-Lopez, Concepcion; Rodriguez-Navarro, Alejandro; Gonzalez-Muñoz, Maria Teresa; Rodriguez-Gallego, Manuel

    2007-03-01

    Myxococcus xanthus, a common soil bacterium, plays an active role in the formation of spheroidal vaterite. Bacterial production of CO 2 and NH 3 and the transformation of the NH 3 to NH4+ and OH -, thus increasing solution pH and carbonate alkalinity, set the physicochemical conditions (high supersaturation) leading to vaterite precipitation in the microenvironment around cells, and directly onto the surface of bacterial cells. In the latter case, fossilization of bacteria occurs. Vaterite crystals formed by aggregation of oriented nanocrystals with c-axis normal to the bacterial cell-wall, or to the core of the spherulite when bacteria were not encapsulated. While preferred orientation of vaterite c-axis appears to be determined by electrostatic affinity (ionotropic effect) between vaterite crystal (0001) planes and the negatively charged functional groups of organic molecules on the bacterium cell-wall or on extracellular polymeric substances (EPS), analysis of the changes in the culture medium chemistry as well as high resolution transmission electron microscopy (HRTEM) observations point to polymorph selection by physicochemical (kinetic) factors (high supersaturation) and stabilization by organics, both connected with bacterial activity. The latter is in agreement with inorganic precipitation of vaterite induced by NH 3 and CO 2 addition in the protein-rich sterile culture medium. Our results as well as recent studies on vaterite precipitation in the presence of different types of bacteria suggest that bacterially mediated vaterite precipitation is not strain-specific, and could be more common than previously thought.

  9. HIF‑1 signaling pathway involving iNOS, COX‑2 and caspase‑9 mediates the neuroprotection provided by erythropoietin in the retina of chronic ocular hypertension rats.

    PubMed

    Gui, Dongmei; Li, Yanfeng; Chen, Xiaolong; Gao, Dianwen; Yang, Yang; Li, Xun

    2015-02-01

    This study aimed to investigate the impacts of erythropoietin (EPO) on the electroretinogram b‑wave (ERG‑b), and on the mRNA and protein expression levels of hypoxia‑inducible factor‑1α (HIF‑1α), inducible nitric oxide synthase (iNOS), cyclooxygenase‑2 (COX‑2) and caspase‑9 in chronic ocular hypertension rats. Episcleral vein cauterization (EVC) was used to establish the chronic ocular hypertension rat model based on the intraocular pressure (IOP) value. ERG‑b and mRNA and protein expression levels of HIF‑1α, iNOS, COX‑2 and caspase‑9 in normal, EVC‑treated and EVC combined with EPO (EVC+EPO)‑treated rats were measured by electroretinography, RT‑PCR and western blotting, respectively. Moreover, the correlations of HIF‑1α with IOP, ERG‑b, iNOS, COX‑2 and caspase‑9 were evaluated. The mRNA and protein expression levels of HIF‑1α, iNOS, COX‑2 and caspase‑9 in EVC‑treated rats were increased significantly compared with normal rats. The peak expression levels of HIF‑1α, iNOS, COX‑2 and caspase‑9 were respectively obtained 7, 7, 7 and 14 days postoperatively. Compared with EVC‑treated rats, EPO administration weakened the mRNA and protein expression levels of HIF‑1α, iNOS, COX‑2 and caspase‑9. The mRNA expression level of HIF‑1α demonstrated a significant positive correlation with IOP and ERG‑b. HIF‑1α was positively correlated with iNOS, COX‑2 and caspase‑9 at the mRNA and protein levels. The protective effect of EPO on the retina of chronic ocular hypertension rats may be mediated by the HIF‑1 signaling pathway involving iNOS, COX‑2 and caspase‑9.

  10. HIF‑1 signaling pathway involving iNOS, COX‑2 and caspase‑9 mediates the neuroprotection provided by erythropoietin in the retina of chronic ocular hypertension rats.

    PubMed

    Gui, Dongmei; Li, Yanfeng; Chen, Xiaolong; Gao, Dianwen; Yang, Yang; Li, Xun

    2015-02-01

    This study aimed to investigate the impacts of erythropoietin (EPO) on the electroretinogram b‑wave (ERG‑b), and on the mRNA and protein expression levels of hypoxia‑inducible factor‑1α (HIF‑1α), inducible nitric oxide synthase (iNOS), cyclooxygenase‑2 (COX‑2) and caspase‑9 in chronic ocular hypertension rats. Episcleral vein cauterization (EVC) was used to establish the chronic ocular hypertension rat model based on the intraocular pressure (IOP) value. ERG‑b and mRNA and protein expression levels of HIF‑1α, iNOS, COX‑2 and caspase‑9 in normal, EVC‑treated and EVC combined with EPO (EVC+EPO)‑treated rats were measured by electroretinography, RT‑PCR and western blotting, respectively. Moreover, the correlations of HIF‑1α with IOP, ERG‑b, iNOS, COX‑2 and caspase‑9 were evaluated. The mRNA and protein expression levels of HIF‑1α, iNOS, COX‑2 and caspase‑9 in EVC‑treated rats were increased significantly compared with normal rats. The peak expression levels of HIF‑1α, iNOS, COX‑2 and caspase‑9 were respectively obtained 7, 7, 7 and 14 days postoperatively. Compared with EVC‑treated rats, EPO administration weakened the mRNA and protein expression levels of HIF‑1α, iNOS, COX‑2 and caspase‑9. The mRNA expression level of HIF‑1α demonstrated a significant positive correlation with IOP and ERG‑b. HIF‑1α was positively correlated with iNOS, COX‑2 and caspase‑9 at the mRNA and protein levels. The protective effect of EPO on the retina of chronic ocular hypertension rats may be mediated by the HIF‑1 signaling pathway involving iNOS, COX‑2 and caspase‑9. PMID:25370745

  11. Attenuation of iNOS and COX2 by blueberry polyphenols is mediated through the suppression of NF-KB activation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Treatment of BV2 microglial cells with blueberry extracts has been shown to be effective in reducing lipopolysaccharide (LPS)-induced pro-inflammatory mediators such as nitric oxide (NO), tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1Beta), inducible NO synthase (iNOS), and cyclo-...

  12. iNOS null MRL+/+ mice show attenuation of trichloroethene-mediated autoimmunity: contribution of reactive nitrogen species and lipid-derived reactive aldehydes.

    PubMed

    Wang, Gangduo; Wakamiya, Maki; Wang, Jianling; Ansari, G A S; Firoze Khan, M

    2015-12-01

    Earlier studies from our laboratory in MRL+/+ mice suggest that free radicals, especially overproduction of reactive nitrogen species (RNS) and lipid-derived reactive aldehydes (LDRAs), are associated with trichloroethene (TCE)-mediated autoimmune response. The current study was undertaken to further assess the contribution of RNS and LDRAs in TCE-mediated autoimmunity by using iNOS-null MRL+/+ mice. iNOS-null MRL+/+ mice were obtained by backcrossing iNOS-null mice (B6.129P2-Nos2(tm1Lau)/J) to MRL +/+ mice. Female MRL+/+ and iNOS-null MRL+/+ mice were given TCE (10 mmol/kg, i.p., every 4(th) day) for 6 weeks; their respective controls received corn oil only. TCE exposure led to significantly increased iNOS mRNA in livers, iNOS protein in livers and sera, increased nitrotyrosine (NT) formation in both livers and sera, induction of MDA-/HNE-protein adducts in livers and their respective antibodies in sera along with significant increases in serum antinuclear antibodies (ANA) and anti-dsDNA in MRL+/+ mice. Even though in iNOS-null MRL+/+ mice, the iNOS and NT levels were negligible in both TCE-treated and untreated groups, TCE treatment still led to significant increases in MDA-/HNE-protein adducts and their respective antibodies along with increases in serum ANA and anti-dsDNA compared to controls. Most remarkably, the increases in serum ANA and anti-dsDNA induced by TCE in the iNOS-null MRL+/+ mice were significantly less pronounced compared to that in MRL+/+ mice. Our results provide further evidence that both RNS and LDRAs contribute to TCE-induced autoimmunity in MRL+/+ mice, and iNOS deficiency attenuates this autoimmune response.

  13. Human inducible nitric oxide synthase (iNOS) expression depends on chromosome region maintenance 1 (CRM1)- and eukaryotic translation initiation factor 4E (elF4E)-mediated nucleocytoplasmic mRNA transport.

    PubMed

    Bollmann, Franziska; Fechir, Katrin; Nowag, Sebastian; Koch, Kathrin; Art, Julia; Kleinert, Hartmut; Pautz, Andrea

    2013-04-01

    Human inducible nitric oxide synthase (iNOS) is regulated on the expressional level mostly by post-transcriptional mechanisms modulating the mRNA stability. Another important step in the control of eukaryotic gene expression is the nucleocytoplasmic mRNA transport. Most cellular mRNAs are exported via the TAP/Nxt complex of proteins. However, some mRNAs are transported by a different mechanism involving the nuclear export receptor CRM1. Treatment of DLD-1 cells with the CRM1 inhibitor leptomycin B (LMB) or anti-CRM1 siRNAs reduced cytokine-induced iNOS expression. We could demonstrate that the iNOS mRNA is exported from the nucleus in a CRM1-dependent manner. Since CRM1 itself does not possess any RNA binding affinity, an adapter protein is needed to mediate CRM1-dependent mRNA export. Western blot experiments showed that the eukaryotic translation initiation factor eIF4E is retained in the nucleus after LMB treatment. Blockade of eIF4E by ribavirin or overexpression of the promyelocytic leukemia protein (PML) decreased iNOS expression due to reduced iNOS mRNA export from the nucleus. Transfection experiments provide evidence that the 3'-untranslated region of the iNOS mRNA is involved in eIF4E-mediated iNOS mRNA transport. In summary, CRM1 and eIF4E seem to play an important role in the nucleocytoplasmic export of human iNOS mRNA.

  14. Tetramethylpyrazine attenuates TNF-α-induced iNOS expression in human endothelial cells: Involvement of Syk-mediated activation of PI3K-IKK-IκB signaling pathways

    SciTech Connect

    Zheng, Zhen; Li, Zhiliang; Chen, Song; Pan, Jieyi; Ma, Xiaochun

    2013-08-15

    Endothelial cells produce nitric oxide (NO) by activation of constitutive nitric oxide synthase (NOS) and transcription of inducible NO synthase (iNOS). We explored the effect of tetramethylpyrazine (TMP), a compound derived from chuanxiong, on tumor necrosis factor (TNF)-α-induced iNOS in human umbilical vein endothelial cells (HUVECs) and explored the signal pathways involved by using RT-PCR and Western blot. TMP suppressed TNF-α-induced expression of iNOS by inhibiting IκB kinase (IKK) phosphorylation, IκB degradation and nuclear factor κB (NF-κB) nuclear translocation, which were required for NO gene transcription. Exposure to wortmannin abrogated IKK/IκB/NF-κB-mediated iNOS expression, suggesting activation of such a signal pathway might be phosphoinositide-3-kinase (PI3K) dependent. Spleen tyrosine kinase (Syk) inhibitor piceatannol significantly inhibited NO production. Furthermore, piceatannol obviously suppressed TNF-α-induced IκB phosphorylation and the downstream NF-κB activation, suggesting that Syk is an upstream key regulator in the activation of PI3K/IKK/IκB-mediated signaling. TMP significantly inhibited TNF-α-induced phosphorylation of Syk and PI3K. Our data indicate that TMP might repress iNOS expression, at least in part, through its inhibitory effect of Syk-mediated PI3K phosphorylation in TNF-α-stimulated HUVECs. -- Highlights: •TMP suppressed TNF-α-induced expression of iNOS by inhibiting IKK/IκB/NF-κB pathway. •PI3K inhibitor wortmannin abrogated IKK/IκB/NF-κB-mediated iNOS expression. •Syk inhibitor piceatannol repressed PI3K/IKK/IκB mediated NO production. •Syk is an upstream regulator in the activation of PI3K/IKK/IκB-mediated signaling. •TMP might repress iNOS expression through Syk-mediated PI3K pathway.

  15. The naturally occurring biflavonoid, ochnaflavone, inhibits LPS-induced iNOS expression, which is mediated by ERK1/2 via NF-kappaB regulation, in RAW264.7 cells.

    PubMed

    Suh, Seok-Jong; Chung, Tae-Wook; Son, Min-Jung; Kim, Sung-Hoon; Moon, Tae Chul; Son, Kun Ho; Kim, Hyun Pyo; Chang, Hyeun Wook; Kim, Cheorl-Ho

    2006-03-15

    Ochnaflavone (OC), a naturally occurring biflavonoid with anti-inflammatory activity [S.J. Lee, J.H. Choi, H.W. Chang, S.S. Kang, H.P. Kim. Life Sci. 57(6), 1995, 551-558], was isolated from Lonicera japonica and its effects on inducible nitric oxide synthase (iNOS) gene expression was examined in RAW264.7 cells. U0126, an inhibitor of the extracellular signal-regulated kinase (ERK), significantly down-regulated lipopolysaccharide (LPS)-induced iNOS expression and promoter activity. Transactivation of LPS-stimulated NF-kappaB was inhibited by U0126. These results suggest that the transcription factor NF-kappaB is involved in ERK-mediated iNOS regulation and that activation of the Ras/ERK pathway contributes to the induction of iNOS expression in RAW264.7 cells in response to LPS. OC treatment inhibited the production of nitric oxide in a concentration-dependent manner and also blocked the LPS-induced expression of iNOS. These inhibitory effects were associated with reduced ERK1/2 activity. OC inhibited the phosphorylation of c-Jun NH2-terminal kinase (JNK) and p38 mitogen-activated protein kinase. The findings herein show that the inhibition of LPS-induced ERK1/2 activation may be a contributing factor to the main mechanisms by which OC inhibits RAW264.7. To clarify the mechanistic basis for its ability to inhibit iNOS induction, we examined the effect of OC on the transactivation of the iNOS gene by luciferase reporter activity using the -1588 flanking region. OC potently suppressed reporter gene activity. We also report here, for the first time, that LPS-induced iNOS expression was abolished by OC in RAW264.7 cells through by blocking the inhibition of transcription factor NF-kappaB binding activities. These activities are associated with the down-regulation of inhibitor kappaB (IkappaB) kinase (IKK) activity by OC (6 microM), thus inhibiting LPS-induced phosphorylation as well as the degradation of IkappaBalpha. These findings suggest that the inhibition of LPS

  16. Bacterial floc mediated rapid streamer formation in creeping flows

    PubMed Central

    Hassanpourfard, Mahtab; Nikakhtari, Zahra; Ghosh, Ranajay; Das, Siddhartha; Thundat, Thomas; Liu, Yang; Kumar, Aloke

    2015-01-01

    One of the central puzzles concerning the interaction of low Reynolds number fluid transport with bacterial biomass is the formation of filamentous structures called streamers. In this manuscript, we report our discovery of a new kind of low Re bacterial streamers, which appear from pre-formed bacterial flocs. In sharp contrast to the biofilm-mediated streamers, these streamers form over extremely small timescales (less than a second). Our experiments, carried out in a microchannel with micropillars rely on fluorescence microscopy techniques to illustrate that floc-mediated streamers form when a freely-moving floc adheres to the micropillar wall and gets rapidly sheared by the background flow. We also show that at their inception the deformation of the flocs is dominated by recoverable large strains indicating significant elasticity. These strains subsequently increase tremendously to produce filamentous streamers. Interestingly, we find that these fully formed streamers are not static structures and show viscous response at time scales larger than their formation time scales. Finally, we show that such novel streamer formation can lead to rapid clogging of microfluidic devices. PMID:26278133

  17. Bacterial floc mediated rapid streamer formation in creeping flows.

    PubMed

    Hassanpourfard, Mahtab; Nikakhtari, Zahra; Ghosh, Ranajay; Das, Siddhartha; Thundat, Thomas; Liu, Yang; Kumar, Aloke

    2015-01-01

    One of the central puzzles concerning the interaction of low Reynolds number fluid transport with bacterial biomass is the formation of filamentous structures called streamers. In this manuscript, we report our discovery of a new kind of low Re bacterial streamers, which appear from pre-formed bacterial flocs. In sharp contrast to the biofilm-mediated streamers, these streamers form over extremely small timescales (less than a second). Our experiments, carried out in a microchannel with micropillars rely on fluorescence microscopy techniques to illustrate that floc-mediated streamers form when a freely-moving floc adheres to the micropillar wall and gets rapidly sheared by the background flow. We also show that at their inception the deformation of the flocs is dominated by recoverable large strains indicating significant elasticity. These strains subsequently increase tremendously to produce filamentous streamers. Interestingly, we find that these fully formed streamers are not static structures and show viscous response at time scales larger than their formation time scales. Finally, we show that such novel streamer formation can lead to rapid clogging of microfluidic devices. PMID:26278133

  18. Perspective: Adhesion Mediated Signal Transduction in Bacterial Pathogens.

    PubMed

    Moorthy, Sudha; Keklak, Julia; Klein, Eric A

    2016-01-01

    During the infection process, pathogenic bacteria undergo large-scale transcriptional changes to promote virulence and increase intrahost survival. While much of this reprogramming occurs in response to changes in chemical environment, such as nutrient availability and pH, there is increasing evidence that adhesion to host-tissue can also trigger signal transduction pathways resulting in differential gene expression. Determining the molecular mechanisms of adhesion-mediated signaling requires disentangling the contributions of chemical and mechanical stimuli. Here we highlight recent work demonstrating that surface attachment drives a transcriptional response in bacterial pathogens, including uropathogenic Escherichia coli (E. coli), and discuss the complexity of experimental design when dissecting the specific role of adhesion-mediated signaling during infection.

  19. Perspective: Adhesion Mediated Signal Transduction in Bacterial Pathogens

    PubMed Central

    Moorthy, Sudha; Keklak, Julia; Klein, Eric A.

    2016-01-01

    During the infection process, pathogenic bacteria undergo large-scale transcriptional changes to promote virulence and increase intrahost survival. While much of this reprogramming occurs in response to changes in chemical environment, such as nutrient availability and pH, there is increasing evidence that adhesion to host-tissue can also trigger signal transduction pathways resulting in differential gene expression. Determining the molecular mechanisms of adhesion-mediated signaling requires disentangling the contributions of chemical and mechanical stimuli. Here we highlight recent work demonstrating that surface attachment drives a transcriptional response in bacterial pathogens, including uropathogenic Escherichia coli (E. coli), and discuss the complexity of experimental design when dissecting the specific role of adhesion-mediated signaling during infection. PMID:26901228

  20. TLR2-mediated Cell Stimulation in Bacterial Vaginosis

    PubMed Central

    Mares, Debra; Simoes, Jose A.; Novak, Richard M.; Spear, Gregory T.

    2008-01-01

    Bacterial vaginosis (BV) is associated with preterm labor, pelvic inflammatory disease and increased HIV acquisition, although the pathways that mediate these pathological effects have not been elucidated. To determine the presence of toll-like receptor (TLR)-ligands and their specificity in BV, genital tract fluids were collected from women with and without BV by cervicovaginal lavage (CVL). The CVL samples were evaluated for their ability to stimulate secretion of proinflammatory cytokines and to activate NF κB and the HIV long terminal repeat (LTR), indicators of TLR activation, in human monocytic cells. Stimulation with BV CVLs induced higher levels of IL-8 and TNFα secretion, as well as higher levels of HIV LTR and NF κB activation, than CVLs from women with normal healthy bacterial flora. To identify which TLRs were important in BV, 293 cells expressing specific TLRs were exposed to CVL samples. BV CVLs induced higher IL-8 secretion by cells expressing TLR2 than CVLs from women without BV. Surprisingly, BV CVLs did not stimulate cells expressing TLR4/MD2, although these cells responded to purified LPS, a TLR4 ligand. BV CVLs, in cells expressing TLR2, also activated the HIV LTR. Thus, these studies show that soluble factor(s) present in the lower genital tract of women with BV activate cells via TLR2, identifying a pathway through which BV may mediate adverse effects. PMID:17532476

  1. Magnesium isotope fractionation in bacterial mediated carbonate precipitation experiments

    NASA Astrophysics Data System (ADS)

    Parkinson, I. J.; Pearce, C. R.; Polacskek, T.; Cockell, C.; Hammond, S. J.

    2012-12-01

    Magnesium is an essential component of life, with pivotal roles in the generation of cellular energy as well as in plant chlorophyll [1]. The bio-geochemical cycling of Mg is associated with mass dependant fractionation (MDF) of the three stable Mg isotopes [1]. The largest MDF of Mg isotopes has been recorded in carbonates, with foraminiferal tests having δ26Mg compositions up to 5 ‰ lighter than modern seawater [2]. Magnesium isotopes may also be fractionated during bacterially mediated carbonate precipitation and such carbonates are known to have formed in both modern and ancient Earth surface environments [3, 4], with cyanobacteria having a dominant role in carbonate formation during the Archean. In this study, we aim to better constrain the extent to which Mg isotope fractionation occurs during cellular processes, and to identify when, and how, this signal is transferred to carbonates. To this end we have undertaken biologically-mediated carbonate precipitation experiments that were performed in artificial seawater, but with the molar Mg/Ca ratio set to 0.6 and with the solution spiked with 0.4% yeast extract. The bacterial strain used was marine isolate Halomonas sp. (gram-negative). Experiments were run in the dark at 21 degree C for two to three months and produced carbonate spheres of various sizes up to 300 μm in diameter, but with the majority have diameters of ~100 μm. Control experiments run in sterile controls (`empty` medium without bacteria) yielded no precipitates, indicating a bacterial control on the precipitation. The carbonate spheres are produced are amenable to SEM, EMP and Mg isotopic analysis by MC-ICP-MS. Our new data will shed light on tracing bacterial signals in carbonates from the geological record. [1] Young & Galy (2004). Rev. Min. Geochem. 55, p197-230. [2] Pogge von Strandmann (2008). Geochem. Geophys. Geosys. 9 DOI:10.1029/2008GC002209. [3] Castanier, et al. (1999). Sed. Geol. 126, 9-23. [4] Cacchio, et al. (2003

  2. The bacterial chemical repertoire mediates metabolic exchange within gut microbiomes

    PubMed Central

    Rath, Christopher M.; Dorrestein, Pieter C

    2014-01-01

    Microbial communities in the gut have been hypothesized to play key roles in the health of the host organism. Exploring the relationship between these populations and disease states has been a focus of the human microbiome project. However, the biological roles of the compounds produced by the gut bacteria are largely unknown. We hypothesize that these compounds act as metabolic exchange factors—mediating inter- and intra- species interactions in the microbiome. This view is supported through this review of known bacterial metabolic exchange factors and evidence for uncharacterized metabolic exchange factors in the gut. The impact of model systems and technological developments in exploring this hypothesis are also discussed. Together, these investigations are revolutionizing our understanding of the gut microbiome—presenting the possibility of identifying new strategies for treating disease in the host. PMID:22209085

  3. A fragrant neighborhood: volatile mediated bacterial interactions in soil.

    PubMed

    Schulz-Bohm, Kristin; Zweers, Hans; de Boer, Wietse; Garbeva, Paolina

    2015-01-01

    There is increasing evidence that volatile organic compounds (VOCs) play essential roles in communication and competition between soil microorganisms. Here we assessed volatile-mediated interactions of a synthetic microbial community in a model system that mimics the natural conditions in the heterogeneous soil environment along the rhizosphere. Phylogenetic different soil bacterial isolates (Burkholderia sp., Dyella sp., Janthinobacterium sp., Pseudomonas sp., and Paenibacillus sp.) were inoculated as mixtures or monoculture in organic-poor, sandy soil containing artificial root exudates (ARE) and the volatile profile and growth were analyzed. Additionally, a two-compartment system was used to test if volatiles produced by inter-specific interactions in the rhizosphere can stimulate the activity of starving bacteria in the surrounding, nutrient-depleted soil. The obtained results revealed that both microbial interactions and shifts in microbial community composition had a strong effect on the volatile emission. Interestingly, the presence of a slow-growing, low abundant Paenibacillus strain significantly affected the volatile production by the other abundant members of the bacterial community as well as the growth of the interacting strains. Furthermore, volatiles released by mixtures of root-exudates consuming bacteria stimulated the activity and growth of starved bacteria. Besides growth stimulation, also an inhibition in growth was observed for starving bacteria exposed to microbial volatiles. The current work suggests that volatiles produced during microbial interactions in the rhizosphere have a significant long distance effect on microorganisms in the surrounding, nutrient-depleted soil. PMID:26579111

  4. A fragrant neighborhood: volatile mediated bacterial interactions in soil

    PubMed Central

    Schulz-Bohm, Kristin; Zweers, Hans; de Boer, Wietse; Garbeva, Paolina

    2015-01-01

    There is increasing evidence that volatile organic compounds (VOCs) play essential roles in communication and competition between soil microorganisms. Here we assessed volatile-mediated interactions of a synthetic microbial community in a model system that mimics the natural conditions in the heterogeneous soil environment along the rhizosphere. Phylogenetic different soil bacterial isolates (Burkholderia sp., Dyella sp., Janthinobacterium sp., Pseudomonas sp., and Paenibacillus sp.) were inoculated as mixtures or monoculture in organic-poor, sandy soil containing artificial root exudates (ARE) and the volatile profile and growth were analyzed. Additionally, a two-compartment system was used to test if volatiles produced by inter-specific interactions in the rhizosphere can stimulate the activity of starving bacteria in the surrounding, nutrient-depleted soil. The obtained results revealed that both microbial interactions and shifts in microbial community composition had a strong effect on the volatile emission. Interestingly, the presence of a slow-growing, low abundant Paenibacillus strain significantly affected the volatile production by the other abundant members of the bacterial community as well as the growth of the interacting strains. Furthermore, volatiles released by mixtures of root-exudates consuming bacteria stimulated the activity and growth of starved bacteria. Besides growth stimulation, also an inhibition in growth was observed for starving bacteria exposed to microbial volatiles. The current work suggests that volatiles produced during microbial interactions in the rhizosphere have a significant long distance effect on microorganisms in the surrounding, nutrient-depleted soil. PMID:26579111

  5. Microfabrication services at INO

    NASA Astrophysics Data System (ADS)

    Alain, Christine; Jerominek, Hubert; Topart, Patrice A.; Pope, Timothy D.; Picard, Francis; Cayer, Felix; Larouche, Carl; Leclair, Sebastien; Tremblay, Bruno

    2003-01-01

    MEMS (Micro Electro Mechanical Systems) technology has expanded widely over the last decade in terms of its use in devices and instrumentation for diverse applications. However, access to versatile foundry services for MEMS fabrication is still limited. At INO, the presence of a multidisciplinary team and a complete tool set allow us to offer unique MEMS foundry-type services. These services include: design, prototyping, fabrication, packaging and testing of various MEMS and MOEMS devices. The design of a device starts with the evaluation of different structures adapted to a given application. Computer simulation tools, like IntelliSuite, ANSYS or custom software are used to evaluate the mechanical, optical, thermal and electromechanical performances. Standard IC manufacturing techniques such as metal, dielectric and semiconductor film deposition and etching as well as photolithographic pattern transfer are available. In addition, some unique techniques such as on-wafer lithography by laser writing, gray-scale mask lithography, thick photoresist lithography, selective electroplating, injection moulding and UV-assisted moulding are available to customers. The hermetic packaging and a novel patented wafer-level micropackaging are also applied. This multifaceted expertise has been utilized to manufacturing of several types of MEMS devices as well as complex instruments including micromirror-type devices, microfilters, IR microbolometric detector arrays, complete cameras and multipurpose sensors.

  6. iNOS signaling interacts with COX-2 pathway in colonic fibroblasts.

    PubMed

    Zhu, Yingting; Zhu, Min; Lance, Peter

    2012-10-01

    COX-2 and iNOS are two major inflammatory mediators implicated in colorectal inflammation and cancer. Previously, the role of colorectal fibroblasts involved in regulation of COX-2 and iNOS expression was largely ignored. In addition, the combined interaction of COX-2 and iNOS signalings and their significance in the progression of colorectal inflammation and cancer within the fibroblasts have received little investigation. To address those issues, we investigated the role of colonic fibroblasts in the regulation of COX-2 and iNOS gene expression, and explored possible mechanisms of interaction between COX-2 and iNOS signalings using a colonic CCD-18Co fibroblast line and LPS, a potential stimulator of COX-2 and iNOS. Our results clearly demonstrated that LPS activated COX-2 gene expression and enhanced PGE(2) production, stimulated iNOS gene expression and promoted NO production in the fibroblasts. Interestingly, activation of COX-2 signaling by LPS was not involved in activation of iNOS signaling, while activation of iNOS signaling by LPS contributed in part to activation of COX-2 signaling. Further analysis indicated that PKC plays a major role in the activation and interaction of COX-2 and iNOS signalings induced by LPS in the fibroblasts. PMID:22683859

  7. Curli mediate bacterial adhesion to fibronectin via tensile multiple bonds

    PubMed Central

    Oh, Yoo Jin; Hubauer-Brenner, Michael; Gruber, Hermann J.; Cui, Yidan; Traxler, Lukas; Siligan, Christine; Park, Sungsu; Hinterdorfer, Peter

    2016-01-01

    Many enteric bacteria including pathogenic Escherichia coli and Salmonella strains produce curli fibers that bind to host surfaces, leading to bacterial internalization into host cells. By using a nanomechanical force-sensing approach, we obtained real-time information about the distribution of molecular bonds involved in the adhesion of curliated bacteria to fibronectin. We found that curliated E. coli and fibronectin formed dense quantized and multiple specific bonds with high tensile strength, resulting in tight bacterial binding. Nanomechanical recognition measurements revealed that approximately 10 bonds were disrupted either sequentially or simultaneously under force load. Thus the curli formation of bacterial surfaces leads to multi-bond structural components of fibrous nature, which may explain the strong mechanical binding of curliated bacteria to host cells and unveil the functions of these proteins in bacterial internalization and invasion. PMID:27652888

  8. Oxidative stress increases hepatocyte iNOS gene transcription and promoter activity.

    PubMed

    Kuo, P C; Abe, K Y; Schroeder, R A

    1997-05-19

    Hepatocyte expression of inducible nitric oxide synthase (iNOS) is initiated by the presence of pro-inflammatory cytokines, such as interleukin-1beta (IL-1). In the presence of oxidative stress, IL-1beta mediated hepatocyte iNOS expression and NO synthesis are significantly increased. To determine the underlying molecular mechanism responsible for oxidative stress augmentation of hepatocyte iNOS expression, rat hepatocytes in primary culture were stimulated with IL-1beta (250 U/mL) in the presence and absence of benzenetriol (BZT, 0-100 microM), an autocatalytic source of superoxide at physiologic pH. Nuclear runon analysis demonstrated that BZT was associated with increased iNOS gene transcription in the setting of IL-1 stimulation. Transient transfection of a plasmid construct composed of the rat hepatocyte iNOS promoter and a chloramphenicol transferase reporter gene demonstrated that the combination of BZT and IL-1 significantly increased iNOS promoter activity in comparison to that of IL-1beta alone. These data indicate that BZT-mediated oxidative stress increases IL-1beta induced iNOS gene transcription and iNOS promoter activity.

  9. A common fold mediates vertebrate defense and bacterial attack.

    PubMed

    Rosado, Carlos J; Buckle, Ashley M; Law, Ruby H P; Butcher, Rebecca E; Kan, Wan-Ting; Bird, Catherina H; Ung, Kheng; Browne, Kylie A; Baran, Katherine; Bashtannyk-Puhalovich, Tanya A; Faux, Noel G; Wong, Wilson; Porter, Corrine J; Pike, Robert N; Ellisdon, Andrew M; Pearce, Mary C; Bottomley, Stephen P; Emsley, Jonas; Smith, A Ian; Rossjohn, Jamie; Hartland, Elizabeth L; Voskoboinik, Ilia; Trapani, Joseph A; Bird, Phillip I; Dunstone, Michelle A; Whisstock, James C

    2007-09-14

    Proteins containing membrane attack complex/perforin (MACPF) domains play important roles in vertebrate immunity, embryonic development, and neural-cell migration. In vertebrates, the ninth component of complement and perforin form oligomeric pores that lyse bacteria and kill virus-infected cells, respectively. However, the mechanism of MACPF function is unknown. We determined the crystal structure of a bacterial MACPF protein, Plu-MACPF from Photorhabdus luminescens, to 2.0 angstrom resolution. The MACPF domain reveals structural similarity with poreforming cholesterol-dependent cytolysins (CDCs) from Gram-positive bacteria. This suggests that lytic MACPF proteins may use a CDC-like mechanism to form pores and disrupt cell membranes. Sequence similarity between bacterial and vertebrate MACPF domains suggests that the fold of the CDCs, a family of proteins important for bacterial pathogenesis, is probably used by vertebrates for defense against infection.

  10. A Common Fold Mediates Vertebrate Defense and Bacterial Attack

    SciTech Connect

    Rosado, Carlos J.; Buckle, Ashley M.; Law, Ruby H.P.; Butcher, Rebecca E.; Kan, Wan-Ting; Bird, Catherina H.; Ung, Kheng; Browne, Kylie A.; Baran, Katherine; Bashtannyk-Puhalovich, Tanya A.; Faux, Noel G.; Wong, Wilson; Porter, Corrine J.; Pike, Robert N.; Ellisdon, Andrew M.; Pearce, Mary C.; Bottomley, Stephen P.; Emsley, Jonas; Smith, A. Ian; Rossjohn, Jamie; Hartland, Elizabeth L.; Voskoboinik, Ilia; Trapani, Joseph A.; Bird, Phillip I.; Dunstone, Michelle A.; Whisstock, James C.

    2008-10-02

    Proteins containing membrane attack complex/perforin (MACPF) domains play important roles in vertebrate immunity, embryonic development, and neural-cell migration. In vertebrates, the ninth component of complement and perforin form oligomeric pores that lyse bacteria and kill virus-infected cells, respectively. However, the mechanism of MACPF function is unknown. We determined the crystal structure of a bacterial MACPF protein, Plu-MACPF from Photorhabdus luminescens, to 2.0 angstrom resolution. The MACPF domain reveals structural similarity with poreforming cholesterol-dependent cytolysins (CDCs) from Gram-positive bacteria. This suggests that lytic MACPF proteins may use a CDC-like mechanism to form pores and disrupt cell membranes. Sequence similarity between bacterial and vertebrate MACPF domains suggests that the fold of the CDCs, a family of proteins important for bacterial pathogenesis, is probably used by vertebrates for defense against infection.

  11. TAL effector-mediated susceptibility to bacterial blight of cotton

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bacterial blight of cotton (BBC) caused by Xanthomonas campestris pv. malvacearum (Xcm) is a destructive disease that has recently re-emerged in the U.S. Xcm injects transcription activator-like (TAL) effectors that directly induce the expression of host susceptibility (S) or resistance (R) genes. ...

  12. Receptors, Mediators, and Mechanisms Involved in Bacterial Sepsis and Septic Shock

    PubMed Central

    Van Amersfoort, Edwin S.; Van Berkel, Theo J. C.; Kuiper, Johan

    2003-01-01

    Bacterial sepsis and septic shock result from the overproduction of inflammatory mediators as a consequence of the interaction of the immune system with bacteria and bacterial wall constituents in the body. Bacterial cell wall constituents such as lipopolysaccharide, peptidoglycans, and lipoteichoic acid are particularly responsible for the deleterious effects of bacteria. These constituents interact in the body with a large number of proteins and receptors, and this interaction determines the eventual inflammatory effect of the compounds. Within the circulation bacterial constituents interact with proteins such as plasma lipoproteins and lipopolysaccharide binding protein. The interaction of the bacterial constituents with receptors on the surface of mononuclear cells is mainly responsible for the induction of proinflammatory mediators by the bacterial constituents. The role of individual receptors such as the toll-like receptors and CD14 in the induction of proinflammatory cytokines and adhesion molecules is discussed in detail. In addition, the roles of a number of other receptors that bind bacterial compounds such as scavenger receptors and their modulating role in inflammation are described. Finally, the therapies for the treatment of bacterial sepsis and septic shock are discussed in relation to the action of the aforementioned receptors and proteins. PMID:12857774

  13. Redox-stable cyclic peptide inhibitors of the SPSB2-iNOS interaction.

    PubMed

    Yap, Beow Keat; Harjani, Jitendra R; Leung, Eleanor W W; Nicholson, Sandra E; Scanlon, Martin J; Chalmers, David K; Thompson, Philip E; Baell, Jonathan B; Norton, Raymond S

    2016-03-01

    SPSB2 mediates the proteasomal degradation of iNOS. Inhibitors of SPSB2-iNOS interaction are expected to prolong iNOS lifetime and thereby enhance killing of persistent pathogens. Here, we describe the synthesis and characterization of two redox-stable cyclized peptides containing the DINNN motif required for SPSB2 binding. Both analogues bind with low nanomolar affinity to the iNOS binding site on SPSB, as determined by SPR and (19)F NMR, and efficiently displace full-length iNOS from binding to SPSB2 in macrophage cell lysates. These peptides provide a foundation for future development of redox-stable, potent ligands for SPSB proteins as a potential novel class of anti-infectives. PMID:26921848

  14. Redox-stable cyclic peptide inhibitors of the SPSB2-iNOS interaction.

    PubMed

    Yap, Beow Keat; Harjani, Jitendra R; Leung, Eleanor W W; Nicholson, Sandra E; Scanlon, Martin J; Chalmers, David K; Thompson, Philip E; Baell, Jonathan B; Norton, Raymond S

    2016-03-01

    SPSB2 mediates the proteasomal degradation of iNOS. Inhibitors of SPSB2-iNOS interaction are expected to prolong iNOS lifetime and thereby enhance killing of persistent pathogens. Here, we describe the synthesis and characterization of two redox-stable cyclized peptides containing the DINNN motif required for SPSB2 binding. Both analogues bind with low nanomolar affinity to the iNOS binding site on SPSB, as determined by SPR and (19)F NMR, and efficiently displace full-length iNOS from binding to SPSB2 in macrophage cell lysates. These peptides provide a foundation for future development of redox-stable, potent ligands for SPSB proteins as a potential novel class of anti-infectives.

  15. Bacteriocin-Mediated Competitive Interactions of Bacterial Populations and Communities

    NASA Astrophysics Data System (ADS)

    Riley, Margaret A.

    Explaining the coexistence of competing species is a major challenge in community ecology. In bacterial systems, competition is often driven by the production of bacteriocins; narrow spectrum proteinaceous toxins that serve to kill closely related species providing the producer better access to limited resources. Bacteriocin producers have been shown to competitively exclude sensitive, nonproducing strains. However, the interaction dynamics between bacteriocin producers, each lethal to its competitor, are largely unknown. Several recent studies have revealed some of the complexity of these interactions, employing a suite of in vitro, in vivo, and in silico bacterial model systems. This chapter describes the current state of knowledge regarding the population and community ecology of this potent family of toxins.

  16. Insulin inhibits hepatocyte iNOS expression induced by cytokines by an Akt-dependent mechanism.

    PubMed

    Harbrecht, Brian G; Nweze, Ikenna; Smith, Jason W; Zhang, Baochun

    2012-01-01

    Hepatocyte inducible nitric oxide synthese (iNOS) expression is a tightly controlled pathway that mediates hepatic inflammation and hepatocyte injury in a variety of disease states. We have shown that cyclic adenosine monophosphate (cAMP) regulates cytokine-induced hepatocyte iNOS expression through mechanisms that involve protein kinase B/Akt. We hypothesized that insulin, which activates Akt signaling in hepatocytes, as well as signaling through p38 and MAPK p42/p44, would regulate iNOS expression during inflammation. In primary rat hepatocytes, insulin inhibited cytokine-stimulated nitrite accumulation and iNOS expression in a dose-dependent manner. Inhibition of MAPK p42/p44 with PD98059 had no effect on iNOS activation, whereas SB203580 to block p38 reversed insulin's inhibitory effect. However, insulin did not increase p38 activation and inhibition of p38 signaling with a dominant negative p38 plasmid had no effect on cytokine- or insulin-mediated effects on iNOS. We found that SB203580 blocked insulin-induced Akt activation. Inhibition of Akt signaling with LY294002 or a dominant negative Akt plasmid increased cytokine-stimulated nitrite production and iNOS protein expression and blocked the inhibitory effects of insulin. NF-κB induces iNOS expression and can be regulated by Akt, but insulin had no effect on cytokine-mediated IκBα levels or NF-κB p65 translocation. Our data demonstrate that insulin inhibits cytokine-stimulated hepatocyte iNOS expression and does so through effects on Akt-mediated signaling. PMID:22038823

  17. CRISPR-mediated control of the bacterial initiation of replication

    PubMed Central

    Wiktor, Jakub; Lesterlin, Christian; Sherratt, David J.; Dekker, Cees

    2016-01-01

    Programmable control of the cell cycle has been shown to be a powerful tool in cell-biology studies. Here, we develop a novel system for controlling the bacterial cell cycle, based on binding of CRISPR/dCas9 to the origin-of-replication locus. Initiation of replication of bacterial chromosomes is accurately regulated by the DnaA protein, which promotes the unwinding of DNA at oriC. We demonstrate that the binding of CRISPR/dCas9 to any position within origin or replication blocks the initiation of replication. Serial-dilution plating, single-cell fluorescence microscopy, and flow-cytometry experiments show that ongoing rounds of chromosome replication are finished upon CRISPR/dCas9 binding, but no new rounds are initiated. Upon arrest, cells stay metabolically active and accumulate cell mass. We find that elevating the temperature from 37 to 42°C releases the CRISR/dCas9 replication inhibition, and we use this feature to recover cells from the arrest. Our simple and robust method of controlling the bacterial cell cycle is a useful asset for synthetic biology and DNA-replication studies in particular. The inactivation of CRISPR/dCas9 binding at elevated temperatures may furthermore be of wide interest for CRISPR/Cas9 applications in genomic engineering. PMID:27036863

  18. Zooplankton-mediated changes of bacterial community structure.

    PubMed

    Jürgens, K; Arndt, H; Rothhaupt, K O

    1994-01-01

    Enclosure experiments in the mesotrophic Schöhsee in northern Germany were designed to study the impact of metazooplankton on components of the microbial food web (bacteria, flagellates, ciliates). Zooplankton was manipulated in 500-liter epilimnetic mesocosms so that either Daphnia or copepods were dominating, or metazooplankton was virtually absent. The bacterial community responded immediately to changes in zooplankton composition. Biomass, productivity, and especially the morphology of the bacteria changed drastically in the different treatments. Cascading predation effects on the bacterioplankton were transmitted mainly by phagotrophic protozoans which had changed in species composition and biomass. When Daphnia dominated, protozoans were largely suppressed and the original morphological structure of the bacteria (mainly small rods and cocci) remained throughout the experiment. Dominance of copepods or the absence of metazoan predators resulted in a mass appearance of bacterivorous protists (flagellates and ciliates). They promoted a fast decline of bacterial abundance and a shift to the predominance of morphologically inedible forms, mainly long filaments. After 3 days they formed 80-90% of the bacterial biomass. The results indicate that metazooplankton predation on phagotrophic protozoans is a key mechanism for the regulation of bacterioplankton density and community structure.

  19. A simple and novel modification of comet assay for determination of bacteriophage mediated bacterial cell lysis.

    PubMed

    Khairnar, Krishna; Sanmukh, Swapnil; Chandekar, Rajshree; Paunikar, Waman

    2014-07-01

    The comet assay is the widely used method for in vitro toxicity testing which is also an alternative to the use of animal models for in vivo testing. Since, its inception in 1984 by Ostling and Johansson, it is being modified frequently for a wide range of application. In spite of its wide applicability, unfortunately there is no report of its application in bacteriophages research. In this study, a novel application of comet assay for the detection of bacteriophage mediated bacterial cell lysis was described. The conventional methods in bacteriophage research for studying bacterial lysis by bacteriophages are plaque assay method. It is time consuming, laborious and costly. The lytic activity of bacteriophage devours the bacterial cell which results in the release of bacterial genomic material that gets detected by ethidium bromide staining method by the comet assay protocol. The objective of this study was to compare efficacy of comet assay with different assay used to study phage mediated bacterial lysis. The assay was performed on culture isolates (N=80 studies), modified comet assay appear to have relatively higher sensitivity and specificity than other assay. The results of the study showed that the application of comet assay can be an economical, time saving and less laborious alternative to conventional plaque assay for the detection of bacteriophage mediated bacterial cell lysis.

  20. Surface Proteoglycans as Mediators in Bacterial Pathogens Infections

    PubMed Central

    García, Beatriz; Merayo-Lloves, Jesús; Martin, Carla; Alcalde, Ignacio; Quirós, Luis M.; Vazquez, Fernando

    2016-01-01

    Infectious diseases remain an important global health problem. The interaction of a wide range of pathogen bacteria with host cells from many different tissues is frequently mediated by proteoglycans. These compounds are ubiquitous complex molecules which are not only involved in adherence and colonization, but can also participate in other steps of pathogenesis. To overcome the problem of microbial resistance to antibiotics new therapeutic agents could be developed based on the characteristics of the interaction of pathogens with proteoglycans. PMID:26941735

  1. Assembly of the Arp5 (Actin-related Protein) Subunit Involved in Distinct INO80 Chromatin Remodeling Activities.

    PubMed

    Yao, Wei; Beckwith, Sean L; Zheng, Tina; Young, Thomas; Dinh, Van T; Ranjan, Anand; Morrison, Ashby J

    2015-10-16

    ATP-dependent chromatin remodeling, which repositions and restructures nucleosomes, is essential to all DNA-templated processes. The INO80 chromatin remodeling complex is an evolutionarily conserved complex involved in diverse cellular processes, including transcription, DNA repair, and replication. The functional diversity of the INO80 complex can, in part, be attributed to specialized activities of distinct subunits that compose the complex. Furthermore, structural analyses have identified biochemically discrete subunit modules that assemble along the Ino80 ATPase scaffold. Of particular interest is the Saccharomyces cerevisiae Arp5-Ies6 module located proximal to the Ino80 ATPase and the Rvb1-Rvb2 helicase module needed for INO80-mediated in vitro activity. In this study we demonstrate that the previously uncharacterized Ies2 subunit is required for Arp5-Ies6 association with the catalytic components of the INO80 complex. In addition, Arp5-Ies6 module assembly with the INO80 complex is dependent on distinct conserved domains within Arp5, Ies6, and Ino80, including the spacer region within the Ino80 ATPase domain. Arp5-Ies6 interacts with chromatin via assembly with the INO80 complex, as IES2 and INO80 deletion results in loss of Arp5-Ies6 chromatin association. Interestingly, ectopic addition of the wild-type Arp5-Ies6 module stimulates INO80-mediated ATP hydrolysis and nucleosome sliding in vitro. However, the addition of mutant Arp5 lacking unique insertion domains facilitates ATP hydrolysis in the absence of nucleosome sliding. Collectively, these results define the requirements of Arp5-Ies6 assembly, which are needed to couple ATP hydrolysis to productive nucleosome movement.

  2. Bacterially Mediated Breakdown of Cinnabar and Metacinnabar and Environmental Implications

    NASA Astrophysics Data System (ADS)

    Jew, A. D.; Rogers, S. B.; Rytuba, J.; Spormann, A. M.; Brown, G. E.

    2006-12-01

    Mercury in the forms of cinnabar (α-HgS) and metacinnabar (β-HgS) is considered by the EPA to be unreactive and of little environmental concern because of their relatively low solubilities. To determine if this current belief is valid, a consortium of bacteria (including a Thiomonas intermedia-like bacterium) was taken from the acid mine drainage (AMD) pond at the New Idria Hg Mine, San Benito Co., CA, and inoculated into filter-sterilized AMD pond water containing either ground cinnabar or metacinnabar crystals (<45μm in diameter), with sampling occurring approximately every 3 days. Under aerobic conditions the samples showed a pronounced increase in aqueous Hg concentration over background water concentrations (390(±20)ng/L). Bacteria growing on α-HgS increased the Hg concentration to 297(±10)μg/L, while bacteria growing on β-HgS resulted in levels of 4.6(±0.2)mg/L; both maxima occurred at 18 days incubation. Experiments conducted with (1) α-HgS or β-HgS in the presence of killed bacteria (anaerobic), (2) α-HgS with pond water (abiotic), and (3) β-HgS with pond water (abiotic) showed drops in aqueous Hg to below the detection limit (0.1ng/L) within 12 days. Anaerobic growth of the bacterial consortium showed a pattern similar to those of the water and HgS experiments, except that Hg levels dropped below detection limit within 6 days. These combined results suggest that HgS degradation by this bacterial consortium is an aerobic process. Killed bacteria incubated aerobically showed a slight increase in Hg levels over background water levels (<10x increase) then dropped below detection limit. This observation suggests that enzymes might be involved in the dissolution of HgS and were still viable for ~6 days after sterilization. The New Idria AMD pond consists of an inlet stream and an outlet pipe, separated from each other by ~3m. The Hg concentration in the ferrihydrite-rich sediments at the inlet is 37mg/kg, dry weight, while the concentration at the

  3. Impact of flow on ligand-mediated bacterial flocculation

    PubMed Central

    Sircar, Sarthok; Bortz, David M.

    2013-01-01

    To understand the adhesion-fragmentation dynamics of bacterial aggregates (i.e., flocs), we model the aggregates as two ligand-covered rigid spheres. We develop and investigate a model for the attachment/detachment dynamics in a fluid subject to a homogeneous planar shear-flow. The binding ligands on the surface of the flocs experience attractive and repulsive surface forces in an ionic medium and exhibit finite resistance to rotation (via bond tilting). For certain range of material and fluid parameters, our results predict a nonlinear or hysteretic relationship between the binding/unbinding of the floc surface and the net floc velocity (translational plus rotational velocity). We show that the surface adhesion is promoted by increased fluid flow until a critical value, beyond which the bonds starts to yield. Moreover, adhesion is not promoted in a medium with low ionic strength, or flocs with bigger size or higher binder stiffness. The numerical simulations of floc-aggregate number density studies support these findings. PMID:23917245

  4. Impact of flow on ligand-mediated bacterial flocculation.

    PubMed

    Sircar, Sarthok; Bortz, David M

    2013-10-01

    To understand the adhesion-fragmentation dynamics of bacterial aggregates (i.e., flocs), we model the aggregates as two ligand-covered rigid spheres. We develop and investigate a model for the attachment/detachment dynamics in a fluid subject to a homogeneous planar shear-flow. The binding ligands on the surface of the flocs experience attractive and repulsive surface forces in an ionic medium and exhibit finite resistance to rotation (via bond tilting). For certain range of material and fluid parameters, our results predict a nonlinear or hysteretic relationship between the binding/unbinding of the floc surface and the net floc velocity (translational plus rotational velocity). We show that the surface adhesion is promoted by increased fluid flow until a critical value, beyond which the bonds starts to yield. Moreover, adhesion is not promoted in a medium with low ionic strength, or flocs with bigger size or higher binder stiffness. The numerical simulations of floc-aggregate number density studies support these findings.

  5. Influence of calcium in extracellular DNA mediated bacterial aggregation and biofilm formation.

    PubMed

    Das, Theerthankar; Sehar, Shama; Koop, Leena; Wong, Yie Kuan; Ahmed, Safia; Siddiqui, Khawar Sohail; Manefield, Mike

    2014-01-01

    Calcium (Ca(2+)) has an important structural role in guaranteeing the integrity of the outer lipopolysaccharide layer and cell walls of bacterial cells. Extracellular DNA (eDNA) being part of the slimy matrix produced by bacteria promotes biofilm formation through enhanced structural integrity of the matrix. Here, the concurrent role of Ca(2+) and eDNA in mediating bacterial aggregation and biofilm formation was studied for the first time using a variety of bacterial strains and the thermodynamics of DNA to Ca(2+) binding. It was found that the eDNA concentrations under both planktonic and biofilm growth conditions were different among bacterial strains. Whilst Ca(2+) had no influence on eDNA release, presence of eDNA by itself favours bacterial aggregation via attractive acid-base interactions in addition, its binding with Ca(2+) at biologically relevant concentrations was shown further increase in bacterial aggregation via cationic bridging. Negative Gibbs free energy (ΔG) values in iTC data confirmed that the interaction between DNA and Ca(2+) is thermodynamically favourable and that the binding process is spontaneous and exothermic owing to its highly negative enthalpy. Removal of eDNA through DNase I treatment revealed that Ca(2+) alone did not enhance cell aggregation and biofilm formation. This discovery signifies the importance of eDNA and concludes that existence of eDNA on bacterial cell surfaces is a key facilitator in binding of Ca(2+) to eDNA thereby mediating bacterial aggregation and biofilm formation.

  6. Interleukin-10 Gene Therapy-Mediated Amelioration of Bacterial Pneumonia

    PubMed Central

    Morrison, Daniel F.; Foss, Dennis L.; Murtaugh, Michael P.

    2000-01-01

    Respiratory infection by Actinobacillus pleuropneumoniae causes a highly pathogenic necrotizing pleuropneumonia with severe edema, hemorrhage and fever. Acute infection is characterized by expression of inflammatory cytokines, including interleukin-1 (IL-1), IL-6 and IL-8. To determine if high level production of inflammatory cytokines contributed to disease pathogenesis, we investigated if inhibiting macrophage activation with adenovirus type 5-expressed IL-10 (Ad-5/IL-10) reduced the severity of acute disease. Porcine tracheal epithelial cells infected with Ad-5/IL-10 produced bioactive human IL-10. When pigs were intratracheally infected with A. pleuropneumoniae, pigs pretreated with Ad-5/IL-10 showed a significant reduction in the amount of lung damage when compared to adenovirus type 5-expressing β-galactosidase (Ad-5/β-Gal)-treated and untreated pigs. In addition, serum zinc levels were unchanged, the lung weight/body weight ratio (an indicator of vascular leakage) was significantly reduced, and lung pathology scores were reduced. Myeloperoxidase activity in lung lavage fluid samples, an indicator of neutrophil invasion, was decreased to levels similar to that seen in pigs not infected with A. pleuropneumoniae. Reduction in inflammatory cytokine levels in lung lavage fluid samples correlated with the clinical observations in that pigs pretreated with Ad-5/IL-10 showed a corresponding reduction of IL-1 and tumor necrosis factor (TNF) compared with untreated and Ad-5/β-Gal-treated pigs. IL-6 levels were unaffected by pretreatment with Ad-5/IL-10, consistent with observations that IL-6 was not derived from alveolar macrophages. Since inflammatory cytokines are expressed at high levels in acute bacterial pleuropneumonia, these results indicate that macrophage activation, involving overproduction of IL-1 and TNF, is a prime factor in infection-related cases of massive lung injury. PMID:10899882

  7. In Vivo Transcriptional Profiling of Yersinia pestis Reveals a Novel Bacterial Mediator of Pulmonary Inflammation

    PubMed Central

    Pechous, Roger D.; Broberg, Christopher A.; Stasulli, Nikolas M.; Miller, Virginia L.

    2015-01-01

    ABSTRACT Inhalation of Yersinia pestis results in primary pneumonic plague, a highly lethal and rapidly progressing necrotizing pneumonia. The disease begins with a period of extensive bacterial replication in the absence of disease symptoms, followed by the sudden onset of inflammatory responses that ultimately prove fatal. Very little is known about the bacterial and host factors that contribute to the rapid biphasic progression of pneumonic plague. In this work, we analyzed the in vivo transcription kinetics of 288 bacterial open reading frames previously shown by microarray analysis to be dynamically regulated in the lung. Using this approach combined with bacterial genetics, we were able to identify five Y. pestis genes that contribute to the development of pneumonic plague. Deletion of one of these genes, ybtX, did not alter bacterial survival but attenuated host inflammatory responses during late-stage disease. Deletion of ybtX in another lethal respiratory pathogen, Klebsiella pneumoniae, also resulted in diminished host inflammation during infection. Thus, our in vivo transcriptional screen has identified an important inflammatory mediator that is common to two Gram-negative bacterial pathogens that cause severe pneumonia. PMID:25691593

  8. TRPA1 channels mediate acute neurogenic inflammation and pain produced by bacterial endotoxins

    NASA Astrophysics Data System (ADS)

    Meseguer, Victor; Alpizar, Yeranddy A.; Luis, Enoch; Tajada, Sendoa; Denlinger, Bristol; Fajardo, Otto; Manenschijn, Jan-Albert; Fernández-Peña, Carlos; Talavera, Arturo; Kichko, Tatiana; Navia, Belén; Sánchez, Alicia; Señarís, Rosa; Reeh, Peter; Pérez-García, María Teresa; López-López, José Ramón; Voets, Thomas; Belmonte, Carlos; Talavera, Karel; Viana, Félix

    2014-01-01

    Gram-negative bacterial infections are accompanied by inflammation and somatic or visceral pain. These symptoms are generally attributed to sensitization of nociceptors by inflammatory mediators released by immune cells. Nociceptor sensitization during inflammation occurs through activation of the Toll-like receptor 4 (TLR4) signalling pathway by lipopolysaccharide (LPS), a toxic by-product of bacterial lysis. Here we show that LPS exerts fast, membrane delimited, excitatory actions via TRPA1, a transient receptor potential cation channel that is critical for transducing environmental irritant stimuli into nociceptor activity. Moreover, we find that pain and acute vascular reactions, including neurogenic inflammation (CGRP release) caused by LPS are primarily dependent on TRPA1 channel activation in nociceptive sensory neurons, and develop independently of TLR4 activation. The identification of TRPA1 as a molecular determinant of direct LPS effects on nociceptors offers new insights into the pathogenesis of pain and neurovascular responses during bacterial infections and opens novel avenues for their treatment.

  9. TRPA1 channels mediate acute neurogenic inflammation and pain produced by bacterial endotoxins.

    PubMed

    Meseguer, Victor; Alpizar, Yeranddy A; Luis, Enoch; Tajada, Sendoa; Denlinger, Bristol; Fajardo, Otto; Manenschijn, Jan-Albert; Fernández-Peña, Carlos; Talavera, Arturo; Kichko, Tatiana; Navia, Belén; Sánchez, Alicia; Señarís, Rosa; Reeh, Peter; Pérez-García, María Teresa; López-López, José Ramón; Voets, Thomas; Belmonte, Carlos; Talavera, Karel; Viana, Félix

    2014-01-01

    Gram-negative bacterial infections are accompanied by inflammation and somatic or visceral pain. These symptoms are generally attributed to sensitization of nociceptors by inflammatory mediators released by immune cells. Nociceptor sensitization during inflammation occurs through activation of the Toll-like receptor 4 (TLR4) signalling pathway by lipopolysaccharide (LPS), a toxic by-product of bacterial lysis. Here we show that LPS exerts fast, membrane delimited, excitatory actions via TRPA1, a transient receptor potential cation channel that is critical for transducing environmental irritant stimuli into nociceptor activity. Moreover, we find that pain and acute vascular reactions, including neurogenic inflammation (CGRP release) caused by LPS are primarily dependent on TRPA1 channel activation in nociceptive sensory neurons, and develop independently of TLR4 activation. The identification of TRPA1 as a molecular determinant of direct LPS effects on nociceptors offers new insights into the pathogenesis of pain and neurovascular responses during bacterial infections and opens novel avenues for their treatment.

  10. Advantages of mixing bioinformatics and visualization approaches for analyzing sRNA-mediated regulatory bacterial networks

    PubMed Central

    Bourqui, Romain; Benchimol, William; Gaspin, Christine; Sirand-Pugnet, Pascal; Uricaru, Raluca; Dutour, Isabelle

    2015-01-01

    The revolution in high-throughput sequencing technologies has enabled the acquisition of gigabytes of RNA sequences in many different conditions and has highlighted an unexpected number of small RNAs (sRNAs) in bacteria. Ongoing exploitation of these data enables numerous applications for investigating bacterial transacting sRNA-mediated regulation networks. Focusing on sRNAs that regulate mRNA translation in trans, recent works have noted several sRNA-based regulatory pathways that are essential for key cellular processes. Although the number of known bacterial sRNAs is increasing, the experimental validation of their interactions with mRNA targets remains challenging and involves expensive and time-consuming experimental strategies. Hence, bioinformatics is crucial for selecting and prioritizing candidates before designing any experimental work. However, current software for target prediction produces a prohibitive number of candidates because of the lack of biological knowledge regarding the rules governing sRNA–mRNA interactions. Therefore, there is a real need to develop new approaches to help biologists focus on the most promising predicted sRNA–mRNA interactions. In this perspective, this review aims at presenting the advantages of mixing bioinformatics and visualization approaches for analyzing predicted sRNA-mediated regulatory bacterial networks. PMID:25477348

  11. The trafficking of bacterial type rhodopsins into the Chlamydomonas eyespot and flagella is IFT mediated

    PubMed Central

    Awasthi, Mayanka; Ranjan, Peeyush; Sharma, Komal; Veetil, Sindhu Kandoth; Kateriya, Suneel

    2016-01-01

    The bacterial type rhodopsins are present in all the three domains of life. In contrast to the animal type rhodopsin that performs mainly sensory functions in higher eukaryotes, the bacterial type rhodopsin could function as ion channel, pumps and as sensory proteins. The functioning of rhodopsin in higher eukaryotes requires the transport of rhodopsin from its site of synthesis to the ciliated outer segment of the photoreceptive cells. However, the trafficking of bacterial type rhodopsin from its site of synthesis to the position of action is not characterized. Here we present the first report for the existence of an IFT-interactome mediated trafficking of the bacterial type rhodopsins into eyespot and flagella of the Chlamydomonas. We show that there is a light-dependent, dynamic localization of rhodopsins between flagella and eyespot of Chlamydomonas. The involvement of IFT components in the rhodopsin trafficking was elucidated by the use of conditional IFT mutants. We found that rhodopsin can be co-immunoprecipitated with the components of IFT machinery and with other protein components required for the IFT-cargo complex formation. These findings show that light-regulated localization of rhodopsin is not restricted to animals thereby suggesting that rhodopsin trafficking is an IFT dependent ancient process. PMID:27694882

  12. Flexible and monolithic zinc oxide bionanocomposite foams by a bacterial cellulose mediated approach for antibacterial applications.

    PubMed

    Wang, Peipei; Zhao, Jun; Xuan, Ruifei; Wang, Yun; Zou, Chen; Zhang, Zhiquan; Wan, Yizao; Xu, Yan

    2014-05-14

    The use of self-assembled biomacromolecules in the development of functional bionanocomposite foams is one of the best lessons learned from nature. Here, we show that monolithic, flexible and porous zinc oxide bionanocomposite foams with a hierarchical architecture can be assembled through the mediation of bacterial cellulose. The assembly is achieved by controlled hydrolysis and solvothermal crystallization using a bacterial cellulose aerogel as a template in a non-aqueous polar medium. The bionanocomposite foam with a maximum zinc oxide loading of 70 wt% is constructed of intimately packed spheres of aggregated zinc oxide nanocrystals exhibiting a BET surface area of 92 m(2) g(-1). The zinc oxide bionanocomposite foams show excellent antibacterial activity, which give them potential value as self-supporting wound dressing and water sterilization materials.

  13. Exposure to diesel exhaust up-regulates iNOS expression in ApoE knockout mice

    SciTech Connect

    Bai Ni; Kido, Takashi; Kavanagh, Terrance J.; Kaufman, Joel D.; Rosenfeld, Michael E.; Breemen, Cornelis van; Eeden, Stephan F. van

    2011-09-01

    Traffic related particulate matter air pollution is a risk factor for cardiovascular events; however, the biological mechanisms are unclear. We hypothesize that diesel exhaust (DE) inhalation induces up-regulation of inducible nitric oxide synthase (iNOS), which is known to contribute to vascular dysfunction, progression of atherosclerosis and ultimately cardiovascular morbidity and mortality. Methods: ApoE knockout mice (30-week) were exposed to DE (at 200 {mu}g/m{sup 3} of particulate matter) or filtered-air (control) for 7 weeks (6 h/day, 5 days/week). iNOS expression in the blood vessels and heart was evaluated by immunohistochemistry and western blotting analysis. To examine iNOS activity, thoracic aortae were mounted in a wire myograph, and vasoconstriction stimulated by phenylephrine (PE) was measured with and without the presence of the specific inhibitor for iNOS (1400 W). NF-{kappa}B (p65) activity was examined by ELISA. The mRNA expression of iNOS and NF-{kappa}B (p65) was determined by real-time PCR. Results: DE exposure significantly enhanced iNOS expression in the thoracic aorta (4-fold) and heart (1.5 fold). DE exposure significantly attenuated PE-stimulated vasoconstriction by {approx} 20%, which was partly reversed by 1400 W. The mRNA expression of iNOS and NF-{kappa}B was significantly augmented after DE exposure. NF-{kappa}B activity was enhanced 2-fold after DE inhalation, and the augmented NF-{kappa}B activity was positively correlated with iNOS expression (R{sup 2} = 0.5998). Conclusions: We show that exposure to DE increases iNOS expression and activity possibly via NF-{kappa}B-mediated pathway. We suspect that DE exposure-caused up-regulation of iNOS contributes to vascular dysfunction and atherogenesis, which could ultimately lead to urban air pollution-associated cardiovascular morbidity and mortality. - Highlights: > Exposed ApoE knockout mice (30-week) to diesel exhaust (DE) for 7 weeks. > Examine iNOS expression and activity in the

  14. Exposure to Diesel Exhaust Up-regulates iNOS Expression in ApoE Knockout Mice

    PubMed Central

    Bai, Ni; Kido, Takashi; Kavanagh, Terrance J.; Kaufman, Joel D.; Rosenfeld, Michael E.; van Breemen, Cornelis; van Eeden, Stephan F.

    2012-01-01

    Traffic related particulate matter air pollution is a risk factor for cardiovascular events; however, the biological mechanisms are unclear. We hypothesize that diesel exhaust (DE) inhalation induces up-regulation of inducible nitric oxide synthase (iNOS), which is known to contribute to vascular dysfunction, progression of atherosclerosis and ultimately cardiovascular morbidity and mortality. Methods ApoE knockout mice (30-week) were exposed to DE (at 200µg/m3 of particulate matter) or filtered-air (control) for 7 weeks (6h/day, 5days/week). iNOS expression in the blood vessels and heart was evaluated by immunohistochemistry and western blotting analysis. To examine iNOS activity, thoracic aortae were mounted in a wire myograph, and vasoconstriction stimulated by phenylephrine (PE) was measured with and without the presence of the specific inhibitor for iNOS (1400W). NF-κB (p65) activity was examined by ELISA. The mRNA expression of iNOS and NF-κB (p65) was determined by real-time PCR. Results DE exposure significantly enhanced iNOS expression in the thoracic aorta (4-fold) and heart (1.5 fold). DE exposure significantly attenuated PE-stimulated vasoconstriction by ~20%, which was partly reversed by 1400W. The mRNA expression of iNOS and NF-κB was significantly augmented after DE exposure. NF-κB activity was enhanced 2-fold after DE inhalation, and the augmented NF-κB activity was positively correlated with iNOS expression (R2= 0.5998). Conclusions We show that exposure to DE increases iNOS expression and activity possibly via NF-κB-mediated pathway. We suspect that DE exposure-caused up-regulation of iNOS contributes to vascular dysfunction and atherogenesis, which could ultimately lead to urban air pollution-associated cardiovascular morbidity and mortality. PMID:21722660

  15. Environmental changes affect the assembly of soil bacterial community primarily by mediating stochastic processes.

    PubMed

    Zhang, Ximei; Johnston, Eric R; Liu, Wei; Li, Linghao; Han, Xingguo

    2016-01-01

    Both 'species fitness difference'-based deterministic processes, such as competitive exclusion and environmental filtering, and 'species fitness difference'-independent stochastic processes, such as birth/death and dispersal/colonization, can influence the assembly of soil microbial communities. However, how both types of processes are mediated by anthropogenic environmental changes has rarely been explored. Here we report a novel and general pattern that almost all anthropogenic environmental changes that took place in a grassland ecosystem affected soil bacterial community assembly primarily through promoting or restraining stochastic processes. We performed four experiments mimicking 16 types of environmental changes and separated the compositional variation of soil bacterial communities caused by each environmental change into deterministic and stochastic components, with a recently developed method. Briefly, because the difference between control and treatment communities is primarily caused by deterministic processes, the deterministic change was quantified as (mean compositional variation between treatment and control) - (mean compositional variation within control). The difference among replicate treatment communities is primarily caused by stochastic processes, so the stochastic change was estimated as (mean compositional variation within treatment) - (mean compositional variation within control). The absolute of the stochastic change was greater than that of the deterministic change across almost all environmental changes, which was robust for both taxonomic and functional-based criterion. Although the deterministic change may become more important as environmental changes last longer, our findings showed that changes usually occurred through mediating stochastic processes over 5 years, challenging the traditional determinism-dominated view.

  16. Characterization of a large human transgene following invasin-mediated delivery in a bacterial artificial chromosome

    PubMed Central

    Gillen, Austin E.; Lucas, Catherine A.; Haussecker, Pei Ling; Kosak, Steven T.; Harris, Ann

    2013-01-01

    Bacterial artificial chromosomes (BACs) are widely used in transgenesis, particularly for the humanization of animal models. Moreover, due to their extensive capacity, BACs provide attractive tools to study distal regulatory elements associated with large gene loci. However, despite their widespread use, little is known about the integration dynamics of these large transgenes in mammalian cells. Here, we investigate the post-integration structure of a ~260 kb BAC carrying the cystic fibrosis transmembrane conductance regulator (CFTR) locus following delivery by bacterial invasion and compare this to the outcome of a more routine lipid-based delivery method. We find substantial variability in integrated copy number and expression levels of the BAC CFTR transgene after bacterial invasion-mediated delivery. Furthermore, we frequently observed variation in the representation of different regions of the CFTR transgene within individual cell clones, indicative of BAC fragmentation. Finally, using fluorescence in situ hybridization (FISH), we observed that the integrated BAC forms extended, megabase-scale structures in some clones that are apparently stably maintained at cell division. These data demonstrate that the utility of large BACs to investigate cis-regulatory elements in the genomic context may be limited by recombination events that complicate their use. PMID:23749207

  17. TRPA1 channels mediate acute neurogenic inflammation and pain produced by bacterial endotoxins.

    PubMed

    Meseguer, Victor; Alpizar, Yeranddy A; Luis, Enoch; Tajada, Sendoa; Denlinger, Bristol; Fajardo, Otto; Manenschijn, Jan-Albert; Fernández-Peña, Carlos; Talavera, Arturo; Kichko, Tatiana; Navia, Belén; Sánchez, Alicia; Señarís, Rosa; Reeh, Peter; Pérez-García, María Teresa; López-López, José Ramón; Voets, Thomas; Belmonte, Carlos; Talavera, Karel; Viana, Félix

    2014-01-01

    Gram-negative bacterial infections are accompanied by inflammation and somatic or visceral pain. These symptoms are generally attributed to sensitization of nociceptors by inflammatory mediators released by immune cells. Nociceptor sensitization during inflammation occurs through activation of the Toll-like receptor 4 (TLR4) signalling pathway by lipopolysaccharide (LPS), a toxic by-product of bacterial lysis. Here we show that LPS exerts fast, membrane delimited, excitatory actions via TRPA1, a transient receptor potential cation channel that is critical for transducing environmental irritant stimuli into nociceptor activity. Moreover, we find that pain and acute vascular reactions, including neurogenic inflammation (CGRP release) caused by LPS are primarily dependent on TRPA1 channel activation in nociceptive sensory neurons, and develop independently of TLR4 activation. The identification of TRPA1 as a molecular determinant of direct LPS effects on nociceptors offers new insights into the pathogenesis of pain and neurovascular responses during bacterial infections and opens novel avenues for their treatment. PMID:24445575

  18. Atorvastatin along with imipenem attenuates acute lung injury in sepsis through decrease in inflammatory mediators and bacterial load.

    PubMed

    Choudhury, Soumen; Kandasamy, Kannan; Maruti, Bhojane Somnath; Addison, M Pule; Kasa, Jaya Kiran; Darzi, Sazad A; Singh, Thakur Uttam; Parida, Subhashree; Dash, Jeevan Ranjan; Singh, Vishakha; Mishra, Santosh Kumar

    2015-10-15

    Lung is one of the vital organs which is affected during the sequential development of multi-organ dysfunction in sepsis. The purpose of the present study was to examine whether combined treatment with atorvastatin and imipenem could attenuate sepsis-induced lung injury in mice. Sepsis was induced by caecal ligation and puncture. Lung injury was assessed by the presence of lung edema, increased vascular permeability, increased inflammatory cell infiltration and cytokine levels in broncho-alveolar lavage fluid (BALF). Treatment with atorvastatin along with imipenem reduced the lung bacterial load and pro-inflammatory cytokines (IL-1β and TNFα) level in BALF. The markers of pulmonary edema such as microvascular leakage and wet-dry weight ratio were also attenuated. This was further confirmed by the reduced activity of MPO and ICAM-1 mRNA expression, indicating the lesser infiltration and adhesion of inflammatory cells to the lungs. Again, expression of mRNA and protein level of iNOS in lungs was also reduced in the combined treatment group. Based on the above findings it can be concluded that, combined treatment with atorvastatin and imipenem dampened the inflammatory response and reduced the bacterial load, thus seems to have promising therapeutic potential in sepsis-induced lung injury in mice. PMID:26375251

  19. Phytoplankton–bacterial interactions mediate micronutrient colimitation at the coastal Antarctic sea ice edge

    PubMed Central

    Bertrand, Erin M.; McCrow, John P.; Moustafa, Ahmed; Zheng, Hong; McQuaid, Jeffrey B.; Delmont, Tom O.; Post, Anton F.; Sipler, Rachel E.; Spackeen, Jenna L.; Xu, Kai; Bronk, Deborah A.; Hutchins, David A.; Allen, Andrew E.

    2015-01-01

    Southern Ocean primary productivity plays a key role in global ocean biogeochemistry and climate. At the Southern Ocean sea ice edge in coastal McMurdo Sound, we observed simultaneous cobalamin and iron limitation of surface water phytoplankton communities in late Austral summer. Cobalamin is produced only by bacteria and archaea, suggesting phytoplankton–bacterial interactions must play a role in this limitation. To characterize these interactions and investigate the molecular basis of multiple nutrient limitation, we examined transitions in global gene expression over short time scales, induced by shifts in micronutrient availability. Diatoms, the dominant primary producers, exhibited transcriptional patterns indicative of co-occurring iron and cobalamin deprivation. The major contributor to cobalamin biosynthesis gene expression was a gammaproteobacterial population, Oceanospirillaceae ASP10-02a. This group also contributed significantly to metagenomic cobalamin biosynthesis gene abundance throughout Southern Ocean surface waters. Oceanospirillaceae ASP10-02a displayed elevated expression of organic matter acquisition and cell surface attachment-related genes, consistent with a mutualistic relationship in which they are dependent on phytoplankton growth to fuel cobalamin production. Separate bacterial groups, including Methylophaga, appeared to rely on phytoplankton for carbon and energy sources, but displayed gene expression patterns consistent with iron and cobalamin deprivation. This suggests they also compete with phytoplankton and are important cobalamin consumers. Expression patterns of siderophore- related genes offer evidence for bacterial influences on iron availability as well. The nature and degree of this episodic colimitation appear to be mediated by a series of phytoplankton–bacterial interactions in both positive and negative feedback loops. PMID:26221022

  20. Phytoplankton-bacterial interactions mediate micronutrient colimitation at the coastal Antarctic sea ice edge.

    PubMed

    Bertrand, Erin M; McCrow, John P; Moustafa, Ahmed; Zheng, Hong; McQuaid, Jeffrey B; Delmont, Tom O; Post, Anton F; Sipler, Rachel E; Spackeen, Jenna L; Xu, Kai; Bronk, Deborah A; Hutchins, David A; Allen, Andrew E

    2015-08-11

    Southern Ocean primary productivity plays a key role in global ocean biogeochemistry and climate. At the Southern Ocean sea ice edge in coastal McMurdo Sound, we observed simultaneous cobalamin and iron limitation of surface water phytoplankton communities in late Austral summer. Cobalamin is produced only by bacteria and archaea, suggesting phytoplankton-bacterial interactions must play a role in this limitation. To characterize these interactions and investigate the molecular basis of multiple nutrient limitation, we examined transitions in global gene expression over short time scales, induced by shifts in micronutrient availability. Diatoms, the dominant primary producers, exhibited transcriptional patterns indicative of co-occurring iron and cobalamin deprivation. The major contributor to cobalamin biosynthesis gene expression was a gammaproteobacterial population, Oceanospirillaceae ASP10-02a. This group also contributed significantly to metagenomic cobalamin biosynthesis gene abundance throughout Southern Ocean surface waters. Oceanospirillaceae ASP10-02a displayed elevated expression of organic matter acquisition and cell surface attachment-related genes, consistent with a mutualistic relationship in which they are dependent on phytoplankton growth to fuel cobalamin production. Separate bacterial groups, including Methylophaga, appeared to rely on phytoplankton for carbon and energy sources, but displayed gene expression patterns consistent with iron and cobalamin deprivation. This suggests they also compete with phytoplankton and are important cobalamin consumers. Expression patterns of siderophore- related genes offer evidence for bacterial influences on iron availability as well. The nature and degree of this episodic colimitation appear to be mediated by a series of phytoplankton-bacterial interactions in both positive and negative feedback loops.

  1. Enhanced Disease Susceptibility1 Mediates Pathogen Resistance and Virulence Function of a Bacterial Effector in Soybean.

    PubMed

    Wang, Jialin; Shine, M B; Gao, Qing-Ming; Navarre, Duroy; Jiang, Wei; Liu, Chunyan; Chen, Qingshan; Hu, Guohua; Kachroo, Aardra

    2014-05-28

    Enhanced disease susceptibility1 (EDS1) and phytoalexin deficient4 (PAD4) are well-known regulators of both basal and resistance (R) protein-mediated plant defense. We identified two EDS1-like (GmEDS1a/GmEDS1b) proteins and one PAD4-like (GmPAD4) protein that are required for resistance signaling in soybean (Glycine max). Consistent with their significant structural conservation to Arabidopsis (Arabidopsis thaliana) counterparts, constitutive expression of GmEDS1 or GmPAD4 complemented the pathogen resistance defects of Arabidopsis eds1 and pad4 mutants, respectively. Interestingly, however, the GmEDS1 and GmPAD4 did not complement pathogen-inducible salicylic acid accumulation in the eds1/pad4 mutants. Furthermore, the GmEDS1a/GmEDS1b proteins were unable to complement the turnip crinkle virus coat protein-mediated activation of the Arabidopsis R protein Hypersensitive reaction to Turnip crinkle virus (HRT), even though both interacted with HRT. Silencing GmEDS1a/GmEDS1b or GmPAD4 reduced basal and pathogen-inducible salicylic acid accumulation and enhanced soybean susceptibility to virulent pathogens. The GmEDS1a/GmEDS1b and GmPAD4 genes were also required for Resistance to Pseudomonas syringae pv glycinea2 (Rpg2)-mediated resistance to Pseudomonas syringae. Notably, the GmEDS1a/GmEDS1b proteins interacted with the cognate bacterial effector AvrA1 and were required for its virulence function in rpg2 plants. Together, these results show that despite significant structural similarities, conserved defense signaling components from diverse plants can differ in their functionalities. In addition, we demonstrate a role for GmEDS1 in regulating the virulence function of a bacterial effector.

  2. Protection against lipopolysaccharide-induced endothelial dysfunction in resistance and conduit vasculature of iNOS knockout mice.

    PubMed

    Chauhan, S D; Seggara, G; Vo, P A; Macallister, R J; Hobbs, A J; Ahluwalia, A

    2003-04-01

    Endothelial dysfunction is a characteristic of, and may be pathogenic in, inflammatory cardiovascular diseases, including sepsis. The mechanism underlying inflammation-induced endothelial dysfunction may be related to the expression and activity of inducible nitric oxide synthase (iNOS). This possibility was investigated in isolated resistance (mesenteric) and conduit (aorta) arteries taken from lipopolysaccharide (LPS)-treated (12.5 mg/kg i.v.) or saline-treated iNOS knockout (KO) and wild-type (WT) mice. LPS pretreatment (for 15 h, but not 4 h) profoundly suppressed responses to acetylcholine (ACh) and significantly reduced sensitivity to the NO donor spermine-NONOate (SPER-NO) in aorta and mesenteric arteries of WT mice. This effect was temporally associated with iNOS protein expression in both conduit and resistance arteries and with a 10-fold increase in plasma NOx levels. In contrast, no elevation of plasma NOx was observed in LPS-treated iNOS KO animals, and arteries dissected from these animals did not express iNOS or display hyporeactivity to ACh or SPER-NO. The mechanism underlying this phenomenon may be suppression of eNOS expression, as observed in arteries of WT animals, that was absent in arteries of iNOS KO animals. These results clearly demonstrate that iNOS induction plays an integral role in mediation of the endothelial dysfunction associated with sepsis in both resistance and conduit arteries.

  3. A pathogenic bacterium triggers epithelial signals to form a functional bacterial receptor that mediates actin pseudopod formation.

    PubMed Central

    Rosenshine, I; Ruschkowski, S; Stein, M; Reinscheid, D J; Mills, S D; Finlay, B B

    1996-01-01

    Enteropathogenic E. coli (EPEC) belongs to a group of bacterial pathogens that induce actin accumulation beneath adherent bacteria. We found that EPEC adherence to epithelial cells mediates the formation of fingerlike pseudopods (up to 10 microm) beneath bacteria. These actin-rich structures also contain tyrosine phosphorylated host proteins concentrated at the pseudopod tip beneath adherent EPEC. Intimate bacterial adherence (and pseudopod formation) occurred only after prior bacterial induction of tyrosine phosphorylation of an epithelial membrane protein, Hp90, which then associates directly with an EPEC adhesin, intimin. These interactions lead to cytoskeletal nucleation and pseudopod formation. This is the first example of a bacterial pathogen that triggers signals in epithelial cells which activates receptor binding activity to a specific bacterial ligand and subsequent cytoskeletal rearrangement. Images PMID:8654358

  4. Induction of endothelial iNOS by 4-hydroxyhexenal through NF-kappaB activation.

    PubMed

    Lee, J Y; Je, J H; Jung, K J; Yu, B P; Chung, H Y

    2004-08-15

    Lipid peroxidation and its end-product, 4-hydroxyhexenal (HHE), are known to affect redox balance during aging, which causes various degenerative processes including vascular alterations from endothelial cell deterioration. To better understand the molecular action of HHE in the development of vascular abnormalities during the aging process, we investigated whether the upregulation of inducible endothelial nitric oxide synthase (iNOS) by HHE is mediated through nuclear factor kappaB (NF-kappaB) activation. Results indicate that HHE stimulates iNOS by the transcriptional regulation of NF-kappaB activation through cytosolic kappaB degradation inhibitors (IkappaB). Pretreatment with NF-kappaB inhibitors Bay 11-7082 and N-acetyl cysteine (NAC) suppressed the upregulation of iNOS by blunting IkappaB degradation and NF-kappaB binding activity. Because inflammatory stimuli induce iNOS to generate large amounts of nitric oxide (NO), intracellular NO levels in the presence of Bay 11-7082, NAC, and caffeic acid methyl ester were estimated. These inhibitors significantly suppressed the HHE-induced NO levels to a basal level. These findings strongly suggest that in endothelial cells, HHE induces iNOS gene expression through NF-kappaB activation, which can lead to vascular dysfunction by the activation of various proinflammatory genes.

  5. Characterization of the DNA-Mediated Oxidation of Dps, A Bacterial Ferritin.

    PubMed

    Arnold, Anna R; Zhou, Andy; Barton, Jacqueline K

    2016-09-01

    Dps proteins are bacterial ferritins that protect DNA from oxidative stress and have been implicated in bacterial survival and virulence. In addition to direct oxidation of the Dps iron sites by diffusing oxidants, oxidation from a distance via DNA charge transport (CT), where electrons and electron holes are rapidly transported through the base-pair π-stack, could represent an efficient DNA protection mechanism utilized by Dps. Here, we spectroscopically characterize the DNA-mediated oxidation of ferrous iron-loaded Dps. X-band EPR was used to monitor the oxidation of DNA-bound Dps after DNA photooxidation using an intercalating ruthenium photooxidant and the flash-quench technique. Upon irradiation with poly(dGdC)2, a signal arises with g = 4.3, consistent with the formation of mononuclear high-spin Fe(III) sites of low symmetry, the expected oxidation product of Dps with one iron bound at each ferroxidase site. When poly(dGdC)2 is substituted with poly(dAdT)2, the yield of Dps oxidation is decreased significantly, consistent with guanine radical intermediates facilitating Dps oxidation. We have also explored possible protein electron transfer (ET) intermediates in the DNA-mediated oxidation of ferrous iron-loaded Dps. Dps proteins contain a conserved tryptophan residue in close proximity to the iron-binding ferroxidase site (W52 in E. coli Dps). In EPR studies of the oxidation of ferrous iron-loaded Dps following DNA photooxidation, a W52A Dps mutant was significantly deficient compared to WT Dps in forming the characteristic EPR signal at g = 4.3, consistent with W52 acting as an ET hopping intermediate. This effect is mirrored in vivo in E. coli survival in response to hydrogen peroxide, where mutation of W52 leads to decreased survival under oxidative stress.

  6. Characterization of the DNA-Mediated Oxidation of Dps, A Bacterial Ferritin.

    PubMed

    Arnold, Anna R; Zhou, Andy; Barton, Jacqueline K

    2016-09-01

    Dps proteins are bacterial ferritins that protect DNA from oxidative stress and have been implicated in bacterial survival and virulence. In addition to direct oxidation of the Dps iron sites by diffusing oxidants, oxidation from a distance via DNA charge transport (CT), where electrons and electron holes are rapidly transported through the base-pair π-stack, could represent an efficient DNA protection mechanism utilized by Dps. Here, we spectroscopically characterize the DNA-mediated oxidation of ferrous iron-loaded Dps. X-band EPR was used to monitor the oxidation of DNA-bound Dps after DNA photooxidation using an intercalating ruthenium photooxidant and the flash-quench technique. Upon irradiation with poly(dGdC)2, a signal arises with g = 4.3, consistent with the formation of mononuclear high-spin Fe(III) sites of low symmetry, the expected oxidation product of Dps with one iron bound at each ferroxidase site. When poly(dGdC)2 is substituted with poly(dAdT)2, the yield of Dps oxidation is decreased significantly, consistent with guanine radical intermediates facilitating Dps oxidation. We have also explored possible protein electron transfer (ET) intermediates in the DNA-mediated oxidation of ferrous iron-loaded Dps. Dps proteins contain a conserved tryptophan residue in close proximity to the iron-binding ferroxidase site (W52 in E. coli Dps). In EPR studies of the oxidation of ferrous iron-loaded Dps following DNA photooxidation, a W52A Dps mutant was significantly deficient compared to WT Dps in forming the characteristic EPR signal at g = 4.3, consistent with W52 acting as an ET hopping intermediate. This effect is mirrored in vivo in E. coli survival in response to hydrogen peroxide, where mutation of W52 leads to decreased survival under oxidative stress. PMID:27571139

  7. The Salmonella effector AvrA mediates bacterial intracellular survival during infection in vivo

    PubMed Central

    Wu, Huixia; Jones, Rheinallt M.; Neish, Andrew S.

    2011-01-01

    SUMMARY The enteric pathogen Salmonella typhimurium secretes the preformed AvrA effector protein into host cells. This acetyltransferase has been shown to modulate mammalian intestinal immune and survival responses by inhibition of JNK MAPK. To study the role of this effector in natural enteric infection, we used a mouse model to compare wild type Salmonella typhimurium to an isogenic AvrA null Salmonella mutant. Salmonella lacking AvrA induced increased intestinal inflammation, more intense systemic cytokine responses, and increased apoptosis in epithelial cells. Increased apoptosis was also observed in extra epithelial macrophages. AvrA null infected mice consistently showed higher bacterial burden within mucosal lymphoid tissues, spleen and liver by 5 days post infection, which indicated a more severe clinical course. To study the molecular mechanisms involved, recombinant adenoviruses expressing AvrA or mutant AvrA proteins were constructed, which showed appropriate expression and mediated the expected inhibition of JNK signaling. Cultured epithelial cells and macrophages transduced with AvrA expressing adenovirus were protected from apoptosis induced by exogenous stimuli. In conclusion, the results demonstrated that Salmonella AvrA modulates survival of infected macrophages likely via JNK suppression, and prevents macrophage death and rapid bacterial dissemination. AvrA suppression of apoptosis in infected macrophages may allow for establishment of a stable intracellular niche typical of intracellular pathogens. PMID:21899703

  8. Contribution of reactive oxygen species to (+)-catechin-mediated bacterial lethality.

    PubMed

    Ajiboye, T O; Aliyu, M; Isiaka, I; Haliru, F Z; Ibitoye, O B; Uwazie, J N; Muritala, H F; Bello, S A; Yusuf, I I; Mohammed, A O

    2016-10-25

    The contribution of reactive oxygen species to (+)-catechin-mediated bacterial lethality was investigated. Minimum inhibitory concentrations (MIC) and minimum bactericidal concentration (MBC) of (+)-catechin against E. coli, P. aeruginosa and S. aureus were investigated using 96-well microtitre plate. MIC and MBC of (+)-catechin against E. coli, P. aeruginosa and S. aureus are 600 and 700; 600 and 800; 600 and 800 μg/mL respectively. The optical densities and colony forming units of (+)-catechin-treated bacteria decreased. (+)-Catechin (4× MIC) significantly increased the superoxide anion content of E. coli, P. aeruginosa and S. aureus compared to DMSO. Superoxide dismutase and catalase in (+)-catechin treated E. coli, P. aeruginosa and S. aureus increased significantly. Conversely, level of reduced glutathione in (+)-catechin-treated E. coli, P. aeruginosa and S. aureus decreased significantly while glutathione disulfide increased significantly. Furthermore, malondialdehyde and fragmented DNA increased significantly following exposure to (+)-catechin. From the above findings, (+)-catechin enhanced the generation of reactive oxygen species (superoxide anion radical and hydroxyl radical) in E. coli, P. aeruginosa and S. aureus, possibly by autoxidation, Fenton chemistry and inhibiting electron transport chain resulting into lipid peroxidation and DNA fragmentation and consequentially bacterial cell death. PMID:27634360

  9. Bacterial conjugation protein MobA mediates integration of complex DNA structures into plant cells.

    PubMed

    Bravo-Angel, A M; Gloeckler, V; Hohn, B; Tinland, B

    1999-09-01

    Agrobacterium tumefaciens transfers T-DNA to plant cells, where it integrates into the genome, a property that is ensured by bacterial proteins VirD2 and VirE2. Under natural conditions, the protein MobA mobilizes its encoding plasmid, RSF1010, between different bacteria. A detailed analysis of MobA-mediated DNA mobilization by Agrobacterium to plants was performed. We compared the ability of MobA to transfer DNA and integrate it into the plant genome to that of pilot protein VirD2. MobA was found to be about 100-fold less efficient than VirD2 in conducting the DNA from the pTi plasmid to the plant cell nucleus. However, interestingly, DNAs transferred by the two proteins were integrated into the plant cell genome with similar efficiencies. In contrast, most of the integrated DNA copies transferred from a MobA-containing strain were truncated at the 5' end. Isolation and analysis of the most conserved 5' ends revealed patterns which resulted from the illegitimate integration of one transferred DNA within another. These complex integration patterns indicate a specific deficiency in MobA. The data conform to a model according to which efficiency of T-DNA integration is determined by plant enzymes and integrity is determined by bacterial proteins. PMID:10482518

  10. Interparticle interactions mediated superspin glass to superferromagnetic transition in Ni-bacterial cellulose aerogel nanocomposites

    NASA Astrophysics Data System (ADS)

    Thiruvengadam, V.; Vitta, Satish

    2016-06-01

    The interparticle interactions in the magnetic nanocomposites play a dominant role in controlling phase transitions: superparamagnetic to superspin glass and to superferromagnetic. These interactions can be tuned by controlling the size and number density of nanoparticles. The aerogel composites, 0.3Ni-BC and 0.7Ni-BC, consisting of Ni nanoparticles distributed in the bacterial cellulose have been used as a model system to study these interactions. Contrary to conventional approach, size of Ni-nanoparticles is not controlled and allowed to form naturally in bacterial cellulose template. The uncontrolled growth of Ni results in the formation of nanoparticles with 3 different size distributions - <10 nm particles along the length of fibrils, 50 nm particles in the intermediate spaces between the fibrils, and >100 nm particles in voids formed by reticulate structure. At room temperature, the composites exhibit a weakly ferromagnetic behaviour with a coercivity of 40 Oe, which increases to 160 Oe at 10 K. The transition from weakly ferromagnetic state to superferromagnetic state at low temperatures is mediated by the superspin glass state at intermediate temperatures via the interparticle interactions aided by nanoparticles present along the length of fibres. A temperature dependent microstructural model has been developed to understand the magnetic behaviour of nanocomposite aerogels.

  11. Hyperglycemia Impairs Neutrophil-Mediated Bacterial Clearance in Mice Infected with the Lyme Disease Pathogen

    PubMed Central

    Javid, Ashkan; Zlotnikov, Nataliya; Pětrošová, Helena; Tang, Tian Tian; Zhang, Yang; Bansal, Anil K.; Ebady, Rhodaba; Parikh, Maitry; Ahmed, Mijhgan; Sun, Chunxiang; Newbigging, Susan; Kim, Yae Ram; Santana Sosa, Marianna; Glogauer, Michael

    2016-01-01

    Insulin-insufficient type 1 diabetes is associated with attenuated bactericidal function of neutrophils, which are key mediators of innate immune responses to microbes as well as pathological inflammatory processes. Neutrophils are central to immune responses to the Lyme pathogen Borrelia burgdorferi. The effect of hyperglycemia on host susceptibility to and outcomes of B. burgdorferi infection has not been examined. The present study investigated the impact of sustained obesity-independent hyperglycemia in mice on bacterial clearance, inflammatory pathology and neutrophil responses to B. burgdorferi. Hyperglycemia was associated with reduced arthritis incidence but more widespread tissue colonization and reduced clearance of bacterial DNA in multiple tissues including brain, heart, liver, lung and knee joint. B. burgdorferi uptake and killing were impaired in neutrophils isolated from hyperglycemic mice. Thus, attenuated neutrophil function in insulin-insufficient hyperglycemia was associated with reduced B. burgdorferi clearance in target organs. These data suggest that investigating the effects of comorbid conditions such as diabetes on outcomes of B. burgdorferi infections in humans may be warranted. PMID:27340827

  12. Hyperglycemia Impairs Neutrophil-Mediated Bacterial Clearance in Mice Infected with the Lyme Disease Pathogen.

    PubMed

    Javid, Ashkan; Zlotnikov, Nataliya; Pětrošová, Helena; Tang, Tian Tian; Zhang, Yang; Bansal, Anil K; Ebady, Rhodaba; Parikh, Maitry; Ahmed, Mijhgan; Sun, Chunxiang; Newbigging, Susan; Kim, Yae Ram; Santana Sosa, Marianna; Glogauer, Michael; Moriarty, Tara J

    2016-01-01

    Insulin-insufficient type 1 diabetes is associated with attenuated bactericidal function of neutrophils, which are key mediators of innate immune responses to microbes as well as pathological inflammatory processes. Neutrophils are central to immune responses to the Lyme pathogen Borrelia burgdorferi. The effect of hyperglycemia on host susceptibility to and outcomes of B. burgdorferi infection has not been examined. The present study investigated the impact of sustained obesity-independent hyperglycemia in mice on bacterial clearance, inflammatory pathology and neutrophil responses to B. burgdorferi. Hyperglycemia was associated with reduced arthritis incidence but more widespread tissue colonization and reduced clearance of bacterial DNA in multiple tissues including brain, heart, liver, lung and knee joint. B. burgdorferi uptake and killing were impaired in neutrophils isolated from hyperglycemic mice. Thus, attenuated neutrophil function in insulin-insufficient hyperglycemia was associated with reduced B. burgdorferi clearance in target organs. These data suggest that investigating the effects of comorbid conditions such as diabetes on outcomes of B. burgdorferi infections in humans may be warranted. PMID:27340827

  13. Gustatory-mediated avoidance of bacterial lipopolysaccharides via TRPA1 activation in Drosophila

    PubMed Central

    Soldano, Alessia; Alpizar, Yeranddy A; Boonen, Brett; Franco, Luis; López-Requena, Alejandro; Liu, Guangda; Mora, Natalia; Yaksi, Emre; Voets, Thomas; Vennekens, Rudi; Hassan, Bassem A; Talavera, Karel

    2016-01-01

    Detecting pathogens and mounting immune responses upon infection is crucial for animal health. However, these responses come at a high metabolic price (McKean and Lazzaro, 2011, Kominsky et al., 2010), and avoiding pathogens before infection may be advantageous. The bacterial endotoxins lipopolysaccharides (LPS) are important immune system infection cues (Abbas et al., 2014), but it remains unknown whether animals possess sensory mechanisms to detect them prior to infection. Here we show that Drosophila melanogaster display strong aversive responses to LPS and that gustatory neurons expressing Gr66a bitter receptors mediate avoidance of LPS in feeding and egg laying assays. We found the expression of the chemosensory cation channel dTRPA1 in these cells to be necessary and sufficient for LPS avoidance. Furthermore, LPS stimulates Drosophila neurons in a TRPA1-dependent manner and activates exogenous dTRPA1 channels in human cells. Our findings demonstrate that flies detect bacterial endotoxins via a gustatory pathway through TRPA1 activation as conserved molecular mechanism. DOI: http://dx.doi.org/10.7554/eLife.13133.001 PMID:27296646

  14. The Salmonella effector AvrA mediates bacterial intracellular survival during infection in vivo.

    PubMed

    Wu, Huixia; Jones, Rheinallt M; Neish, Andrew S

    2012-01-01

    The enteric pathogen Salmonella typhimurium secretes the preformed AvrA effector protein into host cells. This acetyltransferase has been shown to modulate mammalian intestinal immune and survival responses by inhibition of JNK MAPK. To study the role of this effector in natural enteric infection, we used a mouse model to compare wild-type S. typhimurium to an isogenic AvrA null Salmonella mutant. Salmonella lacking AvrA induced increased intestinal inflammation, more intense systemic cytokine responses, and increased apoptosis in epithelial cells. Increased apoptosis was also observed in extra epithelial macrophages. AvrA null-infected mice consistently showed higher bacterial burden within mucosal lymphoid tissues, spleen and liver by 5 days post infection, which indicated a more severe clinical course. To study the molecular mechanisms involved, recombinant adenoviruses expressing AvrA or mutant AvrA proteins were constructed, which showed appropriate expression and mediated the expected inhibition of JNK signalling. Cultured epithelial cells and macrophages transduced with AvrA expressing adenovirus were protected from apoptosis induced by exogenous stimuli. In conclusion, the results demonstrated that Salmonella AvrA modulates survival of infected macrophages likely via JNK suppression, and prevents macrophage death and rapid bacterial dissemination. AvrA suppression of apoptosis in infected macrophages may allow for establishment of a stable intracellular niche typical of intracellular pathogens.

  15. Acacia ferruginea inhibits tumor progression by regulating inflammatory mediators-(TNF-a, iNOS, COX-2, IL-1β, IL-6, IFN-γ, IL-2, GM-CSF) and pro-angiogenic growth factor- VEGF.

    PubMed

    Sakthivel, Kunnathur Murugesan; Guruvayoorappan, Chandrasekaran

    2013-01-01

    The aim of the present investigation was to evaluate the effect of A ferruginea extract on Dalton's lymphoma ascites (DLA) induced tumours in BALB/c mice. Experimental animals received A ferruginea extract (10 mg/ kg.b.wt) intraperitoneally for 14 consecutive days after DLA tumor challenge. Treatment with extract significantly increased the life span, total white blood cell (WBC) count and haemoglobin (Hb) content and decreased the level of serum aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), gamma glutamyl transferase (γ-GT) and nitric oxide (NO) in DLA bearing ascites tumor models. In addition, administration of extract significantly decreased the tumour volume and body weight in a DLA bearing solid tumor model. The levels of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), interleukin-6 (IL-6) and granulocyte monocyte-colony stimulating factor (GM-CSF), as well as pro-angiogenic growth factors such as vascular endothelial growth factor (VEGF) and inducible nitric oxide synthase (iNOS) were elevated in solid tumour controls, but significantly reduced by A ferruginea administration. On the other hand, the extract stimulated the production of interleukin-2 (IL-2) and interferon-gamma (IFN-γ) in animals with DLA induced solid tumours. Increase in CD4+ T-cell population suggested strong immunostimulant activity for this extract. GC/MS and LC/MS analysis showed quinone, quinoline, imidazolidine, pyrrolidine, cyclopentenone, thiazole, pyrazole, catechin and coumarin derivatives as major compounds present in the A ferruginea methanolic extract. Thus, the outcome of the present study suggests that A ferruginea extract has immunomodulatory and tumor inhibitory activities and has the potential to be developed as a natural anticancer agent. PMID:23886206

  16. A visual assay to monitor T6SS-mediated bacterial competition.

    PubMed

    Hachani, Abderrahman; Lossi, Nadine S; Filloux, Alain

    2013-03-20

    Type VI secretion systems (T6SSs) are molecular nanomachines allowing Gram-negative bacteria to transport and inject proteins into a wide variety of target cells(1,2). The T6SS is composed of 13 core components and displays structural similarities with the tail-tube of bacteriophages(3). The phage uses a tube and a puncturing device to penetrate the cell envelope of target bacteria and inject DNA. It is proposed that the T6SS is an inverted bacteriophage device creating a specific path in the bacterial cell envelope to drive effectors and toxins to the surface. The process could be taken further and the T6SS device could perforate other cells with which the bacterium is in contact, thus injecting the effectors into these targets. The tail tube and puncturing device parts of the T6SS are made with Hcp and VgrG proteins, respectively(4,5). The versatility of the T6SS has been demonstrated through studies using various bacterial pathogens. The Vibrio cholerae T6SS can remodel the cytoskeleton of eukaryotic host cells by injecting an "evolved" VgrG carrying a C-terminal actin cross-linking domain(6,7). Another striking example was recently documented using Pseudomonas aeruginosa which is able to target and kill bacteria in a T6SS-dependent manner, therefore promoting the establishment of bacteria in specific microbial niches and competitive environment(8,9,10). In the latter case, three T6SS-secreted proteins, namely Tse1, Tse2 and Tse3 have been identified as the toxins injected in the target bacteria (Figure 1). The donor cell is protected from the deleterious effect of these effectors via an anti-toxin mechanism, mediated by the Tsi1, Tsi2 and Tsi3 immunity proteins(8,9,10). This antimicrobial activity can be monitored when T6SS-proficient bacteria are co-cultivated on solid surfaces in competition with other bacterial species or with T6SS-inactive bacteria of the same species(8,11,12,13). The data available emphasized a numerical approach to the bacterial

  17. Hemozoin Regulates iNOS Expression by Modulating the Transcription Factor NF-κB in Macrophages

    PubMed Central

    Ranjan, Ravi; Karpurapu, Manjula; Rani, Asha; Chishti, Athar H; Christman, John W

    2016-01-01

    Hemozoin (Hz) is released from ruptured erythrocytes during malaria infection caused by Plasmodium sp., in addition the malaria infected individuals are prone to bacterial sepsis. The molecular interactions between Hz, bacterial components and macrophages remains poorly investigated. In this report, we investigated the combinatorial immune-modulatory effects of phagocytosed Hz, Interferon gamma (IFNγ) or lipopolysaccharide (LPS) in macrophages. Macrophages were treated with various concentrations of commercial synthetic Hz, and surprisingly it did not result in inducible nitric oxide synthase (iNOS) expression. However, when macrophages were pretreated with Hz and then challenged with IFNγ or LPS, there was a differential impact on iNOS expression. There was an increase in iNOS expression when macrophages were pre-treated with Hz and subsequently treated with IFNγ when compared to IFNγ alone. Whereas iNOS expression was reduced when Hz phagocytosed macrophages were stimulated with LPS compared to LPS alone. Furthermore, there was an increased activation of NF-κB in Hz phagocytosed macrophages that were challenged with IFNγ. The interaction between Hz and macrophages has an impact on iNOS expression.

  18. Downregulation of inducible nitric oxide synthase (iNOS) expression is implicated in the antiviral activity of acetylsalicylic acid in HCV-expressing cells.

    PubMed

    Ríos-Ibarra, Clara Patricia; Lozano-Sepulveda, Sonia; Muñoz-Espinosa, Linda; Rincón-Sánchez, Ana Rosa; Cordova-Fletes, Carlos; Rivas-Estilla, Ana María G

    2014-12-01

    Previously, we described that acetylsalicylic acid (ASA) decreases HCV expression, but the mechanisms involved have not been clearly established. We evaluated the participation of inducible nitric oxide synthase (iNOS) in the regulation of HCV-RNA induced by ASA. Huh7 cells expressing non-structural HCV proteins were exposed to 4 mM ASA and incubated at the same times we reported HCV downregulation (24-72 h), and iNOS mRNA and protein levels were then measured by real-time PCR and Western blot, respectively. Nitric oxide levels were measured at the same time. Inhibition of iNOS mRNA by small interfering RNAs (siRNA) and activation of the iNOS gene promoter by ASA treatment were evaluated. In Huh7 replicon cells treated with ASA, we found decreased levels of iNOS mRNA, iNOS protein and nitrosylated protein levels at 48-72 h. ASA exposure also reduced the transactivation of the iNOS promoter in HCV replicon cells at 48 h, and this was partly due to the decrease in the affinity of transcription factor C/EBP-β for its binding site in the iNOS promoter. siRNA silencing of iNOS decreased HCV-RNA expression (65 %) and potentiated the antiviral effect (80 %) of ASA compared with control cells. ASA reduces iNOS expression by downregulating promoter activity, mRNA and protein levels at the same time that it decreases HCV expression. These findings suggest that the antiviral activity of ASA is mediated partially through the modulation of iNOS.

  19. Cosmic ray test of INO RPC stack

    NASA Astrophysics Data System (ADS)

    Bhuyan, M.; Datar, V. M.; Kalmani, S. D.; Lahamge, S. M.; Mondal, N. K.; Nagaraj, P.; Pal, S.; Reddy, L. V.; Redij, A.; Samuel, D.; Saraf, M. N.; Satyanarayana, B.; Shinde, R. R.; Verma, P.

    2012-01-01

    The India-based Neutrino Observatory (INO) collaboration is planning to build a 50 kt magnetised iron calorimeter (ICAL) detector using glass Resistive Plate Chambers (RPCs) as active detector elements. A stack of 12 such glass RPCs of 1 m ×1 m in area is tracking cosmic ray muons for over three years. In this paper, we will review the constructional aspects of the stack and discuss the performance of the RPCs using this cosmic ray data.

  20. A bacterial cyclic dinucleotide activates the cytosolic surveillance pathway and mediates innate resistance to tuberculosis

    PubMed Central

    Dey, Bappaditya; Dey, Ruchi Jain; Cheung, Laurene S.; Pokkali, Supriya; Guo, Haidan; Lee, Jong-Hee; Bishai, William R.

    2015-01-01

    Detection of cyclic-di-adenosine monophosphate (c-di-AMP), a bacterial second messenger, by the host cytoplasmic surveillance pathway (CSP) is known to elicit Type I interferon responses critical for antimicrobial defense1–3. However, the mechanisms and role of c-di-AMP signaling in Mycobacterium tuberculosis virulence remain unclear. Here we show that resistance to tuberculosis (TB) requires CSP-mediated detection of c-di-AMP produced by M. tuberculosis and that levels of c-di-AMP modulate the fate of infection. We found that a di-adenylate cyclase (disA or dacA)4 over-expressing M. tuberculosis strain that secretes excess c-di-AMP activates the interferon regulatory factor (IRF) pathway with enhanced levels of IFN-β, elicits increased macrophage autophagy, and exhibits significant attenuation in mice. We show that c-di-AMP-mediated IFN-β induction during M. tuberculosis infection requires stimulator of interferon genes (STING)5-signaling. We observed that c-di-AMP induction of IFN-β is independent of the cytosolic nucleic acid receptor cyclic-GMP-AMP (cGAMP) synthase (cGAS)6–7, but cGAS nevertheless contributes substantially to the overall IFN-β response to M. tuberculosis infection. In sum, our results reveal c-di-AMP to be a key mycobacterial pathogen associated molecular pattern (PAMP) driving host Type I IFN responses and autophagy. These findings suggest that modulating the levels of this small molecule may lead to novel immunotherapeutic strategies against TB. PMID:25730264

  1. MEMS/MOEMS foundry services at INO

    NASA Astrophysics Data System (ADS)

    García-Blanco, Sonia; Ilias, Samir; Williamson, Fraser; Généreux, Francis; Le Noc, Loïc; Poirier, Michel; Proulx, Christian; Tremblay, Bruno; Provençal, Francis; Desroches, Yan; Caron, Jean-Sol; Larouche, Carl; Beaupré, Patrick; Fortin, Benoit; Topart, Patrice; Picard, Francis; Alain, Christine; Pope, Timothy; Jerominek, Hubert

    2010-06-01

    In the MEMS manufacturing world, the "fabless" model is getting increasing importance in recent years as a way for MEMS manufactures and startups to minimize equipment costs and initial capital investment. In order for this model to be successful, the fabless company needs to work closely with a MEMS foundry service provider. Due to the lack of standardization in MEMS processes, as opposed to CMOS microfabrication, the experience in MEMS development processes and the flexibility of the MEMS foundry are of vital importance. A multidisciplinary team together with a complete microfabrication toolset allows INO to offer unique MEMS foundry services to fabless companies looking for low to mid-volume production. Companies that benefit from their own microfabrication facilities can also be interested in INO's assistance in conducting their research and development work during periods where production runs keep their whole staff busy. Services include design, prototyping, fabrication, packaging, and testing of various MEMS and MOEMS devices on wafers fully compatible with CMOS integration. Wafer diameters ranging typically from 1 inch to 6 inches can be accepted while 8-inch wafers can be processed in some instances. Standard microfabrication techniques such as metal, dielectric, and semiconductor film deposition and etching as well as photolithographic pattern transfer are available. A stepper permits reduction of the critical dimension to around 0.4 μm. Metals deposited by vacuum deposition methods include Au, Ag, Al, Al alloys, Ti, Cr, Cu, Mo, MoCr, Ni, Pt, and V with thickness varying from 5 nm to 2 μm. Electroplating of several materials including Ni, Au and In is also available. In addition, INO has developed and built a gold black deposition facility to answer customer's needs for broadband microbolometric detectors. The gold black deposited presents specular reflectance of less than 10% in the wavelength range from 0.2 μm to 100 μm with thickness ranging from

  2. Inflammatory bowel disease: an immunity-mediated condition triggered by bacterial infection with Helicobacter hepaticus.

    PubMed Central

    Cahill, R J; Foltz, C J; Fox, J G; Dangler, C A; Powrie, F; Schauer, D B

    1997-01-01

    Inflammatory bowel disease (IBD) is thought to result from either an abnormal immunological response to enteric flora or a normal immunological response to a specific pathogen. No study to date has combined both factors. The present studies were carried out with an immunologically manipulated mouse model of IBD. Mice homozygous for the severe combined immunodeficiency (scid) mutation develop IBD with adoptive transfer of CD4+ T cells expressing high levels of CD45RB (CD45RB(high) CD4+ T cells). These mice do not develop IBD in germfree conditions, implicating undefined intestinal flora in the pathogenesis of lesions. In controlled duplicate studies, the influence of a single murine pathogen, Helicobacter hepaticus, in combination with the abnormal immunological response on the development of IBD was assessed. The combination of H. hepaticus infection and CD45RB(high) CD4+ T-cell reconstitution resulted in severe disease expression similar to that observed in human IBD. This study demonstrates that IBD develops in mice as a consequence of an abnormal immune response in the presence of a single murine pathogen, H. hepaticus. The interaction of host immunity and a single pathogen in this murine system provides a novel model of human IBD, an immunity-mediated condition triggered by bacterial infection. PMID:9234764

  3. Possible role of alpha-1-microglobulin in mediating bacterial attachment to model surfaces.

    PubMed

    Wassall, M A; Santin, M; Peluso, G; Denyer, S P

    1998-06-01

    Urine proteins in the molecular weight range of 9-137 kDa deposit to an equal extent from pooled human urine onto glass (12.7 +/- 1.9 micrograms/cm) and polystyrene (11.8 +/- 1.8 micrograms/cm). Selective desorption of the proteins was achieved by washing with water or water/isopropanol mixtures. Irrespective of the washing process, proteins of molecular weight greater than 90 kDa remained associated with both surfaces while water washings alone removed most low molecular weight material. A 29 kDa protein, alpha-1-microglobulin, was removed from glass by water washing but required a 30% (v/v) isopropanol wash to desorb from polystyrene, implying attachment via hydrophobic bonding. The adhesion to polystyrene surfaces of Pseudomonas aeruginosa B4, a clinical isolate from a urinary tract infection (UTI), was strongly associated with the presence of alpha-1-microglobulin, which may be acting as a mediator of bacterial adhesion.

  4. The functional diversity of Drosophila Ino80 in development.

    PubMed

    Ghasemi, Mohsen; Pawar, Hema; Mishra, Rakesh K; Brahmachari, Vani

    2015-11-01

    Ino80 is well known as a chromatin remodeling protein with the catalytic function of DNA dependent ATPase and is highly conserved across phyla. Ino80 in human and Drosophila is known to form the Ino80 complex in association with the DNA binding protein Ying-Yang 1 (YY1)/Pleiohomeotic (Pho) the Drosophila homologue. We have earlier reported that Ino80 sub-family of proteins has two functional domains, namely, the DNA dependent ATPase and the DNA binding domain. In the background of the essential role of dIno80 in development, we provide evidence of Pho independent function of dIno80 in development and analyze the dual role of dIno80 in activation as well as repression in the context of the homeotic gene Scr (sex combs reduced) in imaginal discs. This differential effect of dIno80 in different imaginal discs suggests the contextual function of dIno80 as an Enhancer of Trithorax and Polycomb (ETP). We speculate on the role of dIno80 as a chromatin remodeler on one hand and a potential recruiter of epigenetic regulatory complexes on the other.

  5. A comprehensive study on algal-bacterial communities shift during thiocyanate degradation in a microalga-mediated process.

    PubMed

    Ryu, Byung-Gon; Kim, Woong; Nam, Kibok; Kim, Sungwhan; Lee, Bongsoo; Park, Min S; Yang, Ji-Won

    2015-09-01

    Changes in algal and bacterial communities during thiocyanate (SCN(-)) decomposition in a microalga-mediated process were studied. Pyrosequencing indicated that Thiobacillus bacteria and Micractinium algae predominated during SCN(-) hydrolysis, even after its complete degradation. Principal components analysis and evenness profiles (based on the Pareto-Lorenz curve) suggested that the changes in the bacterial communities were driven by nitrogen and sulfur oxidation, pH changes, and photoautotrophic conditions. The populations of predominant microalgae remained relatively stable during SCN(-) hydrolysis, but the proportion of bacteria - especially nitrifying bacteria - fluctuated. Thus, the initial microalgal population may be crucial in determining which microorganisms dominate when the preferred nitrogen source becomes limited. The results also demonstrated that microalgae and SCN(-)-hydrolyzing bacteria can coexist, that microalgae can be effectively used with these bacteria to completely treat SCN(-), and that the structure of the algal-bacterial community is more stable than the community of nitrifying bacteria alone during SCN(-) degradation.

  6. A comprehensive study on algal-bacterial communities shift during thiocyanate degradation in a microalga-mediated process.

    PubMed

    Ryu, Byung-Gon; Kim, Woong; Nam, Kibok; Kim, Sungwhan; Lee, Bongsoo; Park, Min S; Yang, Ji-Won

    2015-09-01

    Changes in algal and bacterial communities during thiocyanate (SCN(-)) decomposition in a microalga-mediated process were studied. Pyrosequencing indicated that Thiobacillus bacteria and Micractinium algae predominated during SCN(-) hydrolysis, even after its complete degradation. Principal components analysis and evenness profiles (based on the Pareto-Lorenz curve) suggested that the changes in the bacterial communities were driven by nitrogen and sulfur oxidation, pH changes, and photoautotrophic conditions. The populations of predominant microalgae remained relatively stable during SCN(-) hydrolysis, but the proportion of bacteria - especially nitrifying bacteria - fluctuated. Thus, the initial microalgal population may be crucial in determining which microorganisms dominate when the preferred nitrogen source becomes limited. The results also demonstrated that microalgae and SCN(-)-hydrolyzing bacteria can coexist, that microalgae can be effectively used with these bacteria to completely treat SCN(-), and that the structure of the algal-bacterial community is more stable than the community of nitrifying bacteria alone during SCN(-) degradation. PMID:25911193

  7. Activity of quinolone CP-115,955 against bacterial and human type II topoisomerases is mediated by different interactions.

    PubMed

    Aldred, Katie J; Schwanz, Heidi A; Li, Gangqin; Williamson, Benjamin H; McPherson, Sylvia A; Turnbough, Charles L; Kerns, Robert J; Osheroff, Neil

    2015-02-10

    CP-115,955 is a quinolone with a 4-hydroxyphenyl at C7 that displays high activity against both bacterial and human type II topoisomerases. To determine the basis for quinolone cross-reactivity between bacterial and human enzymes, the activity of CP-115,955 and a series of related quinolones and quinazolinediones against Bacillus anthracis topoisomerase IV and human topoisomerase IIα was analyzed. Results indicate that the activity of CP-115,955 against the bacterial and human enzymes is mediated by different interactions. On the basis of the decreased activity of quinazolinediones against wild-type and resistant mutant topoisomerase IV and the low activity of quinolones against resistant mutant enzymes, it appears that the primary interaction of CP-115,955 with the bacterial system is mediated through the C3/C4 keto acid and the water-metal ion bridge. In contrast, the drug interacts with the human enzyme primarily through the C7 4-hydroxyphenyl ring and has no requirement for a substituent at C8 in order to attain high activity. Despite the fact that the human type II enzyme is unable to utilize the water-metal ion bridge, quinolones in the CP-115,955 series display higher activity against topoisomerase IIα in vitro and in cultured human cells than the corresponding quinazolinediones. Thus, quinolones may be a viable platform for the development of novel drugs with anticancer potential.

  8. INO prototype detector and data acquisition system

    NASA Astrophysics Data System (ADS)

    Behere, Anita; Bhatia, M. S.; Chandratre, V. B.; Datar, V. M.; Mukhopadhyay, P. K.; Jena, Satyajit; Viyogi, Y. P.; Bhattacharya, Sudeb; Saha, Satyajit; Bhide, Sarika; Kalmani, S. D.; Mondal, N. K.; Nagaraj, P.; Nagesh, B. K.; Rao, Shobha K.; Reddy, L. V.; Saraf, M.; Satyanarayana, B.; Shinde, R. R.; Upadhya, S. S.; Verma, P.; Biswas, Saikat; Chattopadhyay, Subhasish; Sarma, P. R.

    2009-05-01

    India-based Neutrino Observatory (INO) collaboration is proposing to build a 50 kton magnetised iron calorimetric (ICAL) detector in an underground laboratory to be located in South India. Glass resistive plate chambers (RPCs) of about 2 m×2 m in size will be used as active elements for the ICAL detector. As a first step towards building the ICAL detector, a 35 ton prototype of the same is being set up over ground to track cosmic muons. Design and construction details of the prototype detector and its data acquisition system will be discussed. Some of the preliminary results from the detector stack will also be highlighted.

  9. Prophage-mediated dynamics of 'Candidatus Liberibacter asiaticus' populations, the destructive bacterial pathogens of citrus huanglongbing.

    PubMed

    Zhou, Lijuan; Powell, Charles A; Li, Wenbin; Irey, Mike; Duan, Yongping

    2013-01-01

    Prophages are highly dynamic components in the bacterial genome and play an important role in intraspecies variations. There are at least two prophages in the chromosomes of Candidatus Liberibacter asiaticus' (Las) Floridian isolates. Las is both unculturable and the most prevalent species of Liberibacter pathogens that cause huanglongbing (HLB), a worldwide destructive disease of citrus. In this study, seven new prophage variants resulting from two hyper-variable regions were identified by screening clone libraries of infected citrus, periwinkle and psyllids. Among them, Types A and B share highly conserved sequences and localize within the two prophages, FP1 and FP2, respectively. Although Types B and C were abundant in all three libraries, Type A was much more abundant in the libraries from the Las-infected psyllids than from the Las-infected plants, and Type D was only identified in libraries from the infected host plants but not from the infected psyllids. Sequence analysis of these variants revealed that the variations may result from recombination and rearrangement events. Conventional PCR results using type-specific molecular markers indicated that A, B, C and D are the four most abundant types in Las-infected citrus and periwinkle. However, only three types, A, B and C are abundant in Las-infected psyllids. Typing results for Las-infected citrus field samples indicated that mixed populations of Las bacteria present in Floridian isolates, but only the Type D population was correlated with the blotchy mottle symptom. Extended cloning and sequencing of the Type D region revealed a third prophage/phage in the Las genome, which may derive from the recombination of FP1 and FP2. Dramatic variations in these prophage regions were also found among the global Las isolates. These results are the first to demonstrate the prophage/phage-mediated dynamics of Las populations in plant and insect hosts, and their correlation with insect transmission and disease development.

  10. Ligand-free palladium-mediated site-specific protein labeling inside gram-negative bacterial pathogens.

    PubMed

    Li, Jie; Lin, Shixian; Wang, Jie; Jia, Shang; Yang, Maiyun; Hao, Ziyang; Zhang, Xiaoyu; Chen, Peng R

    2013-05-15

    Palladium, a key transition metal in advancing modern organic synthesis, mediates diverse chemical conversions including many carbon-carbon bond formation reactions between organic compounds. However, expanding palladium chemistry for conjugation of biomolecules such as proteins, particularly within their native cellular context, is still in its infancy. Here we report the site-specific protein labeling inside pathogenic Gram-negative bacterial cells via a ligand-free palladium-mediated cross-coupling reaction. Two rationally designed pyrrolysine analogues bearing an aliphatic alkyne or an iodophenyl handle were first encoded in different enteric bacteria, which offered two facial handles for palladium-mediated Sonogashira coupling reaction on proteins within these pathogens. A GFP-based bioorthogonal reaction screening system was then developed, allowing evaluation of both the efficiency and the biocompatibilty of various palladium reagents in promoting protein-small molecule conjugation. The identified simple compound-Pd(NO3)2 exhibited high efficiency and biocompatibility for site-specific labeling of proteins in vitro and inside living E. coli cells. This Pd-mediated protein coupling method was further utilized to label and visualize a Type-III Secretion (T3S) toxin-OspF in Shigella cells. Our strategy may be generally applicable for imaging and tracking various virulence proteins within Gram-negative bacterial pathogens.

  11. Investigation of antibacterial mechanism and identification of bacterial protein targets mediated by antibacterial medicinal plant extracts.

    PubMed

    Yong, Ann-Li; Ooh, Keng-Fei; Ong, Hean-Chooi; Chai, Tsun-Thai; Wong, Fai-Chu

    2015-11-01

    In this paper, we investigated the antibacterial mechanism and potential therapeutic targets of three antibacterial medicinal plants. Upon treatment with the plant extracts, bacterial proteins were extracted and resolved using denaturing gel electrophoresis. Differentially-expressed bacterial proteins were excised from the gels and subjected to sequence analysis by MALDI TOF-TOF mass spectrometry. From our study, seven differentially expressed bacterial proteins (triacylglycerol lipase, N-acetylmuramoyl-L-alanine amidase, flagellin, outer membrane protein A, stringent starvation protein A, 30S ribosomal protein s1 and 60 kDa chaperonin) were identified. Additionally, scanning electron microscope study indicated morphological damages induced on bacterial cell surfaces. To the best of our knowledge, this represents the first time these bacterial proteins are being reported, following treatments with the antibacterial plant extracts. Further studies in this direction could lead to the detailed understanding of their inhibition mechanism and discovery of target-specific antibacterial agents.

  12. The antigen 43 structure reveals a molecular Velcro-like mechanism of autotransporter-mediated bacterial clumping

    PubMed Central

    Heras, Begoña; Totsika, Makrina; Peters, Kate M.; Paxman, Jason J.; Gee, Christine L.; Jarrott, Russell J.; Perugini, Matthew A.; Whitten, Andrew E.; Schembri, Mark A.

    2014-01-01

    Aggregation and biofilm formation are critical mechanisms for bacterial resistance to host immune factors and antibiotics. Autotransporter (AT) proteins, which represent the largest group of outer-membrane and secreted proteins in Gram-negative bacteria, contribute significantly to these phenotypes. Despite their abundance and role in bacterial pathogenesis, most AT proteins have not been structurally characterized, and there is a paucity of detailed information with regard to their mode of action. Here we report the structure–function relationships of Antigen 43 (Ag43a), a prototypic self-associating AT protein from uropathogenic Escherichia coli. The functional domain of Ag43a displays a twisted L-shaped β-helical structure firmly stabilized by a 3D hydrogen-bonded scaffold. Notably, the distinctive Ag43a L shape facilitates self-association and cell aggregation. Combining all our data, we define a molecular “Velcro-like” mechanism of AT-mediated bacterial clumping, which can be tailored to fit different bacterial lifestyles such as the formation of biofilms. PMID:24335802

  13. The INO80 Complex Requires the Arp5-Ies6 Subcomplex for Chromatin Remodeling and Metabolic Regulation

    PubMed Central

    Yao, Wei; King, Devin A.; Beckwith, Sean L.; Gowans, Graeme J.; Yen, Kuangyu; Zhou, Coral

    2016-01-01

    ATP-dependent chromatin remodeling complexes are essential for transcription regulation, and yet it is unclear how these multisubunit complexes coordinate their activities to facilitate diverse transcriptional responses. In this study, we found that the conserved Arp5 and Ies6 subunits of the Saccharomyces cerevisiae INO80 chromatin-remodeler form an abundant and distinct subcomplex in vivo and stimulate INO80-mediated activity in vitro. Moreover, our genomic studies reveal that the relative occupancy of Arp5-Ies6 correlates with nucleosome positioning at transcriptional start sites and expression levels of >1,000 INO80-regulated genes. Notably, these genes are significantly enriched in energy metabolism pathways. Specifically, arp5Δ, ies6Δ, and ino80Δ mutants demonstrate decreased expression of genes involved in glycolysis and increased expression of genes in the oxidative phosphorylation pathway. Deregulation of these metabolic pathways results in constitutively elevated mitochondrial potential and oxygen consumption. Our results illustrate the dynamic nature of the INO80 complex assembly and demonstrate for the first time that a chromatin remodeler regulates glycolytic and respiratory capacity, thereby maintaining metabolic stability. PMID:26755556

  14. Bilirubin inhibits iNOS expression and NO production in response to endotoxin in rats.

    PubMed

    Wang, Weizheng W; Smith, Darcey L H; Zucker, Stephen D

    2004-08-01

    The inducible isoform of heme oxygenase (HO), HO-1, has been shown to play an important role in attenuating tissue injury. Because HO-1 catalyzes the rate-limiting step in bilirubin synthesis, we examined the hypothesis that bilirubin is a key mediator of HO-1 cytoprotection, employing a rat model of endotoxemia. Bilirubin treatment resulted in improved survival and attenuated liver injury in response to lipopolysaccharide infusion. Serum levels of NO and tumor necrosis factor alpha, key mediators of endotoxemia, and hepatic inducible nitric oxide synthase (iNOS) expression were significantly lower in bilirubin-treated rodents versus control animals. Both intraperitoneal and local administration of bilirubin also was found to ameliorate hindpaw inflammation induced by the injection of lambda-carrageenan. Consistent with in vivo results, bilirubin significantly inhibited iNOS expression and suppressed NO production in lipopolysaccharide (LPS)-stimulated RAW 264.7 murine macrophages. In contrast, bilirubin treatment induced a threefold increase in LPS-mediated prostaglandin synthesis in the absence of significant changes in cyclooxygenase expression or activity, suggesting that bilirubin enhances substrate availability for eicosanoid synthesis. Bilirubin had no effect on LPS-mediated activation of nuclear factor kappaB or p38 mitogen-activated protein kinase, consistent with a nuclear factor kappaB-independent mechanism of action. Taken together, these data support a cytoprotective role for bilirubin that is mediated, at least in part, through the inhibition of iNOS expression and, potentially, through stimulation of local prostaglandin E2 production. In conclusion, our findings suggest a role for bilirubin in mollifying tissue injury in response to inflammatory stimuli and support the possibility that the phenomenon of "jaundice of sepsis" represents an adaptive physiological response to endotoxemia. Supplementary material for this article can be found on the

  15. Non-canonical inflammasome activation of caspase-4/caspase-11 mediates epithelial defenses against enteric bacterial pathogens

    PubMed Central

    Knodler, Leigh A.; Crowley, Shauna M.; Sham, Ho Pan; Yang, Hyungjun; Wrande, Marie; Ma, Caixia; Ernst, Robert K.; Steele-Mortimer, Olivia; Celli, Jean; Vallance, Bruce A.

    2014-01-01

    Summary Inflammasome-mediated host defenses have been extensively studied in innate immune cells. Whether inflammasomes function for innate defense in intestinal epithelial cells, which represent the first line of defense against enteric pathogens, remains unknown. We observed enhanced Salmonella enterica serovar Typhimurium colonization in the intestinal epithelium of caspase-11 deficient mice, but not at systemic sites. In polarized epithelial monolayers, siRNA-mediated depletion of caspase-4, a human orthologue of caspase-11, also led to increased bacterial colonization. Decreased rates of pyroptotic cell death, a host defense mechanism that extrudes S. Typhimurium infected cells from the polarized epithelium, accounted for increased pathogen burdens. The caspase-4 inflammasome also governs activation of the proinflammatory cytokine, interleukin (IL)-18, in response to intracellular (S. Typhimurium) and extracellular (enteropathogenic Escherichia coli) enteric pathogens, via intracellular LPS sensing. Therefore an epithelial cell intrinsic non-canonical inflammasome plays a critical role in antimicrobial defense at the intestinal mucosal surface. PMID:25121752

  16. Increased intracellular Ca2+ selectively suppresses IL-1-induced NO production by reducing iNOS mRNA stability

    PubMed Central

    1995-01-01

    This study addresses the role of intracellular calcium (Ca2+) in the expression of iNOS, an IL-1 inducible gene in human articular chondrocytes. The calcium ionophore A23187 and ionomycin did not induce NO release or iNOS expression but inhibited dose dependently IL-1- induced NO release with IC50 of 200 nM and 100 nM, respectively. Increased intracellular Ca2+ induced by thapsigargin or cyclopiazonic acid, inhibitors of the endoplasmic reticulum Ca2+ ATPase, had similar inhibitory effects with IC50 of 1 nM and 3 microM, respectively. LPS and TNF alpha induced NO production were also suppressed by these Ca2+ elevating drugs. Levels of IL-1-induced iNOS protein were reduced by A23187, thapsigargin, and cyclopiazonic acid. These drugs as well as Bay K 8644 and KCl inhibited IL-1-induced iNOS mRNA expression. To analyze the role of Ca2+ in the expression of other IL-1 responsive genes in chondrocytes, these Ca2+ modulating drugs were tested for effects on COXII. In contrast to the inhibitory effects on iNOS mRNA, these drugs induced COXII mRNA expression and in combination with IL-1, enhanced COXII mRNA levels. Ca2+ mediated increases in COXII mRNA expression were associated with an increase in COXII protein. The kinetics of Ca2+ effects on IL-1-induced iNOS mRNA levels suggested a posttranscriptional mechanism. Analysis of iNOS mRNA half life showed that it was 6-7 h in IL-1-stimulated cells and decreased by A23187 to 2- 3 h. In conclusion, these results show that Ca2+ inhibits IL-1-induced NO release, iNOS protein, and mRNA expression in human articular chondrocytes by reducing iNOS mRNA stability. Under identical conditions increased Ca2+ enhances IL-1-induced COXII gene and protein expression. PMID:7540612

  17. Increased iNOS activity is essential for intestinal epithelial tight junction dysfunction in endotoxemic mice.

    PubMed

    Han, Xiaonan; Fink, Mitchell P; Yang, Runkuan; Delude, Russell L

    2004-03-01

    We tested the hypothesis that increased production of nitric oxide (NO.) associated with lipopolysaccharide (LPS)-induced systemic inflammation leads to functionally significant alterations in the expression and/or targeting of key tight junction (TJ) proteins in ileal and colonic epithelium. Wild-type or inducible NO. synthase (iNOS) knockout male C57B1/6J mice were injected intraperitoneally with 2 mg/kg Escherichia coli O111:B4 LPS. iNOS was inhibited using intraperitoneal L-N(6)-(1-iminoethyl)lysine (L-NIL; 5 mg/kg). Immunoblotting of total protein and NP-40 insoluble proteins revealed decreased expression and decreased TJ localization, respectively, of the TJ proteins, zonula occludens (ZO)-1, ZO-2, ZO-3, and/or occludin in ileal mucosa and colonic mucosa (total protein only) after injection of C57B1/6J mice with LPS. Immunohistochemistry showed deranged distribution of ZO-1 and occludin in both tissues from endotoxemic mice. Endotoxemia was associated with evidence of gut epithelial barrier dysfunction evidenced by increased ileal mucosal permeability to fluorescein isothiocyanate-dextran (Mr=4 kDa) and increased bacterial translocation to mesenteric lymph nodes. Pharmacologic inhibition of iNOS activity using L-NIL or genetic ablation of the iNOS gene ameliorated LPS-induced changes in TJ protein expression and gut mucosal barrier function. These results support the view that at least one mechanism contributing to the pathogenesis of gastrointestinal epithelial dysfunction secondary to systemic inflammation is increased iNOS-dependent NO. production leading to altered expression and localization of key TJ proteins.

  18. Rickettsia Sca2 is a bacterial formin-like mediator of actin-based motility

    PubMed Central

    Haglund, Cat M.; Choe, Julie E.; Skau, Colleen T.; Kovar, David R.; Welch, Matthew D.

    2011-01-01

    Diverse intracellular pathogens subvert the host actin polymerization machinery to drive movement within and between cells during infection. Rickettsia in the spotted fever group (SFG) are Gram-negative, obligate intracellular bacterial pathogens that undergo actin-based motility and assemble distinctive ‘comet tails’ that consist of long, unbranched actin filaments1,2. Despite this distinct organization, it was proposed that actin in Rickettsia comet tails is nucleated by the host Arp2/3 complex and the bacterial protein RickA, which assemble branched actin networks3,4. However, a second bacterial gene, sca2, was recently implicated in actin tail formation by R. rickettsii5. Here, we demonstrate that Sca2 is a bacterial actin-assembly factor that functionally mimics eukaryotic formin proteins. Sca2 nucleates unbranched actin filaments, processively associates with growing barbed ends, requires profilin for efficient elongation, and inhibits the activity of capping protein, all properties shared with formins. Sca2 localizes to the Rickettsia surface and is sufficient to promote the assembly of actin filaments in cytoplasmic extract. These results suggest that Sca2 mimics formins to determine the unique organization of actin filaments in Rickettsia tails and drive bacterial motility, independently of host nucleators. PMID:20972427

  19. Bacterial Membrane Vesicles Mediate the Release of Mycobacterium tuberculosis Lipoglycans and Lipoproteins from Infected Macrophages.

    PubMed

    Athman, Jaffre J; Wang, Ying; McDonald, David J; Boom, W Henry; Harding, Clifford V; Wearsch, Pamela A

    2015-08-01

    Mycobacterium tuberculosis is an intracellular pathogen that infects lung macrophages and releases microbial factors that regulate host defense. M. tuberculosis lipoproteins and lipoglycans block phagosome maturation, inhibit class II MHC Ag presentation, and modulate TLR2-dependent cytokine production, but the mechanisms for their release during infection are poorly defined. Furthermore, these molecules are thought to be incorporated into host membranes and released from infected macrophages within exosomes, 40-150-nm extracellular vesicles that derive from multivesicular endosomes. However, our studies revealed that extracellular vesicles released from infected macrophages include two distinct, largely nonoverlapping populations: one containing host cell markers of exosomes (CD9, CD63) and the other containing M. tuberculosis molecules (lipoglycans, lipoproteins). These vesicle populations are similar in size but have distinct densities, as determined by separation on sucrose gradients. Release of lipoglycans and lipoproteins from infected macrophages was dependent on bacterial viability, implicating active bacterial mechanisms in their secretion. Consistent with recent reports of extracellular vesicle production by bacteria (including M. tuberculosis), we propose that bacterial membrane vesicles are secreted by M. tuberculosis within infected macrophages and subsequently are released into the extracellular environment. Furthermore, extracellular vesicles released from M. tuberculosis-infected cells activate TLR2 and induce cytokine responses by uninfected macrophages. We demonstrate that these activities derive from the bacterial membrane vesicles rather than exosomes. Our findings suggest that bacterial membrane vesicles are the primary means by which M. tuberculosis exports lipoglycans and lipoproteins to impair effector functions of infected macrophages and circulate bacterial components beyond the site of infection to regulate immune responses by uninfected

  20. Bacterial membrane vesicles mediate the release of Mycobacterium tuberculosis lipoglycans and lipoproteins from infected macrophages

    PubMed Central

    Athman, Jaffre J.; Wang, Ying; McDonald, David J.; Boom, W. Henry; Harding, Clifford V.; Wearsch, Pamela A.

    2015-01-01

    Mycobacterium tuberculosis (Mtb) is an intracellular pathogen that infects lung macrophages and releases microbial factors that regulate host defense. Mtb lipoproteins and lipoglycans block phagosome maturation, inhibit MHC-II antigen presentation, and modulate TLR2-dependent cytokine production, but the mechanisms for their release during infection are poorly defined. Furthermore, these molecules are thought to be incorporated into host membranes and released from infected macrophages within exosomes, 40-150 nm extracellular vesicles that derive from multivesicular endosomes. However, our studies revealed that extracellular vesicles released from Mtb-infected macrophages include two distinct, largely non-overlapping populations, one containing host cell markers of exosomes (CD9, CD63) and the other containing Mtb molecules (lipoglycans, lipoproteins). These vesicle populations are similar in size, but have distinct densities as determined by separation on sucrose gradients. Release of Mtb lipoglycans and lipoproteins from infected macrophages was dependent on bacterial viability, implicating active bacterial mechanisms in their genesis. Consistent with recent reports of extracellular vesicle production by bacteria (including Mtb), we propose that bacterial membrane vesicles are secreted by Mtb within infected macrophages and subsequently released into the extracellular environment. Extracellular vesicles released from Mtb-infected cells activate TLR2 and induce cytokine responses by uninfected macrophages. We demonstrate that these activities derive from the bacterial membrane vesicles rather than exosomes. Our findings suggest that bacterial membrane vesicles are the primary means by which Mtb exports lipoglycans and lipoproteins to impair effector functions within infected macrophages and circulate bacterial components beyond the site of infection to regulate immune responses by uninfected cells. PMID:26109643

  1. India-based Neutrino Observatory(INO): A Status Report

    SciTech Connect

    Murthy, M. V. N.

    2011-11-23

    We present a status report on the proposed India-based Neutrino Observatory (INO). Various aspects of the INO project such as its location, the present status of the detector development, physics goals and simulation studies are discussed briefly. In particular we focus on physics studies possible with an iron calorimeter detector (ICAL) and the logistics of constructing this detector at INO. Such a detector would make precision measurements of neutrino oscillation parameters with atmospheric neutrinos in the first phase with the possibility of acting as a far-end detector of a future neutrino factory or beta beam.

  2. Tyrosine-Phosphorylated Caveolin-1 Blocks Bacterial Uptake by Inducing Vav2-RhoA-Mediated Cytoskeletal Rearrangements

    PubMed Central

    Kaushansky, Alexis; Pompaiah, Malvika; Thorn, Hans; Brinkmann, Volker; MacBeath, Gavin; Meyer, Thomas F.

    2010-01-01

    Certain bacterial adhesins appear to promote a pathogen's extracellular lifestyle rather than its entry into host cells. However, little is known about the stimuli elicited upon such pathogen host-cell interactions. Here, we report that type IV pili (Tfp)-producing Neisseria gonorrhoeae (P+GC) induces an immediate recruitment of caveolin-1 (Cav1) in the host cell, which subsequently prevents bacterial internalization by triggering cytoskeletal rearrangements via downstream phosphotyrosine signaling. A broad and unbiased analysis of potential interaction partners for tyrosine-phosphorylated Cav1 revealed a direct interaction with the Rho-family guanine nucleotide exchange factor Vav2. Both Vav2 and its substrate, the small GTPase RhoA, were found to play a direct role in the Cav1-mediated prevention of bacterial uptake. Our findings, which have been extended to enteropathogenic Escherichia coli, highlight how Tfp-producing bacteria avoid host cell uptake. Further, our data establish a mechanistic link between Cav1 phosphorylation and pathogen-induced cytoskeleton reorganization and advance our understanding of caveolin function. PMID:20808760

  3. Responses of plasmid-mediated quinolone resistance genes and bacterial taxa to (fluoro)quinolones-containing manure in arable soil.

    PubMed

    Xiong, Wenguang; Sun, Yongxue; Ding, Xueyao; Zhang, Yiming; Zhong, Xiaoxia; Liang, Wenfei; Zeng, Zhenling

    2015-01-01

    The aim of the present study was to investigate the fate of plasmid-mediated quinolone resistance (PMQR) genes and the disturbance of soil bacterial communities posed by (fluoro)quinolones (FQNs)-containing manure in arable soil. Representative FQNs (enrofloxacin (ENR), ciprofloxacin (CIP) and norfloxacin (NOR)), PMQR genes (qepA, oqxA, oqxB, aac(6')-Ib-cr and qnrS) and bacterial communities in untreated soil, +manure and +manure+FQNs groups were analyzed using culture independent methods. The significantly higher abundance of oqxA, oqxB and aac(6')-Ib-cr, and significantly higher abundance of qnrS in +manure group than those in untreated soil disappeared at day 30 and day 60, respectively. All PMQR genes (oqxA, oqxB, aac(6')-Ib-cr and qnrS) dissipated 1.5-1.7 times faster in +manure group than those in +manure+FQNs group. The disturbance of soil bacterial communities posed by FQNs-containing manure was also found. The results indicated that significant effects of PMQR genes (oqxA, oqxB, aac(6')-Ib and qnrS) on arable soils introduced by manure disappeared 2 month after manure application. FQNs introduced by manure slowed down the dissipation of PMQR genes. The presence of high FQNs provided a selective advantage for species affiliated to the phylum including Acidobacteria, Verrucomicrobia and Planctomycetes while suppressing Proteobacteria and Actinobacteria.

  4. Bacterial Photodynamic Inactivation Mediated by Methylene Blue and Red Light Is Enhanced by Synergistic Effect of Potassium Iodide

    PubMed Central

    Vecchio, Daniela; Gupta, Asheesh; Huang, Liyi; Landi, Giacomo; Avci, Pinar; Rodas, Andrea

    2015-01-01

    The inexorable increase of antibiotic resistance occurring in different bacterial species is increasing the interest in developing new antimicrobial treatments that will be equally effective against multidrug-resistant strains and will not themselves induce resistance. One of these alternatives may be photodynamic inactivation (PDI), which uses a combination of nontoxic dyes, called photosensitizers (PS), excited by harmless visible light to generate reactive oxygen species (ROS) by type 1 (radical) and type 2 (singlet oxygen) pathways. In this study, we asked whether it was possible to improve the efficacy of PDI in vitro and in vivo by addition of the inert salt potassium iodide (KI) to a commonly investigated PS, the phenothiazinium dye methylene blue (MB). By adding KI, we observed a consistent increase of red light-mediated bacterial killing of Gram-positive and Gram-negative species in vitro and in vivo. In vivo, we also observed less bacterial recurrence in wounds in the days posttreatment. The mechanism of action is probably due to formation of reactive iodine species that are produced quickly with a short lifetime. This finding may have a relevant clinical impact by reducing the risk of amputation and, in some cases, the risk of death, leading to improvement in the care of patients affected by localized infections. PMID:26077247

  5. Rapid and sensitive detection of Citrus Bacterial Canker by loop-mediated isothermal amplification combined with simple visual evaluation methods

    PubMed Central

    2010-01-01

    Background Citrus Bacterial Canker (CBC) is a major, highly contagious disease of citrus plants present in many countries in Asia, Africa and America, but not in the Mediterranean area. There are three types of Citrus Bacterial Canker, named A, B, and C that have different genotypes and posses variation in host range within citrus species. The causative agent for type A CBC is Xanthomonas citri subsp. citri, while Xanthomonas fuscans subsp. aurantifolii, strain B causes type B CBC and Xanthomonas fuscans subsp. aurantifolii strain C causes CBC type C. The early and accurate identification of those bacteria is essential for the protection of the citrus industry. Detection methods based on bacterial isolation, antibodies or polymerase chain reaction (PCR) have been developed previously; however, these approaches may be time consuming, laborious and, in the case of PCR, it requires expensive laboratory equipment. Loop-mediated isothermal amplification (LAMP), which is a novel isothermal DNA amplification technique, is sensitive, specific, fast and requires no specialized laboratory equipment. Results A loop-mediated isothermal amplification assay for the diagnosis of Citrus Bacterial Canker (CBC-LAMP) was developed and evaluated. DNA samples were obtained from infected plants or cultured bacteria. A typical ladder-like pattern on gel electrophoresis was observed in all positive samples in contrast to the negative controls. In addition, amplification products were detected by visual inspection using SYBRGreen and using a lateral flow dipstick, eliminating the need for gel electrophoresis. The sensitivity and specificity of the assay were evaluated in different conditions and using several sample sources which included purified DNA, bacterium culture and infected plant tissue. The sensitivity of the CBC-LAMP was 10 fg of pure Xcc DNA, 5 CFU in culture samples and 18 CFU in samples of infected plant tissue. No cross reaction was observed with DNA of other phytopathogenic

  6. Reactive Oxygen Species Mediated Bacterial Biofilm Inhibition via Zinc Oxide Nanoparticles and Their Statistical Determination

    PubMed Central

    Dwivedi, Sourabh; Wahab, Rizwan; Khan, Farheen; Mishra, Yogendra K.; Musarrat, Javed; Al-Khedhairy, Abdulaziz A.

    2014-01-01

    The formation of bacterial biofilm is a major challenge in clinical applications. The main aim of this study is to describe the synthesis, characterization and biocidal potential of zinc oxide nanoparticles (NPs) against bacterial strain Pseudomonas aeruginosa. These nanoparticles were synthesized via soft chemical solution process in a very short time and their structural properties have been investigated in detail by using X-ray diffraction and transmission electron microscopy measurements. In this work, the potential of synthesized ZnO-NPs (∼10–15 nm) has been assessed in-vitro inhibition of bacteria and the formation of their biofilms was observed using the tissue culture plate assays. The crystal violet staining on biofilm formation and its optical density revealed the effect on biofilm inhibition. The NPs at a concentration of 100 µg/mL significantly inhibited the growth of bacteria and biofilm formation. The biofilm inhibition by ZnO-NPs was also confirmed via bio-transmission electron microscopy (Bio-TEM). The Bio-TEM analysis of ZnO-NPs treated bacteria confirmed the deformation and damage of cells. The bacterial growth in presence of NPs concluded the bactericidal ability of NPs in a concentration dependent manner. It has been speculated that the antibacterial activity of NPs as a surface coating material, could be a feasible approach for controlling the pathogens. Additionally, the obtained bacterial solution data is also in agreement with the results from statistical analytical methods. PMID:25402188

  7. L-arginine mediated renaturation enhances yield of human, α6 Type IV collagen non-collagenous domain from bacterial inclusion bodies.

    PubMed

    Gunda, Venugopal; Boosani, Chandra Shekhar; Verma, Raj Kumar; Guda, Chittibabu; Sudhakar, Yakkanti Akul

    2012-10-01

    The anti-angiogenic, carboxy terminal non-collagenous domain (NC1) derived from human Collagen type IV alpha 6 chain, [α6(IV)NC1] or hexastatin, was earlier obtained using different recombinant methods of expression in bacterial systems. However, the effect of L-arginine mediated renaturation in enhancing the relative yields of this protein from bacterial inclusion bodies has not been evaluated. In the present study, direct stirring and on-column renaturation methods using L-arginine and different size exclusion chromatography matrices were applied for enhancing the solubility in purifying the recombinant α6(IV)NC1 from bacterial inclusion bodies. This methodology enabled purification of higher quantities of soluble protein from inclusion bodies, which inhibited endothelial cell proliferation, migration and tube formation. Thus, the scope for L-arginine mediated renaturation in obtaining higher yields of soluble, biologically active NC1 domain from bacterial inclusion bodies was evaluated.

  8. Essential roles for platelets during neutrophil-dependent or lymphocyte-mediated defense against bacterial pathogens.

    PubMed

    Wang, Zheng; Zhao, Qi; Zhang, Dongxia; Sun, Chengming; Bao, Cuixia; Yi, Maoli; Xing, Li; Luo, Deyan

    2016-09-01

    Emerging evidence from animal models suggests that platelets may participate in a wide variety of processes including the immune response against infection. More than 200 whole blood samples from patients and healthy controls were run in the System XE-5000 analyzer, and plasma fractions were separated for the following tests by ELISA, Luminex and light scattering. We describe two mechanisms by which platelets may contribute to immune function against various bacterial pathogens based on increased mean platelet volume in gram-positive bacterial infections and increased platelet counts in gram-negative bacterial infections. Gram-negative bacteria activate platelets to recruit neutrophils, which participate in the immune response against infection. During this process, fractalkine, macrophage inflammatory protein-1β, interleukin-17A, tumor necrosis factor-α and platelet-activating factor were higher in patients infected with Escherichia coli; additionally, giant platelets were observed under the microscope. Meanwhile, we found that platelets played a different role in gram-positive bacterial infections. Specifically, they could actively adhere to gram-positive bacteria in circulation and transfer them to immune sites to promote antibacterial lymphocyte expansion. During this process, complement C3 and factor XI were more highly expressed in patients infected with Staphylococcus aureus; additionally, we detected more small platelets under the microscope. Platelets participate in the immune response against both gram-negative and gram-positive bacteria, although the mechanisms differ. These results will help us understand the complex roles of platelets during infections, and direct our use of antibiotics based on clinical platelet data.

  9. Igg Subclasses Targeting the Flagella of Salmonella enterica Serovar Typhimurium Can Mediate Phagocytosis and Bacterial Killing

    PubMed Central

    Goh, Yun Shan; Armour, Kathryn L; Clark, Michael R; Grant, Andrew J; Mastroeni, Pietro

    2016-01-01

    Invasive non-typhoidal Salmonella are a common cause of invasive disease in immuno-compromised individuals and in children. Multi-drug resistance poses challenges to disease control, with a critical need for effective vaccines. Flagellin is an attractive vaccine candidate due to surface exposure and high epitope copy number, but its potential as a target for opsonophacytic antibodies is unclear. We examined the effect of targeting flagella with different classes of IgG on the interaction between Salmonella Typhimurium and a human phagocyte-like cell line, THP-1. We tagged the FliC flagellar protein with a foreign CD52 mimotope (TSSPSAD) and bacteria were opsonized with a panel of humanised CD52 antibodies with the same antigen-binding V-region, but different constant regions. We found that IgG binding to flagella increases bacterial phagocytosis and reduces viable intracellular bacterial numbers. Opsonisation with IgG3, followed by IgG1, IgG4, and IgG2, resulted in the highest level of bacterial uptake and in the highest reduction in the intracellular load of viable bacteria. Taken together, our data provide proof-of-principle evidence that targeting flagella with antibodies can increase the antibacterial function of host cells, with IgG3 being the most potent subclass. These data will assist the rational design of urgently needed, optimised vaccines against iNTS disease. PMID:27366588

  10. EDS1 mediates pathogen resistance and virulence function of a bacterial effector in soybean

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Enhanced disease susceptibility 1 (EDS1) and phytoalexin deficient 4 (PAD4) are well known regulators of both basal and resistance (R) protein-mediated plant defense. We identified two EDS1- (GmEDS1a/b) and one PAD4-like (GmPAD4) protein that are required for resistance signaling in soybean. Consist...

  11. Amino acid oxidase of leukocytes in relation to H2O2-mediated bacterial killing

    PubMed Central

    Eckstein, Marlene R.; Baehner, Robert L.; Nathan, David G.

    1971-01-01

    D-Amino acid oxidase and L-amino acid oxidase have been measured in sucrose homogenates of polymorphonuclear leukocytes (PMN) obtained from guinea pigs and humans. Subcellular distribution patterns and studies on latency indicate that these oxidases are soluble enzymes. Their hydrogen peroxide-generating capacity was verified. Chronic granulomatous disease PMN, which lack a respiratory burst and fail to generate H2O2 during phagocytosis and do not kill catalase positive bacteria, had peroxide-generating amino acid oxidase activity equal to that found in PMN homogenates from patients with bacterial infections. The precise metabolic and bactericidal role of amino acid oxidases in PMN remains uncertain. PMID:4397948

  12. Inflammatory modulating effects of low level laser therapy on iNOS expression by means of bioluminescence imaging

    NASA Astrophysics Data System (ADS)

    Moriyama, Yumi; Moriyama, Eduardo H.; Blackmore, Kristina; Akens, Margarete K.; Lilge, Lothar

    2005-09-01

    This study investigates the efficacy of low level laser therapy (LLLT) in modulating inducible nitric oxide synthase (iNOS) expression as molecular marker of the inflammation signaling pathway. LLLT was mediated by different therapeutic wavelengths using transgenic animals with the luciferase gene under control of the iNOS gene expression. Inflammation in 30 transgenic mice (iNOS-luc mice, from FVB strain) was induced by intra-articular injection of Zymosan-A in both knee joints. Four experimental groups were treated with one of four different wavelengths (λ=635, 785, 808 and 905nm) and one not laser-irradiated control group. Laser treatment (25 mW cm-2, 5 J cm-2) was applied to the knees 15 minutes after inflammation induction. Measurements of iNOS expression were performed at multiple times (0, 3, 5, 7, 9 and 24h) post-LLLT by measuring the bioluminescence signal using a highly sensitive charge-coupled device (CCD) camera. The responsivity of BLI was sufficient to demonstrate a significant increase in bioluminescence signals after laser irradiation of 635nm when compared to non-irradiated animals and the other LLLT treated groups, showing the wavelength-dependence of LLLT on iNOS expression during the acute inflammatory process.

  13. TLR4-mediated immunomodulatory properties of the bacterial metalloprotease arazyme in preclinical tumor models.

    PubMed

    Pereira, Felipe V; Melo, Amanda C L; de Melo, Filipe M; Mourão-Sá, Diego; Silva, Priscila; Berzaghi, Rodrigo; Herbozo, Carolina C A; Coelho-Dos-Reis, Jordana; Scutti, Jorge A; Origassa, Clarice S T; Pereira, Rosana M; Juliano, Luis; Juliano, Maria Aparecida; Carmona, Adriana K; Câmara, Niels O S; Tsuji, Moriya; Travassos, Luiz R; Rodrigues, Elaine G

    2016-07-01

    Despite the recent approval of new agents for metastatic melanoma, its treatment remains challenging. Moreover, few available immunotherapies induce a strong cellular immune response, and selection of the correct immunoadjuvant is crucial for overcoming this obstacle. Here, we studied the immunomodulatory properties of arazyme, a bacterial metalloprotease, which was previously shown to control metastasis in a murine melanoma B16F10-Nex2 model. The antitumor activity of arazyme was independent of its proteolytic activity, since heat-inactivated protease showed comparable properties to the active enzyme; however, the effect was dependent on an intact immune system, as antitumor properties were lost in immunodeficient mice. The protective response was IFNγ-dependent, and CD8(+) T lymphocytes were the main effector antitumor population, although B and CD4(+) T lymphocytes were also induced. Macrophages and dendritic cells were involved in the induction of the antitumor response, as arazyme activation of these cells increased both the expression of surface activation markers and proinflammatory cytokine secretion through TLR4-MyD88-TRIF-dependent, but also MAPK-dependent pathways. Arazyme was also effective in the murine breast adenocarcinoma 4T1 model, reducing primary and metastatic tumor development, and prolonging survival. To our knowledge, this is the first report of a bacterial metalloprotease interaction with TLR4 and subsequent receptor activation that promotes a proinflammatory and tumor protective response. Our results show that arazyme has immunomodulatory properties, and could be a promising novel alternative for metastatic melanoma treatment. PMID:27622031

  14. Proteasomal Degradation of Nod2 Protein Mediates Tolerance to Bacterial Cell Wall Components*

    PubMed Central

    Lee, Kyoung-Hee; Biswas, Amlan; Liu, Yuen-Joyce; Kobayashi, Koichi S.

    2012-01-01

    The innate immune system serves as the first line of defense by detecting microbes and initiating inflammatory responses. Although both Toll-like receptor (TLR) and nucleotide binding domain and leucine-rich repeat (NLR) proteins are important for this process, their excessive activation is hazardous to hosts; thus, tight regulation is required. Endotoxin tolerance is refractory to repeated lipopolysaccharide (LPS) stimulation and serves as a host defense mechanism against septic shock caused by an excessive TLR4 response during Gram-negative bacterial infection. Gram-positive bacteria as well as their cell wall components also induce shock. However, the mechanism underlying tolerance is not understood. Here, we show that activation of Nod2 by its ligand, muramyl dipeptide (MDP) in the bacterial cell wall, induces rapid degradation of Nod2, which confers MDP tolerance in vitro and in vivo. Nod2 is constitutively associated with a chaperone protein, Hsp90, which is required for Nod2 stability and protects Nod2 from degradation. Upon MDP stimulation, Hsp90 rapidly dissociates from Nod2, which subsequently undergoes ubiquitination and proteasomal degradation. The SOCS-3 protein induced by Nod2 activation further facilitates this degradation process. Therefore, Nod2 protein stability is a key factor in determining responsiveness to MDP stimulation. This indicates that TLRs and NLRs induce a tolerant state through distinct molecular mechanisms that protect the host from septic shock. PMID:23019338

  15. Role of bacterial virulence proteins in Agrobacterium-mediated transformation of Aspergillus awamori.

    PubMed

    Michielse, C B; Ram, A F J; Hooykaas, P J J; Hondel, C A M J J van den

    2004-05-01

    The Agrobacterium-mediated transformation of Aspergillus awamori was optimized using defined co-cultivation conditions, which resulted in a reproducible and efficient transformation system. Optimal co-cultivation conditions were used to study the role of Agrobacterium tumefaciens virulence proteins in T-DNA transfer. This study revealed that inactivation of either of the regulatory proteins (VirA, VirG), any of the transport pore proteins (VirB), proteins involved in generation of the T-strand (VirD, VirC) or T-strand protection and targeting (VirE2) abolishes or severely reduces the formation of transformants. The results indicate that the Agrobacterium-mediated transformation of A. awamori requires an intact T-DNA machinery for efficient transformation; however, the plant host range factors, like VirE3, VirH, and VirF, are not important. PMID:15050546

  16. Bacterial infection of wounds: fibronectin-mediated adherence group A and C streptococci to fibrin thrombi in vitro.

    PubMed

    Chhatwal, G S; Valentin-Weigand, P; Timmis, K N

    1990-09-01

    Adherence of group A, B, and C streptococci to fibrin thrombi was studied by using a novel fluorochrome microassay carried out in microdilution plates in which fibrin thrombi had been prepared by clotting citrated human, cattle, or horse plasma. Substantial adherence was observed with various strains of group A and C streptococci, whereas little was observed with group B streptococci. Adherence of all group A and C streptococcal strains decreased by up to 40% when fibronectin was depleted from the plasmas used for preparing fibrin thrombi, and fibronectin repletion increased adherence of streptococci in a dose-dependent manner. Addition of the 210-kilodalton C-terminal fragment of fibronectin to fibronectin-depleted plasma restored the adherence of group C but not group A streptococci, whereas addition of the 29-kilodalton N-terminal fragment was without any effect for all tested streptococcal strains. Prior incubation of group A and C streptococcal strains with fibronectin markedly increased their adherence, but treatment with proteases abolished their ability to adhere to fibrin thrombi. Adherence of group B streptococci was not affected by either fibronectin depletion or proteolytic digestion. These results indicate that both fibronectin incorporated into the fibrin matrix of thrombi and soluble fibronectin can mediate adherence of group A and C streptococci to fibrin thrombi and that binding sites for fibronectin located on the bacterial surface mediate this adherence.

  17. Two apextrin-like proteins mediate extracellular and intracellular bacterial recognition in amphioxus.

    PubMed

    Huang, Guangrui; Huang, Shengfeng; Yan, Xinyu; Yang, Ping; Li, Jun; Xu, Weiya; Zhang, Lingling; Wang, Ruihua; Yu, Yingcai; Yuan, Shaochun; Chen, Shangwu; Luo, Guangbin; Xu, Anlong

    2014-09-16

    Animals exploit different germ-line-encoded proteins with various domain structures to detect the signature molecules of pathogenic microbes. These molecules are known as pathogen-associated molecular patterns (PAMPs), and the host proteins that react with PAMPs are called pattern recognition proteins (PRPs). Here, we present a novel type of protein domain structure capable of binding to bacterial peptidoglycan (PGN) and the minimal PGN motif muramyl dipeptide (MDP). This domain is designated as apextrin C-terminal domain (ApeC), and its presence was confirmed in several invertebrate phyla and subphyla. Two apextrin-like proteins (ALP1 and ALP2) were identified in a basal chordate, the Japanese amphioxus Branchiostoma japonicum (bj). bjALP1 is a mucosal effector secreted into the gut lumen to agglutinate the Gram-positive bacterium Staphylococcus aureus via PGN binding. Neutralization of secreted bjALP1 by anti-bjALP1 monoclonal antibodies caused serious damage to the gut epithelium and rapid death of the animals after bacterial infection. bjALP2 is an intracellular PGN sensor that binds to TNF receptor-associated factor 6 (TRAF6) and prevents TRAF6 from self-ubiquitination and hence from NF-κB activation. MDP was found to compete with TRAF6 for bjALP2, which released TRAF6 to activate the NF-κB pathway. BjALP1 and bjALP2 therefore play distinct and complementary functions in amphioxus gut mucosal immunity. In conclusion, discovery of the ApeC domain and the functional analyses of amphioxus ALP1 and ALP2 allowed us to define a previously undocumented type of PRP that is represented across different animal phyla.

  18. Two apextrin-like proteins mediate extracellular and intracellular bacterial recognition in amphioxus.

    PubMed

    Huang, Guangrui; Huang, Shengfeng; Yan, Xinyu; Yang, Ping; Li, Jun; Xu, Weiya; Zhang, Lingling; Wang, Ruihua; Yu, Yingcai; Yuan, Shaochun; Chen, Shangwu; Luo, Guangbin; Xu, Anlong

    2014-09-16

    Animals exploit different germ-line-encoded proteins with various domain structures to detect the signature molecules of pathogenic microbes. These molecules are known as pathogen-associated molecular patterns (PAMPs), and the host proteins that react with PAMPs are called pattern recognition proteins (PRPs). Here, we present a novel type of protein domain structure capable of binding to bacterial peptidoglycan (PGN) and the minimal PGN motif muramyl dipeptide (MDP). This domain is designated as apextrin C-terminal domain (ApeC), and its presence was confirmed in several invertebrate phyla and subphyla. Two apextrin-like proteins (ALP1 and ALP2) were identified in a basal chordate, the Japanese amphioxus Branchiostoma japonicum (bj). bjALP1 is a mucosal effector secreted into the gut lumen to agglutinate the Gram-positive bacterium Staphylococcus aureus via PGN binding. Neutralization of secreted bjALP1 by anti-bjALP1 monoclonal antibodies caused serious damage to the gut epithelium and rapid death of the animals after bacterial infection. bjALP2 is an intracellular PGN sensor that binds to TNF receptor-associated factor 6 (TRAF6) and prevents TRAF6 from self-ubiquitination and hence from NF-κB activation. MDP was found to compete with TRAF6 for bjALP2, which released TRAF6 to activate the NF-κB pathway. BjALP1 and bjALP2 therefore play distinct and complementary functions in amphioxus gut mucosal immunity. In conclusion, discovery of the ApeC domain and the functional analyses of amphioxus ALP1 and ALP2 allowed us to define a previously undocumented type of PRP that is represented across different animal phyla. PMID:25187559

  19. Characterization of rhizosphere fungi that mediate resistance in tomato against bacterial wilt disease.

    PubMed

    Jogaiah, Sudisha; Abdelrahman, Mostafa; Tran, Lam-Son Phan; Shin-ichi, Ito

    2013-09-01

    Plant immunization for resistance against a wide variety of phytopathogens is an effective strategy for plant disease management. Seventy-nine plant growth-promoting fungi (PGPFs) were isolated from rhizosphere soil of India. Among them, nine revealed saprophytic ability, root colonization, phosphate solubilization, IAA production, and plant growth promotion. Seed priming with four PGPFs exhibited early seedling emergence and enhanced vigour of a tomato cultivar susceptible to the bacterial wilt pathogen compared to untreated controls. Under greenhouse conditions, TriH_JSB27 and PenC_JSB41 treatments remarkably enhanced the vegetative and reproductive growth parameters. Maximum NPK uptake was noticed in TriH_JSB27-treated plants. A significant disease reduction of 57.3% against Ralstonia solanacearum was observed in tomato plants pretreated with TriH_JSB27. Furthermore, induction of defence-related enzymes and genes was observed in plants pretreated with PGPFs or inoculated with pathogen. The maximum phenylalanine ammonia lyase (PAL) activity (111U) was observed at 24h in seedlings treated with TriH_JSB27 and this activity was slightly reduced (99U) after pathogen inoculation. Activities of peroxidase (POX, 54U) and β-1,3-glucanase (GLU, 15U) were significantly higher in control plants inoculated with pathogen after 24h and remained constant at all time points. A similar trend in gene induction for PAL was evident in PGPFs-treated tomato seedlings with or without pathogen inoculation, whereas POX and GLU were upregulated in control plus pathogen-inoculated tomato seedlings. These results determine that the susceptible tomato cultivar is triggered after perception of potent PGPFs to synthesize PAL, POX, and GLU, which activate defence resistance against bacterial wilt disease, thereby contributing to plant health improvement.

  20. Identification of the bacterial protein FtsX as a unique target of chemokine-mediated antimicrobial activity against Bacillus anthracis

    PubMed Central

    Crawford, Matthew A.; Lowe, David E.; Fisher, Debra J.; Stibitz, Scott; Plaut, Roger D.; Beaber, John W.; Zemansky, Jason; Mehrad, Borna; Glomski, Ian J.; Strieter, Robert M.; Hughes, Molly A.

    2011-01-01

    Chemokines are a family of chemotactic cytokines that function in host defense by orchestrating cellular movement during infection. In addition to this function, many chemokines have also been found to mediate the direct killing of a range of pathogenic microorganisms through an as-yet-undefined mechanism. As an understanding of the molecular mechanism and microbial targets of chemokine-mediated antimicrobial activity is likely to lead to the identification of unique, broad-spectrum therapeutic targets for effectively treating infection, we sought to investigate the mechanism by which the chemokine CXCL10 mediates bactericidal activity against the Gram-positive bacterium Bacillus anthracis, the causative agent of anthrax. Here, we report that disruption of the gene ftsX, which encodes the transmembrane domain of a putative ATP-binding cassette transporter, affords resistance to CXCL10-mediated antimicrobial effects against vegetative B. anthracis bacilli. Furthermore, we demonstrate that in the absence of FtsX, CXCL10 is unable to localize to its presumed site of action at the bacterial cell membrane, suggesting that chemokines interact with specific, identifiable bacterial components to mediate direct microbial killing. These findings provide unique insight into the mechanism of CXCL10-mediated bactericidal activity and establish, to our knowledge, the first description of a bacterial component critically involved in the ability of host chemokines to target and kill a bacterial pathogen. These observations also support the notion of chemokine-mediated antimicrobial activity as an important foundation for the development of innovative therapeutic strategies for treating infections caused by pathogenic, potentially multidrug-resistant microorganisms. PMID:21949405

  1. Identification of the bacterial protein FtsX as a unique target of chemokine-mediated antimicrobial activity against Bacillus anthracis.

    PubMed

    Crawford, Matthew A; Lowe, David E; Fisher, Debra J; Stibitz, Scott; Plaut, Roger D; Beaber, John W; Zemansky, Jason; Mehrad, Borna; Glomski, Ian J; Strieter, Robert M; Hughes, Molly A

    2011-10-11

    Chemokines are a family of chemotactic cytokines that function in host defense by orchestrating cellular movement during infection. In addition to this function, many chemokines have also been found to mediate the direct killing of a range of pathogenic microorganisms through an as-yet-undefined mechanism. As an understanding of the molecular mechanism and microbial targets of chemokine-mediated antimicrobial activity is likely to lead to the identification of unique, broad-spectrum therapeutic targets for effectively treating infection, we sought to investigate the mechanism by which the chemokine CXCL10 mediates bactericidal activity against the Gram-positive bacterium Bacillus anthracis, the causative agent of anthrax. Here, we report that disruption of the gene ftsX, which encodes the transmembrane domain of a putative ATP-binding cassette transporter, affords resistance to CXCL10-mediated antimicrobial effects against vegetative B. anthracis bacilli. Furthermore, we demonstrate that in the absence of FtsX, CXCL10 is unable to localize to its presumed site of action at the bacterial cell membrane, suggesting that chemokines interact with specific, identifiable bacterial components to mediate direct microbial killing. These findings provide unique insight into the mechanism of CXCL10-mediated bactericidal activity and establish, to our knowledge, the first description of a bacterial component critically involved in the ability of host chemokines to target and kill a bacterial pathogen. These observations also support the notion of chemokine-mediated antimicrobial activity as an important foundation for the development of innovative therapeutic strategies for treating infections caused by pathogenic, potentially multidrug-resistant microorganisms.

  2. RPC detector characteristics and performance for INO-ICAL experiment

    NASA Astrophysics Data System (ADS)

    Kumar, A.; Gaur, A.; Hasbuddin, Md.; Naimuddin, Md.

    2016-03-01

    The India-based Neutrino Observatory (INO) is an approved multi-institutional collaboration neutrino physics project, aimed at building an underground laboratory in the southern India. INO will utilize a large magnetized Iron Calorimeter (ICAL) detector to study the atmospheric neutrinos, and to explore the unresolved issues related to neutrinos. The Resistive Plate Chambers (RPCs), interleaved in between iron absorber layers, are going to be used as the active signal readouts for the ICAL experiment at INO. The research and development is carried out to find structural quality and electrical response for RPC electrode materials available within local domain. The assembled 2 mm gap RPCs are tested using cosmic muons for their detection performance. The study also incorporates preliminary results on detector timing and signal induced charge measurements.

  3. [TREATMENT OF EXTREMELY PREMATURE NEWBORN INFANT WITH INO. CLINICAL CASE].

    PubMed

    Radulova, P; Slancheva, B; Marinov, R

    2015-01-01

    Prolonged inhaled nitric oxide (iNO) from birth in preterm neonates with BPD improves endogenous surfactant function as well as lung growth, angiogenesis, and alveologenesis. As a result there is a reduction in the frequency of the "new" form of BPD in neonates under 28 weeks of gestation and birth weight under 1000 gr. Delivery of inhaled nitric oxide is a new method of prevention of chronic lung disease. According to a large number of randomized trials iNO in premature neonates reduces pulmonary morbidity and leads to a reduction of the mortality in this population of patients. This new therapy does not have serious side effects. We represent a clinical case of extremely premature newborn infant with BPD treated with iNO.

  4. Structure and primase-mediated activation of a bacterial dodecameric replicative helicase

    PubMed Central

    Bazin, Alexandre; Cherrier, Mickaël V.; Gutsche, Irina; Timmins, Joanna; Terradot, Laurent

    2015-01-01

    Replicative helicases are essential ATPases that unwind DNA to initiate chromosomal replication. While bacterial replicative DnaB helicases are hexameric, Helicobacter pylori DnaB (HpDnaB) was found to form double hexamers, similar to some archaeal and eukaryotic replicative helicases. Here we present a structural and functional analysis of HpDnaB protein during primosome formation. The crystal structure of the HpDnaB at 6.7 Å resolution reveals a dodecameric organization consisting of two hexamers assembled via their N-terminal rings in a stack-twisted mode. Using fluorescence anisotropy we show that HpDnaB dodecamer interacts with single-stranded DNA in the presence of ATP but has a low DNA unwinding activity. Multi-angle light scattering and small angle X-ray scattering demonstrate that interaction with the DnaG primase helicase-binding domain dissociates the helicase dodecamer into single ringed primosomes. Functional assays on the proteins and associated complexes indicate that these single ringed primosomes are the most active form of the helicase for ATP hydrolysis, DNA binding and unwinding. These findings shed light onto an activation mechanism of HpDnaB by the primase that might be relevant in other bacteria and possibly other organisms exploiting dodecameric helicases for DNA replication. PMID:26264665

  5. RNA Tertiary Interactions Mediate Native Collapse of a Bacterial Group I Ribozyme

    SciTech Connect

    Chauhan, Seema; Caliskan, Gokhan; Briber, Robert M.; Perez-Salas, Ursula; Rangan, Prashanth; Thirumalai, D.; Woodson, Sarah A.

    2010-07-13

    Large RNAs collapse into compact intermediates in the presence of counterions before folding to the native state. We previously found that collapse of a bacterial group I ribozyme correlates with the formation of helices within the ribozyme core, but occurs at Mg{sup 2+} concentrations too low to support stable tertiary structure and catalytic activity. Here, using small-angle X-ray scattering, we show that Mg{sup 2+}-induced collapse is a cooperative folding transition that can be fit by a two-state model. The Mg{sup 2+} dependence of collapse is similar to the Mg{sup 2+} dependence of helix assembly measured by partial ribonuclease T{sub 1} digestion and of an unfolding transition measured by UV hypochromicity. The correspondence between multiple probes of RNA structure further supports a two-state model. A mutation that disrupts tertiary contacts between the L9 tetraloop and its helical receptor destabilized the compact state by 0.8 kcal/mol, while mutations in the central triplex were less destabilizing. These results show that native tertiary interactions stabilize the compact folding intermediates under conditions in which the RNA backbone remains accessible to solvent.

  6. Ankyrin-mediated self-protection during cell invasion by the bacterial predator Bdellovibrio bacteriovorus.

    PubMed

    Lambert, Carey; Cadby, Ian T; Till, Rob; Bui, Nhat Khai; Lerner, Thomas R; Hughes, William S; Lee, David J; Alderwick, Luke J; Vollmer, Waldemar; Sockett, R Elizabeth; Sockett, Elizabeth R; Lovering, Andrew L

    2015-01-01

    Predatory Bdellovibrio bacteriovorus are natural antimicrobial organisms, killing other bacteria by whole-cell invasion. Self-protection against prey-metabolizing enzymes is important for the evolution of predation. Initial prey entry involves the predator's peptidoglycan DD-endopeptidases, which decrosslink cell walls and prevent wasteful entry by a second predator. Here we identify and characterize a self-protection protein from B. bacteriovorus, Bd3460, which displays an ankyrin-based fold common to intracellular pathogens of eukaryotes. Co-crystal structures reveal Bd3460 complexation of dual targets, binding a conserved epitope of each of the Bd3459 and Bd0816 endopeptidases. Complexation inhibits endopeptidase activity and cell wall decrosslinking in vitro. Self-protection is vital - ΔBd3460 Bdellovibrio deleteriously decrosslink self-peptidoglycan upon invasion, adopt a round morphology, and lose predatory capacity and cellular integrity. Our analysis provides the first mechanistic examination of self-protection in Bdellovibrio, documents protection-multiplicity for products of two different genomic loci, and reveals an important evolutionary adaptation to an invasive predatory bacterial lifestyle. PMID:26626559

  7. Bacterial cell wall biogenesis is mediated by SEDS and PBP polymerase families functioning semi-autonomously.

    PubMed

    Cho, Hongbaek; Wivagg, Carl N; Kapoor, Mrinal; Barry, Zachary; Rohs, Patricia D A; Suh, Hyunsuk; Marto, Jarrod A; Garner, Ethan C; Bernhardt, Thomas G

    2016-01-01

    Multi-protein complexes organized by cytoskeletal proteins are essential for cell wall biogenesis in most bacteria. Current models of the wall assembly mechanism assume that class A penicillin-binding proteins (aPBPs), the targets of penicillin-like drugs, function as the primary cell wall polymerases within these machineries. Here, we use an in vivo cell wall polymerase assay in Escherichia coli combined with measurements of the localization dynamics of synthesis proteins to investigate this hypothesis. We find that aPBP activity is not necessary for glycan polymerization by the cell elongation machinery, as is commonly believed. Instead, our results indicate that cell wall synthesis is mediated by two distinct polymerase systems, shape, elongation, division, sporulation (SEDS)-family proteins working within the cytoskeletal machines and aPBP enzymes functioning outside these complexes. These findings thus necessitate a fundamental change in our conception of the cell wall assembly process in bacteria. PMID:27643381

  8. Bacterial Synthesis of Unusual Sulfonamide and Sulfone Antibiotics by Flavoenzyme-Mediated Sulfur Dioxide Capture.

    PubMed

    Baunach, Martin; Ding, Ling; Willing, Karsten; Hertweck, Christian

    2015-11-01

    Sulfa drugs, such as sulfonilamide and dapsone, are classical antibiotics that have been in clinical use worldwide. Despite the relatively simple architectures, practically no natural products are known to feature such aromatic sulfonamide or diarylsulfone substructures. We report the unexpected discovery of three fully unprecedented, sulfonyl-bridged alkaloid dimers (sulfadixiamycins A-C) from recombinant Streptomyces species harboring the entire xiamycin biosynthesis gene cluster. Sulfadixiamycins exhibit moderate antimycobacterial activities and potent antibiotic activities even against multidrug-resistant bacteria. Gene inactivation, complementation, and biotransformation experiments revealed that a flavin-dependent enzyme (XiaH) plays a key role in sulfadixiamycin biosynthesis. XiaH mediates a radical-based, three-component reaction involving two equivalents of xiamycin and sulfur dioxide, which is reminiscent of radical styrene/SO2 copolymerization. PMID:26366473

  9. Impacts of pH-mediated EPS structure on probiotic bacterial pili-whey proteins interactions.

    PubMed

    Burgain, Jennifer; Scher, Joel; Lebeer, Sarah; Vanderleyden, Jos; Corgneau, Magda; Guerin, Justine; Caillet, Céline; Duval, Jérôme F L; Francius, Gregory; Gaiani, Claire

    2015-10-01

    Probiotic bacteria are routinely incorporated into dairy foods because of the health benefits they can provide when consumed. In this work, the marked pH-dependence of the pili/EPS organization at the outer surface of Lactobacillus rhamnosus GG is characterized in detail by Single Cell Force Microscopy and cell electrophoretic mobility measurements analyzed according to formalisms for nanomechanical contact and soft particle electrokinetics, respectively. At pH 6.8, LGG pili are easily accessible by AFM tips functionalized with whey proteins for specific binding, while at pH 4.8 the collapsed EPS surface layer significantly immobilized the LGG pili. This resulted in their reduced accessibility to the specific whey-coated AFM tip, and to stronger whey protein-pili rupture forces. Thus, pili interactions with whey proteins are screened to an extent that depends on the pH-mediated embedment of the pili within the EPS layer. PMID:26209966

  10. Impacts of pH-mediated EPS structure on probiotic bacterial pili-whey proteins interactions.

    PubMed

    Burgain, Jennifer; Scher, Joel; Lebeer, Sarah; Vanderleyden, Jos; Corgneau, Magda; Guerin, Justine; Caillet, Céline; Duval, Jérôme F L; Francius, Gregory; Gaiani, Claire

    2015-10-01

    Probiotic bacteria are routinely incorporated into dairy foods because of the health benefits they can provide when consumed. In this work, the marked pH-dependence of the pili/EPS organization at the outer surface of Lactobacillus rhamnosus GG is characterized in detail by Single Cell Force Microscopy and cell electrophoretic mobility measurements analyzed according to formalisms for nanomechanical contact and soft particle electrokinetics, respectively. At pH 6.8, LGG pili are easily accessible by AFM tips functionalized with whey proteins for specific binding, while at pH 4.8 the collapsed EPS surface layer significantly immobilized the LGG pili. This resulted in their reduced accessibility to the specific whey-coated AFM tip, and to stronger whey protein-pili rupture forces. Thus, pili interactions with whey proteins are screened to an extent that depends on the pH-mediated embedment of the pili within the EPS layer.

  11. The application of loop-mediated isothermal amplification (LAMP) in food testing for bacterial pathogens and fungal contaminants.

    PubMed

    Niessen, Ludwig; Luo, Jie; Denschlag, Carla; Vogel, Rudi F

    2013-12-01

    Bacterial pathogens and toxicants, parasites as well as mycotoxin producing fungi are the major biotic factors influencing the safety of food. Moreover, viral infections and prions may be present as quasi biotic challenging factors. A vast array of culture dependent analytical methods and protocols for food safety testing has been developed during the past decades. Presently, protocols involving molecular biological techniques such as PCR-based nucleic acid amplification and hybridization have become available for many of the known pathogens with their major advantages being rapidness, high sensitivity and specificity. However, this type of assays is still quite labor- and cost intensive and mostly cannot be operated directly in the field. Recently, loop-mediated isothermal amplification (LAMP) of DNA has emerged as an alternative to the use of PCR-based methods not only in food safety testing but also in a wide array of application. Its advantages over PCR-based techniques are even shorter reaction time, no need for specific equipment, high sensitivity and specificity as well as comparably low susceptibility to inhibitors present in sample materials which enables detection of the pathogens in sample materials even without time consuming sample preparation. The present article presents a critical review of the application of LAMP-based methods and their usefulness in detecting and identifying food borne bacterial pathogens and toxicants as well as mycotoxin producing food borne fungi as compared to other methods. Moreover does it elaborate on new developments in the design and automation of LAMP-based assays and their implications for the future developments of food testing.

  12. Host Genetic Background Influences the Response to the Opportunistic Pseudomonas aeruginosa Infection Altering Cell-Mediated Immunity and Bacterial Replication

    PubMed Central

    Lorè, Nicola Ivan; Rossi, Giacomo; Cigana, Cristina; De Fino, Ida; Iraqi, Fuad A.; Bragonzi, Alessandra

    2014-01-01

    Pseudomonas aeruginosa is a common cause of healthcare-associated infections including pneumonia, bloodstream, urinary tract, and surgical site infections. The clinical outcome of P. aeruginosa infections may be extremely variable among individuals at risk and patients affected by cystic fibrosis. However, risk factors for P. aeruginosa infection remain largely unknown. To identify and track the host factors influencing P. aeruginosa lung infections, inbred immunocompetent mouse strains were screened in a pneumonia model system. A/J, BALB/cJ, BALB/cAnNCrl, BALB/cByJ, C3H/HeOuJ, C57BL/6J, C57BL/6NCrl, DBA/2J, and 129S2/SvPasCRL mice were infected with P. aeruginosa clinical strain and monitored for body weight and mortality up to seven days. The most deviant survival phenotypes were observed for A/J, 129S2/SvPasCRL and DBA/2J showing high susceptibility while BALB/cAnNCrl and C3H/HeOuJ showing more resistance to P. aeruginosa infection. Next, one of the most susceptible and resistant mouse strains were characterized for their deviant clinical and immunological phenotype by scoring bacterial count, cell-mediated immunity, cytokines and chemokines profile and lung pathology in an early time course. Susceptible A/J mice showed significantly higher bacterial burden, higher cytokines and chemokines levels but lower leukocyte recruitment, particularly neutrophils, when compared to C3H/HeOuJ resistant mice. Pathologic scores showed lower inflammatory severity, reduced intraluminal and interstitial inflammation extent, bronchial and parenchymal involvement and diminished alveolar damage in the lungs of A/J when compared to C3H/HeOuJ. Our findings indicate that during an early phase of infection a prompt inflammatory response in the airways set the conditions for a non-permissive environment to P. aeruginosa replication and lock the spread to other organs. Host gene(s) may have a role in the reduction of cell-mediated immunity playing a critical role in the control of P

  13. Host genetic background influences the response to the opportunistic Pseudomonas aeruginosa infection altering cell-mediated immunity and bacterial replication.

    PubMed

    De Simone, Maura; Spagnuolo, Lorenza; Lorè, Nicola Ivan; Rossi, Giacomo; Cigana, Cristina; De Fino, Ida; Iraqi, Fuad A; Bragonzi, Alessandra

    2014-01-01

    Pseudomonas aeruginosa is a common cause of healthcare-associated infections including pneumonia, bloodstream, urinary tract, and surgical site infections. The clinical outcome of P. aeruginosa infections may be extremely variable among individuals at risk and patients affected by cystic fibrosis. However, risk factors for P. aeruginosa infection remain largely unknown. To identify and track the host factors influencing P. aeruginosa lung infections, inbred immunocompetent mouse strains were screened in a pneumonia model system. A/J, BALB/cJ, BALB/cAnNCrl, BALB/cByJ, C3H/HeOuJ, C57BL/6J, C57BL/6NCrl, DBA/2J, and 129S2/SvPasCRL mice were infected with P. aeruginosa clinical strain and monitored for body weight and mortality up to seven days. The most deviant survival phenotypes were observed for A/J, 129S2/SvPasCRL and DBA/2J showing high susceptibility while BALB/cAnNCrl and C3H/HeOuJ showing more resistance to P. aeruginosa infection. Next, one of the most susceptible and resistant mouse strains were characterized for their deviant clinical and immunological phenotype by scoring bacterial count, cell-mediated immunity, cytokines and chemokines profile and lung pathology in an early time course. Susceptible A/J mice showed significantly higher bacterial burden, higher cytokines and chemokines levels but lower leukocyte recruitment, particularly neutrophils, when compared to C3H/HeOuJ resistant mice. Pathologic scores showed lower inflammatory severity, reduced intraluminal and interstitial inflammation extent, bronchial and parenchymal involvement and diminished alveolar damage in the lungs of A/J when compared to C3H/HeOuJ. Our findings indicate that during an early phase of infection a prompt inflammatory response in the airways set the conditions for a non-permissive environment to P. aeruginosa replication and lock the spread to other organs. Host gene(s) may have a role in the reduction of cell-mediated immunity playing a critical role in the control of P

  14. Glycoconjugates as Mediators of Nitric Oxide Production upon Exposure to Bacterial Spores by Macrophages

    NASA Astrophysics Data System (ADS)

    Lahiani, Mohamed; Soderberg, Lee; Tarasenko, Olga

    2011-06-01

    Phagocytes generate nitric oxide (NO) in large quantities to combat bacteria. The spore-producing Gram-positive organisms of Bacillus cereus family are causative agents from mild to a life threatening infection in humans and domestic animals. Our group have shown that glycoconjugates (GCs) activate macrophages and enhance killing of Bacillus spores. In this investigation, we will explore the effect of different GCs structures on NO production. The objective of this study is to study effects of GCs 2, 4, 6, 8, 10 on NO release upon exposure to B. cereus and Bacillus anthracis spores by macrophages. Our results demonstrated that GCs activated macrophages and increased NO production using studied GCs ligands compared to macrophage only (p<0.001). GC2 and GC8 were able to further increase NO production in macrophages compared to the B. anthracis spores treated macrophages (p<0.001). Our finding suggests that GCs could be used as potential mediators of NO production in macrophages to fight B. anthracis and other pathogens.

  15. Modeling and validation of autoinducer-mediated bacterial gene expression in microfluidic environments

    PubMed Central

    Austin, Caitlin M.; Stoy, William; Su, Peter; Harber, Marie C.; Bardill, J. Patrick; Hammer, Brian K.; Forest, Craig R.

    2014-01-01

    Biosensors exploiting communication within genetically engineered bacteria are becoming increasingly important for monitoring environmental changes. Currently, there are a variety of mathematical models for understanding and predicting how genetically engineered bacteria respond to molecular stimuli in these environments, but as sensors have miniaturized towards microfluidics and are subjected to complex time-varying inputs, the shortcomings of these models have become apparent. The effects of microfluidic environments such as low oxygen concentration, increased biofilm encapsulation, diffusion limited molecular distribution, and higher population densities strongly affect rate constants for gene expression not accounted for in previous models. We report a mathematical model that accurately predicts the biological response of the autoinducer N-acyl homoserine lactone-mediated green fluorescent protein expression in reporter bacteria in microfluidic environments by accommodating these rate constants. This generalized mass action model considers a chain of biomolecular events from input autoinducer chemical to fluorescent protein expression through a series of six chemical species. We have validated this model against experimental data from our own apparatus as well as prior published experimental results. Results indicate accurate prediction of dynamics (e.g., 14% peak time error from a pulse input) and with reduced mean-squared error with pulse or step inputs for a range of concentrations (10 μM–30 μM). This model can help advance the design of genetically engineered bacteria sensors and molecular communication devices. PMID:25379076

  16. Self-potential and Geochemical Measurements of Microbially Mediated Bacterial Sulfate Reduction in Saturated Sediments

    NASA Astrophysics Data System (ADS)

    Park, S.; Wolf, L. W.; Lee, M.; Saunders, J.

    2004-12-01

    In situ bioremediation is a non-invasive groundwater remediation technique that stimulates microorganisms to catalyze desirable redox reactions. Using a series of laboratory experiments, we explored the suitability of self-potential methods for monitoring bioremediation of metals contamination. Each experiment was designed to quantify the relationship between electrical potential and changing redox conditions and to determine factors influencing this relationship. In the first experiment, we introduced sulfate-reducting bacteria (SRB) into a Plexiglas tank containing autoclaved quartz sand saturated with an iron-rich Desulfovibrio (a sulfate-reducing bacteria) media. An array of non-polarizable electrodes positioned on the sediment surface was used to record electrical potentials both prior to and after inoculation for about 40 days. Changes in water chemistry were determined through a series of samples taken before, during and after the experiments. A significant decrease in total iron occurred after 3 days near the injection site; however, a clearly discernable decrease in electrical potential was not perceived until ~ day 10. Contoured SP data indicate that the redox front migrated away from the injection site over time. This change probably reflects the changing water chemistry as well as bacterial migration, as iron close to the injection site was consumed. The second experiment consisted of 4 glass columns, two of which were inoculated with SRB. The first pair contained sediment similar to the tank experiment saturated with an iron-rich media. The second pair contained the same sediment but was saturated with acid-mine drainage (AMD) collected from a contaminated field site. Each column was identically instrumented with a system of four electrodes. In the active columns, an increase in pH, a decrease in sulfate and a significant decrease in total iron in the media column accompany a decrease in electrical potential after about 10 days. Results of the study

  17. Site-specific bacterial chromosome engineering: ΦC31 integrase mediated cassette exchange (IMCE).

    PubMed

    Heil, John R; Cheng, Jiujun; Charles, Trevor C

    2012-03-16

    The bacterial chromosome may be used to stably maintain foreign DNA in the mega-base range. Integration into the chromosome circumvents issues such as plasmid replication, plasmid stability, plasmid incompatibility, and plasmid copy number variance. This method uses the site-specific integrase from the Streptomyces phage (Φ) C31. The ΦC31 integrase catalyzes a direct recombination between two specific DNA sites: attB and attP (34 and 39 bp, respectively). This recombination is stable and does not revert. A "landing pad" (LP) sequence consisting of a spectinomycin-resistance gene, aadA (SpR), and the E. coli ß-glucuronidase gene (uidA) flanked by attP sites has been integrated into the chromosomes of Sinorhizobium meliloti, Ochrobactrum anthropi, and Agrobacterium tumefaciens in an intergenic region, the ampC locus, and the tetA locus, respectively. S. meliloti is used in this protocol. Mobilizable donor vectors containing attB sites flanking a stuffer red fluorescent protein (rfp) gene and an antibiotic resistance gene have also been constructed. In this example the gentamicin resistant plasmid pJH110 is used. The rfp gene may be replaced with a desired construct using SphI and PstI. Alternatively a synthetic construct flanked by attB sites may be sub-cloned into a mobilizable vector such as pK19mob. The expression of the ΦC31 integrase gene (cloned from pHS62) is driven by the lac promoter, on a mobilizable broad host range plasmid pRK7813. A tetraparental mating protocol is used to transfer the donor cassette into the LP strain thereby replacing the markers in the LP sequence with the donor cassette. These cells are trans-integrants. Trans-integrants are formed with a typical efficiency of 0.5%. Trans-integrants are typically found within the first 500-1,000 colonies screened by antibiotic sensitivity or blue-white screening using 5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid (X-gluc). This protocol contains the mating and selection procedures for

  18. ER stress upregulated PGE2/IFNγ-induced IL-6 expression and down-regulated iNOS expression in glial cells

    NASA Astrophysics Data System (ADS)

    Hosoi, Toru; Honda, Miya; Oba, Tatsuya; Ozawa, Koichiro

    2013-12-01

    The disruption of endoplasmic reticulum (ER) function can lead to neurodegenerative disorders, in which inflammation has also been implicated. We investigated the possible correlation between ER stress and immune function using glial cells. We demonstrated that ER stress synergistically enhanced prostaglandin (PG) E2 + interferon (IFN) γ-induced interleukin (IL)-6 production. This effect was mediated through cAMP. Immune-activated glial cells produced inducible nitric oxide synthase (iNOS). Interestingly, ER stress inhibited PGE2 + IFNγ-induced iNOS expression. Similar results were obtained when cells were treated with dbcAMP + IFNγ. Thus, cAMP has a dual effect on immune reactions; cAMP up-regulated IL-6 expression, but down-regulated iNOS expression under ER stress. Therefore, our results suggest a link between ER stress and immune reactions in neurodegenerative diseases.

  19. Mec1, INO80, and the PAF1 complex cooperate to limit transcription replication conflicts through RNAPII removal during replication stress

    PubMed Central

    Poli, Jérôme; Gerhold, Christian-Benedikt; Tosi, Alessandro; Hustedt, Nicole; Seeber, Andrew; Sack, Ragna; Herzog, Franz; Pasero, Philippe; Shimada, Kenji; Hopfner, Karl-Peter; Gasser, Susan M.

    2016-01-01

    Little is known about how cells ensure DNA replication in the face of RNA polymerase II (RNAPII)-mediated transcription, especially under conditions of replicative stress. Here we present genetic and proteomic analyses from budding yeast that uncover links between the DNA replication checkpoint sensor Mec1–Ddc2 (ATR–ATRIP), the chromatin remodeling complex INO80C (INO80 complex), and the transcription complex PAF1C (PAF1 complex). We found that a subset of chromatin-bound RNAPII is degraded in a manner dependent on Mec1, INO80, and PAF1 complexes in cells exposed to hydroxyurea (HU). On HU, Mec1 triggers the efficient removal of PAF1C and RNAPII from transcribed genes near early firing origins. Failure to evict RNAPII correlates inversely with recovery from replication stress: paf1Δ cells, like ino80 and mec1 mutants, fail to restart forks efficiently after stalling. Our data reveal unexpected synergies between INO80C, Mec1, and PAF1C in the maintenance of genome integrity and suggest a mechanism of RNAPII degradation that reduces transcription–replication fork collision. PMID:26798134

  20. A Bacterial Parasite Effector Mediates Insect Vector Attraction in Host Plants Independently of Developmental Changes.

    PubMed

    Orlovskis, Zigmunds; Hogenhout, Saskia A

    2016-01-01

    Parasites can take over their hosts and trigger dramatic changes in host appearance and behavior that are typically interpreted as extended phenotypes that promote parasite survival and fitness. For example, Toxoplasma gondii is thought to manipulate the behaviors of infected rodents to aid transmission to cats and parasitic trematodes of the genus Ribeiroia alter limb development in their amphibian hosts to facilitate predation of the latter by birds. Plant parasites and pathogens also reprogram host development and morphology. However, whereas some parasite-induced morphological alterations may have a direct benefit to the fitness of the parasite and may therefore be adaptive, other host alterations may be side effects of parasite infections having no adaptive effects on parasite fitness. Phytoplasma parasites of plants often induce the development of leaf-like flowers (phyllody) in their host plants, and we previously found that the phytoplasma effector SAP54 generates these leaf-like flowers via the degradation of plant MADS-box transcription factors (MTFs), which regulate all major aspects of development in plants. Leafhoppers prefer to reproduce on phytoplasma-infected and SAP54-trangenic plants leading to the hypothesis that leafhopper vectors are attracted to plants with leaf-like flowers. Surprisingly, here we show that leafhopper attraction occurs independently of the presence of leaf-like flowers. First, the leafhoppers were also attracted to SAP54 transgenic plants without leaf-like flowers and to single leaves of these plants. Moreover, leafhoppers were not attracted to leaf-like flowers of MTF-mutant plants without the presence of SAP54. Thus, the primary role of SAP54 is to attract leafhopper vectors, which spread the phytoplasmas, and the generation of leaf-like flowers may be secondary or a side effect of the SAP54-mediated degradation of MTFs. PMID:27446117

  1. A Bacterial Parasite Effector Mediates Insect Vector Attraction in Host Plants Independently of Developmental Changes

    PubMed Central

    Orlovskis, Zigmunds; Hogenhout, Saskia A.

    2016-01-01

    Parasites can take over their hosts and trigger dramatic changes in host appearance and behavior that are typically interpreted as extended phenotypes that promote parasite survival and fitness. For example, Toxoplasma gondii is thought to manipulate the behaviors of infected rodents to aid transmission to cats and parasitic trematodes of the genus Ribeiroia alter limb development in their amphibian hosts to facilitate predation of the latter by birds. Plant parasites and pathogens also reprogram host development and morphology. However, whereas some parasite-induced morphological alterations may have a direct benefit to the fitness of the parasite and may therefore be adaptive, other host alterations may be side effects of parasite infections having no adaptive effects on parasite fitness. Phytoplasma parasites of plants often induce the development of leaf-like flowers (phyllody) in their host plants, and we previously found that the phytoplasma effector SAP54 generates these leaf-like flowers via the degradation of plant MADS-box transcription factors (MTFs), which regulate all major aspects of development in plants. Leafhoppers prefer to reproduce on phytoplasma-infected and SAP54-trangenic plants leading to the hypothesis that leafhopper vectors are attracted to plants with leaf-like flowers. Surprisingly, here we show that leafhopper attraction occurs independently of the presence of leaf-like flowers. First, the leafhoppers were also attracted to SAP54 transgenic plants without leaf-like flowers and to single leaves of these plants. Moreover, leafhoppers were not attracted to leaf-like flowers of MTF-mutant plants without the presence of SAP54. Thus, the primary role of SAP54 is to attract leafhopper vectors, which spread the phytoplasmas, and the generation of leaf-like flowers may be secondary or a side effect of the SAP54-mediated degradation of MTFs. PMID:27446117

  2. Loop-Mediated Isothermal Amplification of Specific Endoglucanase Gene Sequence for Detection of the Bacterial Wilt Pathogen Ralstonia solanacearum

    PubMed Central

    Pirc, Manca; Llop, Pablo; Ravnikar, Maja; Dreo, Tanja

    2014-01-01

    The increased globalization of crops production and processing industries also promotes the side-effects of more rapid and efficient spread of plant pathogens. To prevent the associated economic losses, and particularly those related to bacterial diseases where their management relies on removal of the infected material from production, simple, easy-to-perform, rapid and cost-effective tests are needed. Loop-mediated isothermal amplification (LAMP) assays that target 16S rRNA, fliC and egl genes were compared and evaluated as on-site applications. The assay with the best performance was that targeted to the egl gene, which shows high analytical specificity for diverse strains of the betaproteobacterium Ralstonia solanacearum, including its non-European and non-race 3 biovar 2 strains. The additional melting curve analysis provides confirmation of the test results. According to our extensive assessment, the egl LAMP assay requires minimum sample preparation (a few minutes of boiling) for the identification of pure cultures and ooze from symptomatic material, and it can also be used in a high-throughput format in the laboratory. This provides sensitive and reliable detection of R. solanacearum strains of different phylotypes. PMID:24763488

  3. Ambient UV-B exposure reduces the binding of ofloxacin with bacterial DNA gyrase and induces DNA damage mediated apoptosis.

    PubMed

    Singh, Jyoti; Dwivedi, Ashish; Mujtaba, Syed Faiz; Singh, Krishna P; Pal, Manish Kumar; Chopra, Deepti; Goyal, Shruti; Srivastav, Ajeet K; Dubey, Divya; Gupta, Shailendra K; Haldar, Chandana; Ray, Ratan Singh

    2016-04-01

    Ofloxacin (OFLX) is a broad spectrum antibiotic, which generates photo-products under sunlight exposure. Previous studies have failed to explain the attenuated anti-bacterial activity of OFLX. The study was extended to explore the unknown molecular mechanism of photogenotoxicity on human skin cell line (HaCaT) under environmental UV-B irradiation. Photochemically OFLX generates ROS and caused 2'-dGuO photodegradation. We have addressed the binding affinity of OFLX and its photo-products against DNA gyrase. Significant free radical generation such as (1)O2, O2(•-) and (•)OH reduces antioxidants and demonstrated the ROS mediated OFLX phototoxicity. However, the formation of micronuclei and CPDs showed photogenotoxic potential of OFLX. OFLX induced cell cycle arrest in sub-G1 peak. OFLX triggers apoptosis via permeabilization of mitochondrial membrane with the downregulation of anti-apoptotic Bcl-2 and caspase-3 whereas, upregulation of pro-apoptotic Bax and Cyto-C proteins. Our study illustrated that binding affinity of OFLX photo-products with DNA gyrase was mainly responsible for the attenuated antimicrobial activity. It was proved through molecular docking study. Thus, study suggests that sunlight exposure should avoid by drug users especially during peak hours for their safety from photosensitivity. Clinicians may guide patients regarding the safer use of photosensitive drugs during treatment.

  4. Ambient UV-B exposure reduces the binding of ofloxacin with bacterial DNA gyrase and induces DNA damage mediated apoptosis.

    PubMed

    Singh, Jyoti; Dwivedi, Ashish; Mujtaba, Syed Faiz; Singh, Krishna P; Pal, Manish Kumar; Chopra, Deepti; Goyal, Shruti; Srivastav, Ajeet K; Dubey, Divya; Gupta, Shailendra K; Haldar, Chandana; Ray, Ratan Singh

    2016-04-01

    Ofloxacin (OFLX) is a broad spectrum antibiotic, which generates photo-products under sunlight exposure. Previous studies have failed to explain the attenuated anti-bacterial activity of OFLX. The study was extended to explore the unknown molecular mechanism of photogenotoxicity on human skin cell line (HaCaT) under environmental UV-B irradiation. Photochemically OFLX generates ROS and caused 2'-dGuO photodegradation. We have addressed the binding affinity of OFLX and its photo-products against DNA gyrase. Significant free radical generation such as (1)O2, O2(•-) and (•)OH reduces antioxidants and demonstrated the ROS mediated OFLX phototoxicity. However, the formation of micronuclei and CPDs showed photogenotoxic potential of OFLX. OFLX induced cell cycle arrest in sub-G1 peak. OFLX triggers apoptosis via permeabilization of mitochondrial membrane with the downregulation of anti-apoptotic Bcl-2 and caspase-3 whereas, upregulation of pro-apoptotic Bax and Cyto-C proteins. Our study illustrated that binding affinity of OFLX photo-products with DNA gyrase was mainly responsible for the attenuated antimicrobial activity. It was proved through molecular docking study. Thus, study suggests that sunlight exposure should avoid by drug users especially during peak hours for their safety from photosensitivity. Clinicians may guide patients regarding the safer use of photosensitive drugs during treatment. PMID:26812543

  5. Loop-mediated isothermal amplification of specific endoglucanase gene sequence for detection of the bacterial wilt pathogen Ralstonia solanacearum.

    PubMed

    Lenarčič, Rok; Morisset, Dany; Pirc, Manca; Llop, Pablo; Ravnikar, Maja; Dreo, Tanja

    2014-01-01

    The increased globalization of crops production and processing industries also promotes the side-effects of more rapid and efficient spread of plant pathogens. To prevent the associated economic losses, and particularly those related to bacterial diseases where their management relies on removal of the infected material from production, simple, easy-to-perform, rapid and cost-effective tests are needed. Loop-mediated isothermal amplification (LAMP) assays that target 16S rRNA, fliC and egl genes were compared and evaluated as on-site applications. The assay with the best performance was that targeted to the egl gene, which shows high analytical specificity for diverse strains of the betaproteobacterium Ralstonia solanacearum, including its non-European and non-race 3 biovar 2 strains. The additional melting curve analysis provides confirmation of the test results. According to our extensive assessment, the egl LAMP assay requires minimum sample preparation (a few minutes of boiling) for the identification of pure cultures and ooze from symptomatic material, and it can also be used in a high-throughput format in the laboratory. This provides sensitive and reliable detection of R. solanacearum strains of different phylotypes. PMID:24763488

  6. Caries-risk assessment with a chairside optical spectroscopic sensor by monitoring bacterial-mediated acidogenic-profile of saliva in children

    PubMed Central

    Shrestha, Annie; Mohamed- Tahir, MA; Hegde, Jayshree; Azarpazhooh, Amir; Kishen, Anil

    2011-01-01

    Objective: This study aimed to evaluate the ability of an optical spectroscopic sensor (OSS) to monitor bacterial-mediated acidogenic-profile of saliva resulting from bacteria-sucrose interaction. Materials and Methods: Stage-1, characterization experiments were conducted to standardize the OSS. Stage-2 clinical experiments were carried out on stimulated saliva samples from 70 children of age-group 1-12 years. The bacterial-mediated acidogenic-profile of saliva mixed with sucrose was monitored using the OSS for 180 minutes. Results: The clinical patients were categorized based on the dmfs score as caries-active, caries-inactive and caries-free. The bacterial-mediated acidogenic-profile measured in terms of t1/2 monitored using the OSS was significantly different between the caries-free and caries-active (P<0.05); and caries-free and caries-inactive groups (P<0.005). Conclusions: The significant correlation of the acidogenic-profile determined using the OSS and the caries-status highlighted the OSS as a sensitive and rapid chairside tool for the quantification of the acidogenic-profile of saliva that could help in monitoring the caries-risk in children. PMID:22144811

  7. Polysaccharide capsule and sialic acid-mediated regulation promote biofilm-like intracellular bacterial communities during cystitis.

    PubMed

    Anderson, Gregory G; Goller, Carlos C; Justice, Sheryl; Hultgren, Scott J; Seed, Patrick C

    2010-03-01

    Uropathogenic Escherichia coli (UPEC) is the leading cause of urinary tract infections (UTIs). A murine UTI model has revealed an infection cascade whereby UPEC undergoes cycles of invasion of the bladder epithelium, intracellular proliferation in polysaccharide-containing biofilm-like masses called intracellular bacterial communities (IBC), and then dispersal into the bladder lumen to initiate further rounds of epithelial colonization and invasion. We predicted that the UPEC K1 polysaccharide capsule is a key constituent of the IBC matrix. Compared to prototypic E. coli K1 strain UTI89, a capsule assembly mutant had a fitness defect in functionally TLR4(+) and TLR4(-) mice, suggesting a protective role of capsule in inflamed and noninflamed hosts. K1 capsule assembly and synthesis mutants had dramatically reduced IBC formation, demonstrating the common requirement for K1 polysaccharide in IBC development. The capsule assembly mutant appeared dispersed in the cytoplasm of the bladder epithelial cells and failed to undergo high-density intracellular replication during later stages of infection, when the wild-type strain continued to form serial generations of IBC. Deletion of the sialic acid regulator gene nanR partially restored IBC formation in the capsule assembly mutant. These data suggest that capsule is necessary for efficient IBC formation and that aberrant sialic acid accumulation, resulting from disruption of K1 capsule assembly, produces a NanR-mediated defect in intracellular proliferation and IBC development. Together, these data demonstrate the complex but important roles of UPEC polysaccharide encapsulation and sialic acid signaling in multiple stages of UTI pathogenesis.

  8. Stimulation of Microbially Mediated Arsenic Release in Bangladesh Aquifers by Young Carbon Indicated by Radiocarbon Analysis of Sedimentary Bacterial Lipids.

    PubMed

    Whaley-Martin, K J; Mailloux, B J; van Geen, A; Bostick, B C; Silvern, R F; Kim, C; Ahmed, K M; Choudhury, I; Slater, G F

    2016-07-19

    The sources of reduced carbon driving the microbially mediated release of arsenic to shallow groundwater in Bangladesh remain poorly understood. Using radiocarbon analysis of phospholipid fatty acids (PLFAs) and potential carbon pools, the abundance and carbon sources of the active, sediment-associated, in situ bacterial communities inhabiting shallow aquifers (<30 m) at two sites in Araihazar, Bangladesh, were investigated. At both sites, sedimentary organic carbon (SOC) Δ(14)C signatures of -631 ± 54‰ (n = 12) were significantly depleted relative to dissolved inorganic carbon (DIC) of +24 ± 30‰ and dissolved organic carbon (DOC) of -230 ± 100‰. Sediment-associated PLFA Δ(14)C signatures (n = 10) at Site F (-167‰ to +20‰) and Site B (-163‰ to +21‰) were highly consistent and indicated utilization of carbon sources younger than the SOC, likely from the DOC pool. Sediment-associated PLFA Δ(14)C signatures were consistent with previously determined Δ(14)C signatures of microbial DNA sampled from groundwater at Site F indicating that the carbon source for these two components of the subsurface microbial community is consistent and is temporally stable over the two years between studies. These results demonstrate that the utilization of relatively young carbon sources by the subsurface microbial community occurs at sites with varying hydrology. Further they indicate that these young carbon sources drive the metabolism of the more abundant sediment-associated microbial communities that are presumably more capable of Fe reduction and associated release of As. This implies that an introduction of younger carbon to as of yet unaffected sediments (such as those comprising the deeper Pleistocene aquifer) could stimulate microbial communities and result in arsenic release.

  9. Stimulation of Microbially Mediated Arsenic Release in Bangladesh Aquifers by Young Carbon Indicated by Radiocarbon Analysis of Sedimentary Bacterial Lipids.

    PubMed

    Whaley-Martin, K J; Mailloux, B J; van Geen, A; Bostick, B C; Silvern, R F; Kim, C; Ahmed, K M; Choudhury, I; Slater, G F

    2016-07-19

    The sources of reduced carbon driving the microbially mediated release of arsenic to shallow groundwater in Bangladesh remain poorly understood. Using radiocarbon analysis of phospholipid fatty acids (PLFAs) and potential carbon pools, the abundance and carbon sources of the active, sediment-associated, in situ bacterial communities inhabiting shallow aquifers (<30 m) at two sites in Araihazar, Bangladesh, were investigated. At both sites, sedimentary organic carbon (SOC) Δ(14)C signatures of -631 ± 54‰ (n = 12) were significantly depleted relative to dissolved inorganic carbon (DIC) of +24 ± 30‰ and dissolved organic carbon (DOC) of -230 ± 100‰. Sediment-associated PLFA Δ(14)C signatures (n = 10) at Site F (-167‰ to +20‰) and Site B (-163‰ to +21‰) were highly consistent and indicated utilization of carbon sources younger than the SOC, likely from the DOC pool. Sediment-associated PLFA Δ(14)C signatures were consistent with previously determined Δ(14)C signatures of microbial DNA sampled from groundwater at Site F indicating that the carbon source for these two components of the subsurface microbial community is consistent and is temporally stable over the two years between studies. These results demonstrate that the utilization of relatively young carbon sources by the subsurface microbial community occurs at sites with varying hydrology. Further they indicate that these young carbon sources drive the metabolism of the more abundant sediment-associated microbial communities that are presumably more capable of Fe reduction and associated release of As. This implies that an introduction of younger carbon to as of yet unaffected sediments (such as those comprising the deeper Pleistocene aquifer) could stimulate microbial communities and result in arsenic release. PMID:27333443

  10. Prophage-Mediated Dynamics of ‘Candidatus Liberibacter asiaticus’ Populations, the Destructive Bacterial Pathogens of Citrus Huanglongbing

    PubMed Central

    Zhou, Lijuan; Powell, Charles A.; Li, Wenbin; Irey, Mike; Duan, Yongping

    2013-01-01

    Prophages are highly dynamic components in the bacterial genome and play an important role in intraspecies variations. There are at least two prophages in the chromosomes of Candidatus Liberibacter asiaticus’ (Las) Floridian isolates. Las is both unculturable and the most prevalent species of Liberibacter pathogens that cause huanglongbing (HLB), a worldwide destructive disease of citrus. In this study, seven new prophage variants resulting from two hyper-variable regions were identified by screening clone libraries of infected citrus, periwinkle and psyllids. Among them, Types A and B share highly conserved sequences and localize within the two prophages, FP1 and FP2, respectively. Although Types B and C were abundant in all three libraries, Type A was much more abundant in the libraries from the Las-infected psyllids than from the Las-infected plants, and Type D was only identified in libraries from the infected host plants but not from the infected psyllids. Sequence analysis of these variants revealed that the variations may result from recombination and rearrangement events. Conventional PCR results using type-specific molecular markers indicated that A, B, C and D are the four most abundant types in Las-infected citrus and periwinkle. However, only three types, A, B and C are abundant in Las-infected psyllids. Typing results for Las-infected citrus field samples indicated that mixed populations of Las bacteria present in Floridian isolates, but only the Type D population was correlated with the blotchy mottle symptom. Extended cloning and sequencing of the Type D region revealed a third prophage/phage in the Las genome, which may derive from the recombination of FP1 and FP2. Dramatic variations in these prophage regions were also found among the global Las isolates. These results are the first to demonstrate the prophage/phage-mediated dynamics of Las populations in plant and insect hosts, and their correlation with insect transmission and disease

  11. Interaction of the Chromatin Remodeling Protein hINO80 with DNA

    PubMed Central

    Jain, Shruti; Kaur, Taniya; Brahmachari, Vani

    2016-01-01

    The presence of a highly conserved DNA binding domain in INO80 subfamily predicted that INO80 directly interacts with DNA and we demonstrated its DNA binding activity in vitro. Here we report the consensus motif recognized by the DBINO domain identified by SELEX method and demonstrate the specific interaction of INO80 with the consensus motif. We show that INO80 significantly down regulates the reporter gene expression through its binding motif, and the repression is dependent on the presence of INO80 but not YY1 in the cell. The interaction is lost if specific residues within the consensus motif are altered. We identify a large number of potential target sites of INO80 in the human genome through in silico analysis that can grouped into three classes; sites that contain the recognition sequence for INO80 and YY1, only YY1 and only INO80. We demonstrate the binding of INO80 to a representative set of sites in HEK cells and the correlated repressive histone modifications around the binding motif. In the light of the role of INO80 in homeotic gene regulation in Drosophila as an Enhancer of trithorax and polycomb protein (ETP) that can modify the effect of both repressive complexes like polycomb as well as the activating complex like trithorax, it remains to be seen if INO80 can act as a recruiter of chromatin modifying complexes. PMID:27428271

  12. Differential induction of PPAR-gamma by luminal glutamine and iNOS by luminal arginine in the rodent postischemic small bowel.

    PubMed

    Sato, N; Moore, F A; Kone, B C; Zou, L; Smith, M A; Childs, M A; Moore-Olufemi, S; Schultz, S G; Kozar, R A

    2006-04-01

    Using a rodent model of gut ischemia-reperfusion (I/R), we have previously shown that the induction of inducible nitric oxide synthase (iNOS) is harmful, whereas the induction of heme oxygenase 1 (HO-1) and peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is protective. In the present study, we hypothesized that the luminal nutrients arginine and glutamine differentially modulate these molecular events in the postischemic gut. Jejunal sacs were created in rats at laparotomy, filled with either 60 mM glutamine, arginine, or magnesium sulfate (osmotic control) followed by 60 min of superior mesenteric artery occlusion and 6 h of reperfusion, and compared with shams. The jejunum was harvested for histology or myeloperoxidase (MPO) activity (inflammation). Heat shock proteins and iNOS were quantitated by Western blot analysis and PPAR-gamma by DNA binding activity. In some experiments, rats were pretreated with the PPAR-gamma inhibitor G9662 or with the iNOS inhibitor N-[3(aminomethyl)benzyl]acetamidine (1400W). iNOS was significantly increased by arginine but not by glutamine following gut I/R and was associated with increased MPO activity and mucosal injury. On the other hand, PPAR-gamma was significantly increased by glutamine but decreased by arginine, whereas heat shock proteins were similarly increased in all experimental groups. The PPAR-gamma inhibitor G9662 abrogated the protective effects of glutamine, whereas the iNOS inhibitor 1400W attenuated the injurious effects of arginine. We concluded that luminal arginine and glutamine differentially modulate the molecular events that regulate injurious I/R-mediated gut inflammation and injury. The induction of PPAR-gamma by luminal glutamine is a novel protective mechanism, whereas luminal arginine appears harmful to the postischemic gut due to enhanced expression of iNOS.

  13. Inducible nitric oxide synthase (iNOS) expression in monocytes during acute Dengue Fever in patients and during in vitro infection

    PubMed Central

    Neves-Souza, Patrícia CF; Azeredo, Elzinandes L; Zagne, Sonia MO; Valls-de-Souza, Rogério; Reis, Sonia RNI; Cerqueira, Denise IS; Nogueira, Rita MR; Kubelka, Claire F

    2005-01-01

    Abstract Mononuclear phagocytes are considered to be main targets for Dengue Virus (DENV) replication. These cells are activated after infection, producing proinflammatory mediators, including tumour-necrosis factor-α, which has also been detected in vivo. Nitric oxide (NO), usually produced by activated mononuclear phagocytes, has antimicrobial and antiviral activities. Methods The expression of DENV antigens and inducible nitric oxide synthase (iNOS) in human blood isolated monocytes were analysed by flow cytometry using cells either from patients with acute Dengue Fever or after DENV-1 in vitro infection. DENV-1 susceptibility to iNOS inhibition and NO production was investigated using NG-methyl L-Arginine (NGMLA) as an iNOS inhibitor, which was added to DENV-1 infected human monocytes, and sodium nitroprussiate (SNP), a NO donor, added to infected C6/36 mosquito cell clone. Viral antigens after treatments were detected by flow cytometry analysis. Results INOS expression in activated monocytes was observed in 10 out of 21 patients with Dengue Fever and was absent in cells from ten healthy individuals. DENV antigens detected in 25 out of 35 patients, were observed early during in vitro infection (3 days), significantly diminished with time, indicating that virus replicated, however monocytes controlled the infection. On the other hand, the iNOS expression was detected at increasing frequency in in vitro infected monocytes from three to six days, exhibiting an inverse relationship to DENV antigen expression. We demonstrated that the detection of the DENV-1 antigen was enhanced during monocyte treatment with NGMLA. In the mosquito cell line C6/36, virus detection was significantly reduced in the presence of SNP, when compared to that of untreated cells. Conclusion This study is the first to reveal the activation of DENV infected monocytes based on induction of iNOS both in vivo and in vitro, as well as the susceptibility of DENV-1 to a NO production. PMID:16109165

  14. Phosphorylation of human INO80 is involved in DNA damage tolerance

    SciTech Connect

    Kato, Dai; Waki, Mayumi; Umezawa, Masaki; Aoki, Yuka; Utsugi, Takahiko; Ohtsu, Masaya; Murakami, Yasufumi

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer Depletion of hINO80 significantly reduced PCNA ubiquitination. Black-Right-Pointing-Pointer Depletion of hINO80 significantly reduced nuclear dots intensity of RAD18 after UV irradiation. Black-Right-Pointing-Pointer Western blot analyses showed phosphorylated hINO80 C-terminus. Black-Right-Pointing-Pointer Overexpression of phosphorylation mutant hINO80 reduced PCNA ubiquitination. -- Abstract: Double strand breaks (DSBs) are the most serious type of DNA damage. DSBs can be generated directly by exposure to ionizing radiation or indirectly by replication fork collapse. The DNA damage tolerance pathway, which is conserved from bacteria to humans, prevents this collapse by overcoming replication blockages. The INO80 chromatin remodeling complex plays an important role in the DNA damage response. The yeast INO80 complex participates in the DNA damage tolerance pathway. The mechanisms regulating yINO80 complex are not fully understood, but yeast INO80 complex are necessary for efficient proliferating cell nuclear antigen (PCNA) ubiquitination and for recruitment of Rad18 to replication forks. In contrast, the function of the mammalian INO80 complex in DNA damage tolerance is less clear. Here, we show that human INO80 was necessary for PCNA ubiquitination and recruitment of Rad18 to DNA damage sites. Moreover, the C-terminal region of human INO80 was phosphorylated, and overexpression of a phosphorylation-deficient mutant of human INO80 resulted in decreased ubiquitination of PCNA during DNA replication. These results suggest that the human INO80 complex, like the yeast complex, was involved in the DNA damage tolerance pathway and that phosphorylation of human INO80 was involved in the DNA damage tolerance pathway. These findings provide new insights into the DNA damage tolerance pathway in mammalian cells.

  15. The dual role of iNOS in cancer.

    PubMed

    Vannini, Federica; Kashfi, Khosrow; Nath, Niharika

    2015-12-01

    Nitric oxide (NO) is one of the 10 smallest molecules found in nature. It is a simple gaseous free radical whose predominant functions is that of a messenger through cGMP. In mammals, NO is synthesized by the enzyme nitric oxide synthase (NOS) of which there are three isoforms. Neuronal (nNOS, NOS1) and endothelial (eNOS, NOS3) are constitutive calcium-dependent forms of the enzyme that regulate neural and vascular function respectively. The third isoform (iNOS, NOS2), is calcium-independent and is inducible. In many tumors, iNOS expression is high, however, the role of iNOS during tumor development is very complex and quite perplexing, with both promoting and inhibiting actions having been described. This review will aim to summarize the dual actions of iNOS-derived NO showing that the microenvironment of the tumor is a contributing factor to these observations and ultimately to cellular outcomes. PMID:26335399

  16. The dual role of iNOS in cancer☆

    PubMed Central

    Vanini, Frederica; Kashfi, Khosrow; Nath, Niharika

    2015-01-01

    Nitric oxide (NO) is one of the 10 smallest molecules found in nature. It is a simple gaseous free radical whose predominant functions is that of a messenger through cGMP. In mammals, NO is synthesized by the enzyme nitric oxide synthase (NOS) of which there are three isoforms. Neuronal (nNOS, NOS1) and endothelial (eNOS, NOS3) are constitutive calcium-dependent forms of the enzyme that regulate neural and vascular function respectively. The third isoform (iNOS, NOS2), is calcium-independent and is inducible. In many tumors, iNOS expression is high, however, the role of iNOS during tumor development is very complex and quite perplexing, with both promoting and inhibiting actions having been described. This review will aim to summarize the dual actions of iNOS-derived NO showing that the microenvironment of the tumor is a contributing factor to these observations and ultimately to cellular outcomes. PMID:26335399

  17. iNOS Activity Modulates Inflammation, Angiogenesis, and Tissue Fibrosis in Polyether-Polyurethane Synthetic Implants.

    PubMed

    Cassini-Vieira, Puebla; Araújo, Fernanda Assis; da Costa Dias, Filipi Leles; Russo, Remo Castro; Andrade, Silvia Passos; Teixeira, Mauro Martins; Barcelos, Luciola Silva

    2015-01-01

    There is considerable interest in implantation techniques and scaffolds for tissue engineering and, for safety and biocompatibility reasons, inflammation, angiogenesis, and fibrosis need to be determined. The contribution of inducible nitric oxide synthase (iNOS) in the regulation of the foreign body reaction induced by subcutaneous implantation of a synthetic matrix was never investigated. Here, we examined the role of iNOS in angiogenesis, inflammation, and collagen deposition induced by polyether-polyurethane synthetic implants, using mice with targeted disruption of the iNOS gene (iNOS(-/-)) and wild-type (WT) mice. The hemoglobin content and number of vessels were decreased in the implants of iNOS(-/-) mice compared to WT mice 14 days after implantation. VEGF levels were also reduced in the implants of iNOS(-/-) mice. In contrast, the iNOS(-/-) implants exhibited an increased neutrophil and macrophage infiltration. However, no alterations were observed in levels of CXCL1 and CCL2, chemokines related to neutrophil and macrophage migration, respectively. Furthermore, the implants of iNOS(-/-) mice showed boosted collagen deposition. These data suggest that iNOS activity controls inflammation, angiogenesis, and fibrogenesis in polyether-polyurethane synthetic implants and that lack of iNOS expression increases foreign body reaction to implants in mice.

  18. Comparative metabolomic analysis highlights the involvement of sugars and glycerol in melatonin-mediated innate immunity against bacterial pathogen in Arabidopsis.

    PubMed

    Qian, Yongqiang; Tan, Dun-Xian; Reiter, Russel J; Shi, Haitao

    2015-01-01

    Melatonin is an important secondary messenger in plant innate immunity against the bacterial pathogen Pseudomonas syringe pv. tomato (Pst) DC3000 in the salicylic acid (SA)- and nitric oxide (NO)-dependent pathway. However, the metabolic homeostasis in melatonin-mediated innate immunity is unknown. In this study, comparative metabolomic analysis found that the endogenous levels of both soluble sugars (fructose, glucose, melibose, sucrose, maltose, galatose, tagatofuranose and turanose) and glycerol were commonly increased after both melatonin treatment and Pst DC3000 infection in Arabidopsis. Further studies showed that exogenous pre-treatment with fructose, glucose, sucrose, or glycerol increased innate immunity against Pst DC3000 infection in wild type (Col-0) Arabidopsis plants, but largely alleviated their effects on the innate immunity in SA-deficient NahG plants and NO-deficient mutants. This indicated that SA and NO are also essential for sugars and glycerol-mediated disease resistance. Moreover, exogenous fructose, glucose, sucrose and glycerol pre-treatments remarkably increased endogenous NO level, but had no significant effect on the endogenous melatonin level. Taken together, this study highlights the involvement of sugars and glycerol in melatonin-mediated innate immunity against bacterial pathogen in SA and NO-dependent pathway in Arabidopsis.

  19. Pathogenesis of Streptococcus urinary tract infection depends on bacterial strain and β-hemolysin/cytolysin that mediates cytotoxicity, cytokine synthesis, inflammation and virulence

    PubMed Central

    Leclercq, Sophie Y.; Sullivan, Matthew J.; Ipe, Deepak S.; Smith, Joshua P.; Cripps, Allan W.; Ulett, Glen C.

    2016-01-01

    Streptococcus agalactiae can cause urinary tract infection (UTI) including cystitis and asymptomatic bacteriuria (ABU). The early host-pathogen interactions that occur during S. agalactiae UTI and subsequent mechanisms of disease pathogenesis are poorly defined. Here, we define the early interactions between human bladder urothelial cells, monocyte-derived macrophages, and mouse bladder using uropathogenic S. agalactiae (UPSA) 807 and ABU-causing S. agalactiae (ABSA) 834 strains. UPSA 807 adhered, invaded and killed bladder urothelial cells more efficiently compared to ABSA 834 via mechanisms including low-level caspase-3 activation, and cytolysis, according to lactate dehydrogenase release measures and cell viability. Severe UPSA 807-induced cytotoxicity was mediated entirely by the bacterial β-hemolysin/cytolysin (β-H/C) because an β-H/C-deficient UPSA 807 isogenic mutant, UPSA 807ΔcylE, was not cytotoxic in vitro; the mutant was also significantly attenuated for colonization in the bladder in vivo. Analysis of infection-induced cytokines, including IL-8, IL-1β, IL-6 and TNF-α in vitro and in vivo revealed that cytokine and chemokine responses were dependent on expression of β-H/C that also elicited severe bladder neutrophilia. Thus, virulence of UPSA 807 encompasses adhesion to, invasion of and killing of bladder cells, pro-inflammatory cytokine/chemokine responses that elicit neutrophil infiltration, and β-H/C-mediated subversion of innate immune-mediated bacterial clearance from the bladder. PMID:27383371

  20. Comparative metabolomic analysis highlights the involvement of sugars and glycerol in melatonin-mediated innate immunity against bacterial pathogen in Arabidopsis

    PubMed Central

    Qian, Yongqiang; Tan, Dun-Xian; Reiter, Russel J.; Shi, Haitao

    2015-01-01

    Melatonin is an important secondary messenger in plant innate immunity against the bacterial pathogen Pseudomonas syringe pv. tomato (Pst) DC3000 in the salicylic acid (SA)- and nitric oxide (NO)-dependent pathway. However, the metabolic homeostasis in melatonin-mediated innate immunity is unknown. In this study, comparative metabolomic analysis found that the endogenous levels of both soluble sugars (fructose, glucose, melibose, sucrose, maltose, galatose, tagatofuranose and turanose) and glycerol were commonly increased after both melatonin treatment and Pst DC3000 infection in Arabidopsis. Further studies showed that exogenous pre-treatment with fructose, glucose, sucrose, or glycerol increased innate immunity against Pst DC3000 infection in wild type (Col-0) Arabidopsis plants, but largely alleviated their effects on the innate immunity in SA-deficient NahG plants and NO-deficient mutants. This indicated that SA and NO are also essential for sugars and glycerol-mediated disease resistance. Moreover, exogenous fructose, glucose, sucrose and glycerol pre-treatments remarkably increased endogenous NO level, but had no significant effect on the endogenous melatonin level. Taken together, this study highlights the involvement of sugars and glycerol in melatonin-mediated innate immunity against bacterial pathogen in SA and NO-dependent pathway in Arabidopsis. PMID:26508076

  1. New evidence for Cu-decorated binary-oxides mediating bacterial inactivation/mineralization in aerobic media.

    PubMed

    Rtimi, S; Pulgarin, C; Bensimon, M; Kiwi, J

    2016-08-01

    Binary oxide semiconductors TiO2-ZrO2 and Cu-decorated TiO2-ZrO2 (TiO2-ZrO2-Cu) uniform films were sputtered on polyester (PES). These films were irradiated under low intensity solar simulated light and led to bacterial inactivation in aerobic and anaerobic media as evaluated by CFU-plate counting. But bacterial mineralization was only induced by TiO2-ZrO2-Cu in aerobic media. The highly oxidative radicals generated on the films surface under light were identified by the use of appropriate scavengers. The hole generated on the TiO2-ZrO2 films is shown to be the main specie leading to bacterial inactivation. TiO2-ZrO2 and Cu-decorated TiO2-ZrO2 films release Zr and Ti <1ppb and Cu 4.6ppb/cm(2) as determined by inductively coupled plasma mass spectrometry (ICP-MS) This level is far below the citotoxicity permitted level allowed for mammalian cells suggesting that bacterial disinfection proceeds through an oligodynamic effect. By Fourier transform attenuated infrared spectroscopy (ATR-FTIR) the systematic shift of the predominating νs(CH2) vibrational-rotational peak making up most of the bacterial cell-wall content in C was monitored. Based on this evidence a mechanism suggested leading to CH bond stretching followed by cell lysis and cell death. Bacterial inactivation cycling was observed on TiO2-ZrO2-Cu showing the stability of these films leading to bacterial inactivation. PMID:27088192

  2. Acacia ferruginea inhibits inflammation by regulating inflammatory iNOS and COX-2.

    PubMed

    Sakthivel, Kunnathur Murugesan; Guruvayoorappan, Chandrasekaran

    2016-01-01

    Inflammation is a local defensive reaction of a host to cellular injury or infection. Prolonged inflammation can contribute to pathogenesis of many disorders. Identification of naturally occurring phytoconstituents that can suppress inflammatory mediators can lead to the discovery of anti-inflammatory therapeutics. Acacia ferruginea is used traditionally to treat numerous ailments including hemorrhage, irritable bowel syndrome and leprosy. The present study evaluated the anti-inflammatory activity of A. ferruginea extract against acute (carrageenan) and chronic (formaldehyde) inflammation in Balb/c mice. Pre-treatment with A. ferruginea extract (10 mg/kg BW) for 5 consecutive days via intraperitonial (IP) administration significantly inhibited subsequent induction of paw edema in both models; the effects were comparable to that of the standard drug indomethacin. The results also showed the A. ferruginea extract significantly inhibited nitric oxide (NO) synthesis and iNOS expression (as measured in serum), diminished inflammation in - and neutrophil infiltration to - the paw tissues and led to a reduction in the number of COX-2(+) immunoreative cells (as evidenced by histologic and immunohistochemical analyses) in the paws relative to those in paws of mice that received the irritants only. Further, in vitro studies showed the extract could significantly scavenge free radicals generated as in DPPH and NO radical generating assays. Taken together, the results showed that A. ferruginea extract imparted potent anti-oxidant and -inflammatory effects, in part by maintaining oxidative homeostasis, inhibiting NO synthesis and suppressing iNOS and COX-2 expression and so could potentially be exploited as a potential plant-based medication against inflammatory disorders.

  3. Stabilization and targeting of INO80 to replication forks by BAP1 during normal DNA synthesis.

    PubMed

    Lee, Han-Sae; Lee, Shin-Ai; Hur, Shin-Kyoung; Seo, Jeong-Wook; Kwon, Jongbum

    2014-10-06

    The INO80 chromatin-remodelling complex has been implicated in DNA replication during stress in yeast. However, its role in normal DNA replication and its underlying mechanisms remain unclear. Here, we show that INO80 binds to replication forks and promotes fork progression in human cells under unperturbed, normal conditions. We find that Ino80, which encodes the catalytic ATPase of INO80, is essential for mouse embryonic DNA replication and development. Ino80 is recruited to replication forks through interaction with ubiquitinated H2A--aided by BRCA1-associated protein-1 (BAP1), a tumour suppressor and nuclear de-ubiquitinating enzyme that also functions to stabilize Ino80. Importantly, Ino80 is downregulated in BAP1-defective cancer cells due to the lack of an Ino80 stabilization mechanism via BAP1. Our results establish a role for INO80 in normal DNA replication and uncover a mechanism by which this remodeler is targeted to replication forks, suggesting a molecular basis for the tumour-suppressing function of BAP1.

  4. Nitrosyl iodide, INO: A combined ab initio and high-resolution spectroscopic study

    NASA Astrophysics Data System (ADS)

    Bailleux, S.; Duflot, D.; Aiba, S.; Nakahama, S.; Ozeki, H.

    2016-04-01

    In the nitrosyl halides series (XNO, where X = F, Cl, Br, I), INO is the only chemical species whose rotational spectrum has not been reported. Nitrosyl iodide, together with the nitryl (INO2), nitrite (IONO) and nitrate (IONO2) iodides, is believed to impact tropospheric ozone levels. Guided by our quantum chemical calculations, we report the detection of INO in the gas phase by high-resolution spectroscopy for the first time. INO was generated by mixing continuously I2 and NO. The measurement and least-squares analysis of 173 a-type rotational transitions resulted in the accurate determination of molecular parameters.

  5. PEX7 and EBP50 target iNOS to the peroxisome in hepatocytes.

    PubMed

    Loughran, Patricia A; Stolz, Donna B; Barrick, Stacey R; Wheeler, David S; Friedman, Peter A; Rachubinski, Richard A; Watkins, Simon C; Billiar, Timothy R

    2013-05-31

    iNOS localizes to both the cytosol and peroxisomes in hepatocytes in vitro and in vivo. The structural determinants for iNOS localization are not known. One plausible mechanism for iNOS localization to the peroxisome is through the interaction with peroxisomal import proteins PEX5 or PEX7. siRNA knockdown of PEX7 reduced iNOS colocalization with the peroxisomal protein PMP70. Proteomic studies using MALDI-MS identified iNOS association with the 50-kD ezrin binding PDZ protein (EBP50). Confocal microscopy studies and immunoelectron microscopy confirmed iNOS association with EBP50, with greatest colocalization occurring at 8h of cytokine exposure. EBP50 associated with peroxisomes in a PEX5 and PEX7-dependent manner. iNOS localization to peroxisomes was contingent on EBP50 expression in LPS-treated mice. Thus, iNOS targeting to peroxisomes in hepatocytes involves interaction with PEX7 and EBP50. The targeting of iNOS protein to the peroxisome may shift the balance of metabolic processes that rely on heme proteins susceptible to modification by radical oxygen and nitrogen radicals.

  6. PEX7 and EBP50 Target iNOS to the Peroxisome in Hepatocytes

    PubMed Central

    Loughran, Patricia A.; Stolz, Donna B.; Barrick, Stacey R.; Wheeler, David S.; Friedman, Peter A.; Rachubinski, Richard A.; Watkins, Simon C.; Billiar, Timothy R.

    2013-01-01

    iNOS localizes to both the cytosol and peroxisomes in hepatocytes in vitro and in vivo. The structural determinants for iNOS localization are not known. One plausible mechanism for iNOS localization to the peroxisome is through the interaction with peroxisomal import proteins PEX5 or PEX7. siRNA knockdown of PEX7 reduced iNOS colocalization with the peroxisomal protein PMP70. Proteomic studies using MALDI-MS identified iNOS association with the 50-kD ezrin binding PDZ protein (EBP50). Confocal microscopy studies and immunoelectron microscopy confirmed iNOS association with EBP50, with greatest colocalization occurring at 8 hours of cytokine exposure. EBP50 associated with peroxisomes in a PEX5 and PEX7-dependent manner. iNOS localization to peroxisomes was contingent on EBP50 expression in LPS-treated mice. Thus, iNOS targeting to peroxisomes in hepatocytes involves interaction with PEX7 and EBP50. The targeting of iNOS protein to the peroxisome may shift the balance of metabolic processes that rely on heme proteins susceptible to modification by radical oxygen and nitrogen radicals. PMID:23474170

  7. Development of glass resistive plate chambers for INO experiment

    NASA Astrophysics Data System (ADS)

    Datar, V. M.; Jena, Satyajit; Kalmani, S. D.; Mondal, N. K.; Nagaraj, P.; Reddy, L. V.; Saraf, M.; Satyanarayana, B.; Shinde, R. R.; Verma, P.

    2009-05-01

    The India-based Neutrino Observatory (INO) collaboration is planning to build a massive 50 kton magnetised Iron Calorimeter (ICAL) detector, to study atmospheric neutrinos and to make precision measurements of the parameters related to neutrino oscillations. Glass Resistive Plate Chambers (RPCs) of about 2 m×2 m in size are going to be used as active elements for the ICAL detector. We have fabricated a large number of glass RPC prototypes of 1 m×1 m in size and have studied their performance and long term stability. In the process, we have developed and produced a number of materials and components required for fabrication of RPCs. We have also designed and optimised a number of fabrication and quality control procedures for assembling the gas gaps. In this paper we will review our various activities towards development of glass RPCs for the INO ICAL detector. We will present results of the characterisation studies of the RPCs and discuss our plans to prototype 2 m×2 m sized RPCs.

  8. NLRC4 inflammasome-mediated production of IL-1β modulates mucosal immunity in the lung against gram-negative bacterial infection.

    PubMed

    Cai, Shanshan; Batra, Sanjay; Wakamatsu, Nobuko; Pacher, Pal; Jeyaseelan, Samithamby

    2012-06-01

    Bacterial flagellin is critical to mediate NLRC4 inflammasome-dependent caspase-1 activation. However, Shigella flexneri, a nonflagellated bacterium, and a flagellin (fliC) knockout strain of Pseudomonas aeruginosa are known to activate NLRC4 in bone marrow-derived macrophages. Furthermore, the flagellin-deficient fliC strain of P. aeruginosa was used in a mouse model of peritonitis to show the requirement of NLRC4. In a model of pulmonary P. aeruginosa infection, flagellin was shown to be essential for the induction of NLRC4-dependent caspase-1 activation. Moreover, in all P. aeruginosa studies, IL-1β production was attenuated in NLRC4(-/-) mice; however, the role of IL-1β in NLRC4-mediated innate immunity in the lungs against a nonflagellated bacterium was not explored. In this article, we report that NLRC4 is important for host survival and bacterial clearance, as well as neutrophil-mediated inflammation in the lungs following Klebsiella pneumoniae infection. NLRC4 is essential for K. pneumoniae-induced production of IL-1β, IL-17A, and neutrophil chemoattractants (keratinocyte cell-derived chemokines, MIP-2, and LPS-induced CXC chemokines) in the lungs. NLRC4 signaling in hematopoietic cells contributes to K. pneumoniae-induced lung inflammation. Furthermore, exogenous IL-1β, but not IL-18 or IL-17A, partially rescued survival, neutrophil accumulation, and cytokine/chemokine expression in the lungs of NLRC4(-/-) mice following infectious challenge. Furthermore, IL-1R1(-/-) mice displayed a decrease in neutrophilic inflammation in the lungs postinfection. Taken together, these findings provide novel insights into the role of NLRC4 in host defense against K. pneumoniae infection.

  9. Plasma membrane association of three classes of bacterial toxins is mediated by a basic-hydrophobic motif.

    PubMed

    Geissler, Brett; Ahrens, Sebastian; Satchell, Karla J F

    2012-02-01

    Plasma membrane targeting is essential for the proper function of many bacterial toxins. A conserved fourhelical bundle membrane localization domain (4HBM) was recently identified within three diverse families of toxins: clostridial glucosylating toxins, MARTX toxins and Pasteurella multocida-like toxins. When expressed in tissue culture cells or in yeast, GFP fusions to at least one 4HBM from each toxin family show significant peripheral membrane localization but with differing profiles. Both in vivo expression and in vitro binding studies reveal that the ability of these domains to localize to the plasma membrane and bind negatively charged phospholipids requires a basic-hydrophobic motif formed by the L1 and L3 loops. The different binding capacity of each 4HBM is defined by the hydrophobicity of an exposed residue within the motif. This study establishes that bacterial effectors utilize a normal host cell mechanism to locate the plasma membrane where they can then access their intracellular targets.

  10. Microbubble-mediated ultrasound promotes accumulation of bone marrow mesenchymal stem cell to the prostate for treating chronic bacterial prostatitis in rats

    PubMed Central

    Yi, Shanhong; Han, Guangwei; Shang, Yonggang; Liu, Chengcheng; Cui, Dong; Yu, Shuangjiang; Liao, Bin; Ao, Xiang; Li, Guangzhi; Li, Longkun

    2016-01-01

    Chronic bacterial prostatitis (CBP) is an intractable disease. Although bone marrow mesenchymal stem cells (BMMSCs) are able to regulate inflammation in CBP, the effect of microbubble-mediated ultrasound- induced accumulation of BMMSCs on CBP remains unclear. To address this gap, a model of CBP was established in SD rats, which were then treated with BMMSCs alone (BMMSC group), BMMSCs with ultrasound (ultrasound group), BMMSCs with microbubble-mediated ultrasound (MMUS group) and compared with a healthy control group. A therapeutic-ultrasound apparatus was used to treat the prostate in the presence of circulating microbubbles and BMMSCs. The BMMSC distribution was assessed with in vivo imaging, and the prostate structure with light microscopy. Real-time quantitative RT-PCR, ELISA, and immunohistochemistry were used to assess the expressions of TNF-α and IL-1β. More BMMSCs were found in the prostate in the MMUS group than in the CBP, ultrasound, and BMMSC groups. Inflammatory cell infiltration, fibrous tissue hyperplasia, and tumor-like epithelial proliferation were significantly reduced in the MMUS group, as were the mRNA and protein expressions of TNF-α and IL-1β. Microbubble-mediated ultrasound-induced accumulation of BMMSCs can inhibit inflammation and decrease TNF-α and IL-1β expressions in the prostate of CBP rats, suggesting that this method may be therapeutic for CPB. PMID:26797392

  11. SIV Infection-Mediated Changes in Gastrointestinal Bacterial Microbiome and Virome Are Associated with Immunodeficiency and Prevented by Vaccination.

    PubMed

    Handley, Scott A; Desai, Chandni; Zhao, Guoyan; Droit, Lindsay; Monaco, Cynthia L; Schroeder, Andrew C; Nkolola, Joseph P; Norman, Megan E; Miller, Andrew D; Wang, David; Barouch, Dan H; Virgin, Herbert W

    2016-03-01

    AIDS caused by simian immunodeficiency virus (SIV) infection is associated with gastrointestinal disease, systemic immune activation, and, in cross-sectional studies, changes in the enteric virome. Here we performed a longitudinal study of a vaccine cohort to define the natural history of changes in the fecal metagenome in SIV-infected monkeys. Matched rhesus macaques were either uninfected or intrarectally challenged with SIV, with a subset receiving the Ad26 vaccine, an adenovirus vector expressing the viral Env/Gag/Pol antigens. Progression of SIV infection to AIDS was associated with increased detection of potentially pathogenic viruses and bacterial enteropathogens. Specifically, adenoviruses were associated with an increased incidence of gastrointestinal disease and AIDS-related mortality. Viral and bacterial enteropathogens were largely absent from animals protected by the vaccine. These data suggest that the SIV-associated gastrointestinal disease is associated with the presence of both viral and bacterial enteropathogens and that protection against SIV infection by vaccination prevents enteropathogen emergence. PMID:26962943

  12. Latest developments in active remote sensing at INO

    NASA Astrophysics Data System (ADS)

    Babin, F.; Forest, R.; Bourliaguet, B.; Cantin, D.; Cottin, P.; Pancrati, O.; Turbide, S.; Lambert-Girard, S.; Cayer, F.; Lemieux, D.; Cormier, J.-F.; Châteauneuf, F.

    2012-09-01

    Remote sensing or stand-off detection using controlled light sources is a well known and often used technique for atmospheric and surface spatial mapping. Today, ground based, vehicle-borne and airborne systems are able to cover large areas with high accuracy and good reliability. This kind of detection based on LiDAR (Light Detection and Ranging) or active Differential Optical Absorption Spectroscopy (DOAS) technologies, measures optical responses from controlled illumination of targets. Properties that can be recorded include volume back-scattering, surface reflectivity, molecular absorption, induced fluorescence and Raman scattering. The various elastic and inelastic backscattering responses allow the identification or characterization of content of the target volumes or surfaces. INO has developed instrumentations to measure distance to solid targets and monitor particles suspended in the air or in water in real time. Our full waveform LiDAR system is designed for use in numerous applications in environmental or process monitoring such as dust detection systems, aerosol (pesticide) drift monitoring, liquid level sensing or underwater bathymetric LiDARs. Our gated imaging developments are used as aids in visibility enhancement or in remote sensing spectroscopy. Furthermore, when coupled with a spectrograph having a large number of channels, the technique becomes active multispectral/hyperspectral detection or imaging allowing measurement of ultra-violet laser induced fluorescence (UV LIF), time resolved fluorescence (in the ns to ms range) as well as gated Raman spectroscopy. These latter techniques make possible the stand-off detection of bio-aerosols, drugs, explosives as well as the identification of mineral content for geological survey. This paper reviews the latest technology developments in active remote sensing at INO and presents on-going projects conducted to address future applications in environmental monitoring.

  13. Highly selective detection of bacterial alarmone ppGpp with an off-on fluorescent probe of copper-mediated silver nanoclusters.

    PubMed

    Zhang, Pu; Wang, Yi; Chang, Yong; Xiong, Zu Hong; Huang, Cheng Zhi

    2013-11-15

    In this study, a facile strategy for highly selective and sensitive detection of bacterial alarmone, ppGpp, which is generated when bacteria face stress circumstances such as nutritional deprivation, has been established by developing an off-on fluorescent probe of Cu(2+)-mediated silver nanoclusters (Ag NCs). This work not only achieves highly selective detection of ppGpp in a broad range concentration of 2-200 μM, but also improves our understanding of the specific recognitions among DNA-Ag NCs, Cu(2+), and ppGpp. The present strategy, together with other reports on the Ag NCs-related analytical methods, has also identified that Ag NCs functionalized with different molecules on their surfaces can be engineered fluorescent probes for a wide range of applications such as biosensing and bioimaging.

  14. Packaging of live Legionella pneumophila into pellets expelled by Tetrahymena spp. does not require bacterial replication and depends on a Dot/Icm-mediated survival mechanism.

    PubMed

    Berk, Sharon G; Faulkner, Gary; Garduño, Elizabeth; Joy, Mark C; Ortiz-Jimenez, Marco A; Garduño, Rafael A

    2008-04-01

    The freshwater ciliate Tetrahymena sp. efficiently ingested, but poorly digested, virulent strains of the gram-negative intracellular pathogen Legionella pneumophila. Ciliates expelled live legionellae packaged in free spherical pellets. The ingested legionellae showed no ultrastructural indicators of cell division either within intracellular food vacuoles or in the expelled pellets, while the number of CFU consistently decreased as a function of time postinoculation, suggesting a lack of L. pneumophila replication inside Tetrahymena. Pulse-chase feeding experiments with fluorescent L. pneumophila and Escherichia coli indicated that actively feeding ciliates maintain a rapid and steady turnover of food vacuoles, so that the intravacuolar residence of the ingested bacteria was as short as 1 to 2 h. L. pneumophila mutants with a defective Dot/Icm virulence system were efficiently digested by Tetrahymena sp. In contrast to pellets of virulent L. pneumophila, the pellets produced by ciliates feeding on dot mutants contained very few bacterial cells but abundant membrane whorls. The whorls became labeled with a specific antibody against L. pneumophila OmpS, indicating that they were outer membrane remnants of digested legionellae. Ciliates that fed on genetically complemented dot mutants produced numerous pellets containing live legionellae, establishing the importance of the Dot/Icm system to resist digestion. We thus concluded that production of pellets containing live virulent L. pneumophila depends on bacterial survival (mediated by the Dot/Icm system) and occurs in the absence of bacterial replication. Pellets of virulent L. pneumophila may contribute to the transmission of Legionnaires' disease, an issue currently under investigation. PMID:18245233

  15. Siderophore-Mediated Iron Acquisition Influences Motility and Is Required for Full Virulence of the Xylem-Dwelling Bacterial Phytopathogen Pantoea stewartii subsp. stewartii

    PubMed Central

    Burbank, Lindsey; Mohammadi, Mojtaba

    2014-01-01

    Iron is a key micronutrient for microbial growth but is often present in low concentrations or in biologically unavailable forms. Many microorganisms overcome this challenge by producing siderophores, which are ferric-iron chelating compounds that enable the solubilization and acquisition of iron in a bioactive form. Pantoea stewartii subsp. stewartii, the causal agent of Stewart's wilt of sweet corn, produces a siderophore under iron-limiting conditions. The proteins involved in the biosynthesis and export of this siderophore are encoded by the iucABCD-iutA operon, which is homologous to the aerobactin biosynthetic gene cluster found in a number of enteric pathogens. Mutations in iucA and iutA resulted in a decrease in surface-based motility that P. stewartii utilizes during the early stages of biofilm formation, indicating that active iron acquisition impacts surface motility for P. stewartii. Furthermore, bacterial movement in planta is also dependent on a functional siderophore biosynthesis and uptake pathway. Most notably, siderophore-mediated iron acquisition is required for full virulence in the sweet corn host, indicating that active iron acquisition is essential for pathogenic fitness for this important xylem-dwelling bacterial pathogen. PMID:25326304

  16. Mechanism of UCH-L5 Activation and Inhibition by DEUBAD Domains in RPN13 and INO80G

    PubMed Central

    Sahtoe, Danny D.; van Dijk, Willem J.; El Oualid, Farid; Ekkebus, Reggy; Ovaa, Huib; Sixma, Titia K.

    2015-01-01

    Summary Deubiquitinating enzymes (DUBs) control vital processes in eukaryotes by hydrolyzing ubiquitin adducts. Their activities are tightly regulated, but the mechanisms remain elusive. In particular, the DUB UCH-L5 can be either activated or inhibited by conserved regulatory proteins RPN13 and INO80G, respectively. Here we show how the DEUBAD domain in RPN13 activates UCH-L5 by positioning its C-terminal ULD domain and crossover loop to promote substrate binding and catalysis. The related DEUBAD domain in INO80G inhibits UCH-L5 by exploiting similar structural elements in UCH-L5 to promote a radically different conformation, and employs molecular mimicry to block ubiquitin docking. In this process, large conformational changes create small but highly specific interfaces that mediate activity modulation of UCH-L5 by altering the affinity for substrates. Our results establish how related domains can exploit enzyme conformational plasticity to allosterically regulate DUB activity. These allosteric sites may present novel insights for pharmaceutical intervention in DUB activity. PMID:25702870

  17. The mammalian INO80 chromatin remodeling complex is required for replication stress recovery

    PubMed Central

    Vassileva, Ivelina; Yanakieva, Iskra; Peycheva, Michaela; Gospodinov, Anastas; Anachkova, Boyka

    2014-01-01

    A number of studies have implicated the yeast INO80 chromatin remodeling complex in DNA replication, but the function of the human INO80 complex during S phase remains poorly understood. Here, we have systematically investigated the involvement of the catalytic subunit of the human INO80 complex during unchallenged replication and under replication stress by following the effects of its depletion on cell survival, S-phase checkpoint activation, the fate of individual replication forks, and the consequences of fork collapse. We report that INO80 was specifically needed for efficient replication elongation, while it was not required for initiation of replication. In the absence of the Ino80 protein, cells became hypersensitive to hydroxyurea and displayed hyperactive ATR-Chk1 signaling. Using bulk and fiber labeling of DNA, we found that cells deficient for Ino80 and Arp8 had impaired replication restart after treatment with replication inhibitors and accumulated double-strand breaks as evidenced by the formation of γ-H2AX and Rad51 foci. These data indicate that under conditions of replication stress mammalian INO80 protects stalled forks from collapsing and allows their subsequent restart. PMID:25016522

  18. Rapid and efficient introduction of a foreign gene into bacterial artificial chromosome-cloned varicella vaccine by Tn7-mediated site-specific transposition

    SciTech Connect

    Somboonthum, Pranee; Koshizuka, Tetsuo; Okamoto, Shigefumi; Matsuura, Masaaki; Gomi, Yasuyuki; Takahashi, Michiaki; Yamanishi, Koichi; Mori, Yasuko

    2010-06-20

    Using a rapid and reliable system based on Tn7-mediated site-specific transposition, we have successfully constructed a recombinant Oka varicella vaccine (vOka) expressing the mumps virus (MuV) fusion protein (F). The backbone of the vector was our previously reported vOka-BAC (bacterial artificial chromosome) genome. We inserted the transposon Tn7 attachment sequence, LacZ{alpha}-mini-attTn7, into the region between ORF12 and ORF13 to generate a vOka-BAC-Tn genome. The MuV-F expressing cassette was transposed into the vOka-BAC genome at the mini-attTn7 transposition site. MuV-F protein was expressed in recombinant virus, rvOka-F infected cells. In addition, the MuV-F protein was cleaved in the rvOka-F infected cells as in MuV-infected cells. The growth of rvOka-F was similar to that of the original recombinant vOka without the F gene. Thus, we show that Tn7-mediated transposition is an efficient method for introducing a foreign gene expression cassette into the vOka-BAC genome as a live virus vector.

  19. The peroxidase-mediated biodegradation of petroleum hydrocarbons in a H2O2-induced SBR using in-situ production of peroxidase: Biodegradation experiments and bacterial identification.

    PubMed

    Shekoohiyan, Sakine; Moussavi, Gholamreza; Naddafi, Kazem

    2016-08-01

    A bacterial peroxidase-mediated oxidizing process was developed for biodegrading total petroleum hydrocarbons (TPH) in a sequencing batch reactor (SBR). Almost complete biodegradation (>99%) of high TPH concentrations (4g/L) was attained in the bioreactor with a low amount (0.6mM) of H2O2 at a reaction time of 22h. A specific TPH biodegradation rate as high as 44.3mgTPH/gbiomass×h was obtained with this process. The reaction times required for complete biodegradation of TPH concentrations of 1, 2, 3, and 4g/L were 21, 22, 28, and 30h, respectively. The catalytic activity of hydrocarbon catalyzing peroxidase was determined to be 1.48U/mL biomass. The biodegradation of TPH in seawater was similar to that in fresh media (no salt). A mixture of bacteria capable of peroxidase synthesis and hydrocarbon biodegradation including Pseudomonas spp. and Bacillus spp. were identified in the bioreactor. The GC/MS analysis of the effluent indicated that all classes of hydrocarbons could be well-degraded in the H2O2-induced SBR. Accordingly, the peroxidase-mediated process is a promising method for efficiently biodegrading concentrated TPH-laden saline wastewater. PMID:27060866

  20. Immunoglobulin isotype isolated from human placental extract does not interfere in complement-mediated bacterial opsonization within the wound milieu

    PubMed Central

    Sharma, Kanika; Bhattacharyya, Debasish

    2015-01-01

    The wound healing potency of an aqueous extract of placenta can be evaluated through the presence of numerous regulatory components. The presence of glycans was detected by thin layer chromatography and fluorophore-assisted carbohydrate electrophoresis. Mass spectrometric analysis revealed the existence of multiple fragments of immunoglobulin G (IgG). IgG was present in the extract at a concentration of 25.2 ± 3.97 μg/ml. IgG possesses anti-complementary activity by diverting the complement activation from target surface. Thus, effect of placental IgG on complement–bacteria interaction was investigated through classical and alternative pathway and the preparation was ascertained to be safe with respect to their interference in the process of bacterial opsonization. PMID:25984442

  1. Use of bacterial and firefly luciferases as reporter genes in DEAE-dextran-mediated transfection of mammalian cells.

    PubMed

    Pazzagli, M; Devine, J H; Peterson, D O; Baldwin, T O

    1992-08-01

    The aim of this study was to compare three different luciferase genes by placing them in a single reporter vector and expressing them in the same mammalian cell type. The luciferase genes investigated were the luc genes from the fireflies Photinus pyralis (PP) and Luciola mingrelica (LM) and the lux AB5 gene, a translational fusion of the two subunits of the bacterial luciferase from Vibrio harveyi (VH). The chloramphenicol acetyltransferase (CAT) gene was also included in this study for comparison. The performances of the assay methods of the corresponding enzymes were evaluated using reference materials and the results of the expressed enzymes following transfection were calculated using calibration curves. All of the bioluminescent assays possess high reproducibility both within and between the batches (less than 15%). The comparison of the assay methods shows that firefly luciferases have the highest detection sensitivity (0.05 and 0.08 amol for PP and LM, respectively) whereas the VH bacterial luciferase has 5 amol and CAT 100 amol. On the other hand, the transfection of the various plasmids shows that the content of the expressed enzyme within the cells is much higher for CAT than for the other luciferase genes. VH luciferase is expressed at very low levels in mammalian cells due to the relatively high temperature of growing of the mammalian cells that seems to impair the correct folding of the active enzyme. PP and LM luciferases are both expressed at picomolar level but usually 10 to 70 times less in content with respect to CAT within the transfected cells. On the basis of these results the overall improvement in sensitivity related to the use of firefly luciferases as reporter genes in mammalian cells is about 30 to 50 times with respect to that of CAT. PMID:1443530

  2. India-based neutrino observatory (INO): Physics reach and status report

    SciTech Connect

    Indumathi, D.

    2015-07-15

    We present a review of the physics reach and current status of the proposed India-based Neutrino Observatory (INO). We briefly outline details of the INO location and the present status of detector development. We then present the physics goals and simulation studies of the main detector, the magnetised Iron Calorimeter (ICAL) detector, to be housed in INO. The ICAL detector would make precision measurements of neutrino oscillation parameters with atmospheric neutrinos including a measurement of the neutrino mass hierarchy. Additional synergies with other experiments due to the complete insensitivity of ICAL to the CP phase are also discussed.

  3. Urinary tract infection in iNOS-deficient mice with focus on bacterial sensitivity to nitric oxide.

    PubMed

    Poljakovic, Mirjana; Persson, Katarina

    2003-01-01

    Inducible nitric oxide synthase (iNOS)-deficient mice were used to examine the role of iNOS in Escherichia coli-induced urinary tract infection (UTI). The toxicity of nitric oxide (NO)/peroxynitrite to bacteria and host was also investigated. The nitrite levels in urine of iNOS+/+ but not iNOS/ mice increased after infection. No differences in bacterial clearance or persistence were noted between the genotypes. In vitro, the uropathogenic E. coli 1177 was sensitive to 3-morpholinosydnonimine, whereas the avirulent E. coli HB101 was sensitive to both NO and 3-morpholinosydnonimine. E. coli HB101 was statistically (P < 0.05) more sensitive to peroxynitrite than E. coli 1177. Nitrotyrosine immunoreactivity was observed in infected bladders of both genotypes and in infected kidneys of iNOS+/+ mice. Myeloperoxidase, neuronal (n)NOS, and endothelial (e)NOS immunoreactivity was observed in inflammatory cells of both genotypes. Our results indicate that iNOS/ and iNOS+/+ mice are equally susceptible to E. coli-induced UTI and that the toxicity of NO to E. coli depends on bacterial virulence. Furthermore, myeloperoxidase and nNOS/eNOS may contribute to nitrotyrosine formation in the absence of iNOS.

  4. TNF-Α May Mediate Inflammasome Activation in the Absence of Bacterial Infection in More than One Way

    PubMed Central

    Álvarez, Susana; Muñoz-Fernández, Ma Ángeles

    2013-01-01

    Members of the mammalian nucleotide binding domain, leucine-rich repeat (LRR)-containing receptor family of proteins are key modulators of innate immunity regulating inflammation. To date, microbial pathogen-associated molecules and toxins have been identified as key triggers of activation of inflammasomes. However, recently, environmental, and neurodegenerative stimuli have been identified that lead to IL-1β release by means of inflammasomes. IL-1β plays a crucial role during brain inflammation, and caspase-1 appears to be a key modulator of IL-1β bioactivity and the consequent transcriptional regulation of gene expression within the brain during inflammation. We show here that exposure of a human neuroblastoma cell line (SK-N-MC cells) to TNF-α promotes ROS-mediated caspase-1 activation and IL-1β secretion. The involvement of NF-κB in the regulation of IL-1β synthesis is investigated through specific inhibition of this transcription factor. The effect of TNF-α was abolished in the presence of ROS inhibitors as NAC, or DPI. Remarkably, SK-N-MC cells do not respond to ATP stimulation in spite of P2X7R expression. These results provide a mechanism by which danger signals and particulate matter mediate inflammation via the inflammasome in the absence of microbial infection. PMID:23940760

  5. OMNIS, The Observatory for Multiflavor NeutrInos from Supernovae

    NASA Astrophysics Data System (ADS)

    Murphy, Alexander; Boyd, Richard

    2001-10-01

    OMNIS, the Observatory for Multiflavor NeutrInos from Supernovae will consist of 8 kT of lead and 4 kT of iron which, when irradiated by neutrinos from a supernova, will produce secondary neutrons. Detection of the neutrons in gadolinium loaded liquid scintillators will then signal the arrival of the supernova neutrinos. A supernova at the center of the Galaxy, will produce about 2000 events in OMNIS, mostly from neutral current interactions, thus providing statistically significant tests of the energies and emission time profiles of core-collapse supernova models. Additionally, OMNIS' combination of lead and iron modules gives it sensitivity to several neutrino oscillation scenarios, especially the type ν_μ/τ rightarrow ν_e. Its intrinsic timing capability, better than 1 ms, gives it the capability to measure neutrino mass from the time-of-flight shifts in the luminosity curves of the different neutrino flavors, to about 20 eV/c^2. OMNIS will also be able to detect differences in the luminosity cutoffs of the different flavors in the event of a fairly prompt collapse to a black hole, which might allow charting out of the neutrinospheres and other diagnostics, and a better measurement of neutrino mass ( ~3 eV/c^2).

  6. Inducible nitric oxide synthase (iNOS) in muscle wasting syndrome, sarcopenia, and cachexia

    PubMed Central

    Hall, Derek T.; Ma, Jennifer F.; Di Marco, Sergio; Gallouzi, Imed-Eddine

    2011-01-01

    Muscle atrophy—also known as muscle wasting—is a debilitating syndrome that slowly develops with age (sarcopenia) or rapidly appears at the late stages of deadly diseases such as cancer, AIDS, and sepsis (cachexia). Despite the prevalence and the drastic detrimental effects of these two syndromes, there are currently no widely used, effective treatment options for those suffering from muscle wasting. In an attempt to identify potential therapeutic targets, the molecular mechanisms of sarcopenia and cachexia have begun to be elucidated. Growing evidence suggests that inflammatory cytokines may play an important role in the pathology of both syndromes. As one of the key cytokines involved in both sarcopenic and cachectic muscle wasting, tumor necrosis factor α (TNFα) and its downstream effectors provide an enticing target for pharmacological intervention. However, to date, no drugs targeting the TNFα signaling pathway have been successful as a remedial option for the treatment of muscle wasting. Thus, there is a need to identify new effectors in this important pathway that might prove to be more efficacious targets. Inducible nitric oxide synthase (iNOS) has recently been shown to be an important mediator of TNFα-induced cachectic muscle loss, and studies suggest that it may also play a role in sarcopenia. In addition, investigations into the mechanism of iNOS-mediated muscle loss have begun to reveal potential therapeutic strategies. In this review, we will highlight the potential for targeting the iNOS/NO pathway in the treatment of muscle loss and discuss its functional relevance in sarcopenia and cachexia. PMID:21832306

  7. Treatment with TNF-α or bacterial lipopolysaccharide attenuates endocardial endothelial cell-mediated stimulation of cardiac fibroblasts

    PubMed Central

    Kuruvilla, Leena; Kartha, Cheranellore Chandrasekharan

    2009-01-01

    Background The endocardial endothelium that lines the inner cavity of the heart is distinct from the microvascular endothelial cells and modulates cardiac muscle performance in a manner similar to the vascular endothelial modulation of vascular structure and vasomotor tone. Although the modulatory effects of endocardial endothelium (EE) on cardiomyocytes are firmly established, the regulatory effects of endocardial endothelium on the cardiac interstitium and its cellular components remain ill defined. Methods and Results We investigated whether the stimulatory effect of EE on cardiac fibroblasts would be altered when EECs are activated by the cytokine tumor necrosis factor-α (TNF-α) or the endotoxin bacterial lipopolysaccharide (LPS). Both TNF-α and LPS were found to independently attenuate the stimulatory effect of EE on cardiac fibroblasts. These agents lowered the synthesis or release of ET-1 and increased the secretion of TGF-β and NO. Conclusion The findings of this study using endocardial endothelial cells (EECs) and neonatal cardiac fibroblasts demonstrate that pro-inflammatory cytokines cause altered secretion of paracrine factors by EECs and inhibit proliferation and lower collagen synthesis in fibroblasts. These changes may influence fibroblast response and extra cellular matrix remodeling in pathological conditions of the heart. PMID:19272191

  8. Bacterial endophytes mediate positive feedback effects of early legume termination times on the yield of subsequent durum wheat crops.

    PubMed

    Yang, Chao; Hamel, Chantal; Gan, Yantai; Vujanovic, Vladimir

    2012-12-01

    Field crops influence the biotic properties of the soil, impacting the health and productivity of subsequent crops. Polymerase chain reaction and 454 GS FLX pyrosequencing of amplicons were used to clarify the legacy of chickpea and pea crops on the quality of the bacterial community colonizing the root endosphere of subsequent crops of wheat, in a replicated field study. Similar communities of root endosphere bacteria were formed in durum wheat grown after pea and chickpea crops when chickpea crops were terminated as early as pea (July). Termination of the chickpea crops in September led to the domination of Firmicutes in wheat root endosphere; Actinobacteria dominated the wheat root endosphere following early pulse crop termination. The architecture of wheat plants was correlated with the composition of its root endosphere community. High grain yield was associated with the production of fewer but larger wheat heads, the abundance of endospheric Actinobacteria and Acidobacteria, and the scarcity of endospheric Firmicutes. Pulse termination time affected wheat root endosphere colonization strongly in 2009 but weakly in 2010, an abnormally wet year. This study improved our understanding of the so-called "crop rotation effect" in pulse-wheat systems and showed how this system can be manipulated through agronomic decisions. PMID:23210994

  9. Bacterial endophytes mediate positive feedback effects of early legume termination times on the yield of subsequent durum wheat crops.

    PubMed

    Yang, Chao; Hamel, Chantal; Gan, Yantai; Vujanovic, Vladimir

    2012-12-01

    Field crops influence the biotic properties of the soil, impacting the health and productivity of subsequent crops. Polymerase chain reaction and 454 GS FLX pyrosequencing of amplicons were used to clarify the legacy of chickpea and pea crops on the quality of the bacterial community colonizing the root endosphere of subsequent crops of wheat, in a replicated field study. Similar communities of root endosphere bacteria were formed in durum wheat grown after pea and chickpea crops when chickpea crops were terminated as early as pea (July). Termination of the chickpea crops in September led to the domination of Firmicutes in wheat root endosphere; Actinobacteria dominated the wheat root endosphere following early pulse crop termination. The architecture of wheat plants was correlated with the composition of its root endosphere community. High grain yield was associated with the production of fewer but larger wheat heads, the abundance of endospheric Actinobacteria and Acidobacteria, and the scarcity of endospheric Firmicutes. Pulse termination time affected wheat root endosphere colonization strongly in 2009 but weakly in 2010, an abnormally wet year. This study improved our understanding of the so-called "crop rotation effect" in pulse-wheat systems and showed how this system can be manipulated through agronomic decisions.

  10. The Oligopeptide Permease Opp Mediates Illicit Transport of the Bacterial P-site Decoding Inhibitor GE81112 †

    PubMed Central

    Maio, Alessandro; Brandi, Letizia; Donadio, Stefano; Gualerzi, Claudio O.

    2016-01-01

    GE81112 is a tetrapeptide antibiotic that binds to the 30S ribosomal subunit and specifically inhibits P-site decoding of the mRNA initiation codon by the fMet-tRNA anticodon. GE81112 displays excellent microbiological activity against some Gram-positive and Gram-negative bacteria in both minimal and complete, chemically defined, broth, but is essentially inactive in complete complex media. This is due to the presence of peptides that compete with the antibiotic for the oligopeptide permease system (Opp) responsible for its illicit transport into the bacterial cells as demonstrated in the cases of Escherichia coli and Bacillus subtilis. Mutations that inactivate the Opp system and confer GE81112 resistance arise spontaneously with a frequency of ca. 1 × 10−6, similar to that of the mutants resistant to tri-l-ornithine, a known Opp substrate. On the contrary, cells expressing extrachromosomal copies of the opp genes are extremely sensitive to GE81112 in rich medium and GE81112-resistant mutations affecting the molecular target of the antibiotic were not detected upon examining >109 cells of this type. However, some mutations introduced in the 16S rRNA to confer kasugamycin resistance were found to reduce the sensitivity of the cells to GE81112. PMID:27231947

  11. Ethanol-induced iNOS and COX-2 expression in cultured astrocytes via NF-kappa B.

    PubMed

    Blanco, Ana M; Pascual, María; Valles, Soraya L; Guerri, Consuelo

    2004-03-22

    The CNS is particularly susceptible to the effects of alcohol and toxicity. Astrocytes are immunoactive cells, and the activation of these cells is associated with several neurodegenerative disorders. By using cultured cortical astrocytes, we show that a short ethanol treatment (100 mM) is able to up-regulate both cyclooxygenase 2 (COX-2) and inducible nitric oxide synthase (iNOS) expression, and that these effects are regulated via nuclear factor kappa B (NF-kappa B) as revealed by the inhibition of NF-kappa B activation with pyrrolidine dithiocarbamate (PDTC) or BAY 11-7082. These results suggest that ethanol is able to induce inflammatory mediators in astrocytes through the NF-kappa B activation.

  12. The RSC and INO80 chromatin-remodeling complexes in DNA double-strand break repair.

    PubMed

    Chambers, Anna L; Downs, Jessica A

    2012-01-01

    In eukaryotes, DNA is packaged into chromatin and is therefore relatively inaccessible to DNA repair enzymes. In order to perform efficient DNA repair, ATP-dependent chromatin-remodeling enzymes are required to alter the chromatin structure near the site of damage to facilitate processing and allow access to repair enzymes. Two of the best-studied remodeling complexes involved in repair are RSC (Remodels the Structure of Chromatin) and INO80 from Saccharomyces cerevisiae, which are both conserved in higher eukaryotes. RSC is very rapidly recruited to breaks and mobilizes nucleosomes to promote phosphorylation of H2A S129 and resection. INO80 enrichment at a break occurs later and is dependent on phospho-S129 H2A. INO80 activity at the break site also facilitates resection. Consequently, both homologous recombination and nonhomologous end-joining are defective in rsc mutants, while subsets of these repair pathways are affected in ino80 mutants.

  13. Participation of tumor necrosis factor in the mediation of gram negative bacterial lipopolysaccharide-induced injury in rabbits.

    PubMed Central

    Mathison, J C; Wolfson, E; Ulevitch, R J

    1988-01-01

    Macrophages are induced by LPS to release a number of products that determine the host response during gram negative sepsis. To examine the role of one such substance, tumor necrosis factor (TNF), in mediating LPS-induced injury, we employed a rabbit model of endotoxic shock to (a) determine the kinetics and extent of release of TNF into plasma after injection of LPS, and (b) to evaluate the protective effect of in vivo neutralization of LPS-induced TNF by prior infusion of anti-TNF antibody. TNF was maximally induced 45-100 min after injection of 10 micrograms i.v. parent Salmonella minnesota Re595 LPS or 250 micrograms Re595 LPS-HDL complexes. Maximal induction of TNF by LPS was associated with development of hypotension, focal hepatic necrosis, intravascular fibrin deposition and lethality. Based on (a) the peak levels of TNF observed in serum, 2.5 X 10(3) U/ml, (b) the specific activity of purified rabbit macrophage-derived TNF, 1 X 10(8) U/mg, and (c) the biphasic disappearance of intravenously injected purified TNF (t1/2 = 0.5 min, 11 min) we constructed a kinetic model showing that at least 130 micrograms of TNF (1.3 X 10(7) U) was released into plasma 30-200 min postinjection of LPS. Prior infusion of anti-TNF antibody (30-45 min before LPS injection) resulted in neutralization of the LPS-induced serum TNF activity and provided significant protection from the development of hypotension, fibrin deposition, and lethality. Thus, these results provide further evidence that TNF plays a central role mediating the pathophysiologic changes that occur during gram negative endotoxic shock. Images PMID:3384955

  14. Mechanism for dynamic regulation of iNOS expression after UVB-irradiation.

    PubMed

    Lu, Wei; Wu, Shiyong

    2013-08-01

    Ultraviolet B (UVB) induces an immediate activation of cNOSs, which contributes to the early release of nitric oxide after irradiation. UVB also induces the expression of iNOS, which peaks at both the mRNA and protein level near 24 h post-irradiation. The induced expression of iNOS contributes largely to the late elevation of nitric oxide after UVB irradiation. However, the regulation of iNOS expression in the early stages of UVB irradiation is not well studied. We previously reported that the UVB-induced early release of nitric oxide leads to the activation of PERK and GCN2, which phosphorylate the alpha-subunit of eIF2 and inhibit protein synthesis. In this report, we demonstrate that eIF2 phosphorylation plays a critical role in regulation of iNOS expression in the early-phase (with in 12 h) of UVB irradiation. Our data shows that with an increased phosphorylation of eIF2, the iNOS protein expression was reduced even though the iNOS mRNA expression was linearly increased in HaCaT and MEF cells after UVB irradiation. The UVB-induced dynamic up- and down-regulation of iNOS expression was almost completely lost in MEF(A/A) cells, which contain a nonphosphorylatable S51A mutation on eIF2. Our results suggest that the UVB-induced eIF2 phosphorylation does not only regulate iNOS expression at the translational level, but at the transcriptional level as well. PMID:22430947

  15. An HD-domain phosphodiesterase mediates cooperative hydrolysis of c-di-AMP to affect bacterial growth and virulence

    PubMed Central

    Huynh, TuAnh Ngoc; Luo, Shukun; Pensinger, Daniel; Sauer, John-Demian; Tong, Liang; Woodward, Joshua J.

    2015-01-01

    The nucleotide cyclic di-3′,5′- adenosine monophosphate (c-di-AMP) was recently identified as an essential and widespread second messenger in bacterial signaling. Among c-di-AMP–producing bacteria, altered nucleotide levels result in several physiological defects and attenuated virulence. Thus, a detailed molecular understanding of c-di-AMP metabolism is of both fundamental and practical interest. Currently, c-di-AMP degradation is recognized solely among DHH-DHHA1 domain-containing phosphodiesterases. Using chemical proteomics, we identified the Listeria monocytogenes protein PgpH as a molecular target of c-di-AMP. Biochemical and structural studies revealed that the PgpH His-Asp (HD) domain bound c-di-AMP with high affinity and specifically hydrolyzed this nucleotide to 5′-pApA. PgpH hydrolysis activity was inhibited by ppGpp, indicating a cross-talk between c-di-AMP signaling and the stringent response. Genetic analyses supported coordinated regulation of c-di-AMP levels in and out of the host. Intriguingly, a L. monocytogenes mutant that lacks c-di-AMP phosphodiesterases exhibited elevated c-di-AMP levels, hyperinduced a host type-I IFN response, and was significantly attenuated for infection. Furthermore, PgpH homologs, which belong to the 7TMR-HD family, are widespread among hundreds of c-di-AMP synthesizing microorganisms. Thus, PgpH represents a broadly conserved class of c-di-AMP phosphodiesterase with possibly other physiological functions in this crucial signaling network. PMID:25583510

  16. Triclosan Resistance in a Bacterial Fish Pathogen, Aeromonas salmonicida subsp. salmonicida, is Mediated by an Enoyl Reductase, FabV.

    PubMed

    Khan, Raees; Lee, Myung Hwan; Joo, Hae-Jin; Jung, Yong-Hoon; Ahmad, Shabir; Choi, Jin-Hee; Lee, Seon-Woo

    2015-04-01

    Triclosan, the widely used biocide, specifically targets enoyl-acyl carrier protein reductase (ENR) in the bacterial fatty acid synthesis system. Although the fish pathogen Aeromonas salmonicida subsp. salmonicida exhibits triclosan resistance, the nature of this resistance has not been elucidated. Here, we aimed to characterize the triclosan resistance of A. salmonicida subsp. salmonicida causing furunculosis. The fosmid library of triclosan-resistant A. salmonicida subsp. salmonicida was constructed to select a fosmid clone showing triclosan resistance. With the fosmid clone showing triclosan resistance, a subsequent secondary library search resulted in the selection of subclone pTSR-1. DNA sequence analysis of pTSR-1 revealed the presence of a chromosomal-borne fabV-encoding ENR homolog. The ENR of A. salmonicida (FabVas) exhibited significant homology with previously known FabV, including the catalytic domain YX(8)K. fabVas introduction into E. coli dramatically increased its resistance to triclosan. Heterologous expression of FabVas might functionally replace the triclosan-sensitive FabI in vivo to confer E. coli with triclosan resistance. A genome-wide search for fabVas homologs revealed the presence of an additional fabV gene (fabVas2) paralog in A. salmonicida strains and the fabVas orthologs from other gram-negative fish pathogens. Both of the potential FabV ENRs expressed similarly with or without triclosan supplement. This is the first report about the presence of two potential FabV ENRs in a single pathogenic bacterium. Our result suggests that triclosan-resistant ENRs are widely distributed in various bacteria in nature, and the wide use of this biocide can spread these triclosan-tolerant ENRs among fish pathogens and other pathogenic bacteria.

  17. Green synthesis of bacterial mediated anti-proliferative gold nanoparticles: inducing mitotic arrest (G2/M phase) and apoptosis (intrinsic pathway)

    NASA Astrophysics Data System (ADS)

    Ganesh Kumar, C.; Poornachandra, Y.; Chandrasekhar, Cheemalamarri

    2015-11-01

    The physiochemical and biological properties of microbial derived gold nanoparticles have potential applications in various biomedical domains as well as in cancer therapy. We have fabricated anti-proliferative bacterial mediated gold nanoparticles (b-Au NPs) using a culture supernatant of Streptomyces clavuligerus and later characterized them by UV-visible, TEM, DLS, XRD and FT-IR spectroscopic techniques. The capping agent responsible for the nanoparticle formation was characterized based on SDS-PAGE and MALDI-TOF-MS analyses. They were tested for anticancer activity in A549, HeLa and DU145 cell lines. The biocompatibility and non-toxic nature of the nanoparticles were tested on normal human lung cell line (MRC-5). The b-Au NPs induced the cell cycle arrest in G2/M phase and also inhibited the microtubule assembly in DU145 cells. Mechanistic studies, such as ROS, MMP, Cyt-c, GSH, caspases 9, 8 and 3 activation and the Annexin V-FITC staining, along with the above parameters tested provided sufficient evidence that the b-Au NPs induced apoptosis through the intrinsic pathway. The results supported the use of b-Au NPs for future therapeutic application in cancer therapy and other biomedical applications.The physiochemical and biological properties of microbial derived gold nanoparticles have potential applications in various biomedical domains as well as in cancer therapy. We have fabricated anti-proliferative bacterial mediated gold nanoparticles (b-Au NPs) using a culture supernatant of Streptomyces clavuligerus and later characterized them by UV-visible, TEM, DLS, XRD and FT-IR spectroscopic techniques. The capping agent responsible for the nanoparticle formation was characterized based on SDS-PAGE and MALDI-TOF-MS analyses. They were tested for anticancer activity in A549, HeLa and DU145 cell lines. The biocompatibility and non-toxic nature of the nanoparticles were tested on normal human lung cell line (MRC-5). The b-Au NPs induced the cell cycle arrest in G2

  18. Rutin-Mediated Priming of Plant Resistance to Three Bacterial Pathogens Initiating the Early SA Signal Pathway

    PubMed Central

    Li, Yang; Wang, Yingzi; Li, Ming; Wang, Yong; Ding, Xinhua; Chu, Zhaohui

    2016-01-01

    Flavonoids are ubiquitous in the plant kingdom and have many diverse functions, including UV protection, auxin transport inhibition, allelopathy, flower coloring and insect resistance. Here we show that rutin, a proud member of the flavonoid family, could be functional as an activator to improve plant disease resistances. Three plant species pretreated with 2 mM rutin were found to enhance resistance to Xanthomonas oryzae pv. oryzae, Ralstonia solanacearum, and Pseudomonas syringae pv. tomato strain DC3000 in rice, tobacco and Arabidopsis thaliana respectively. While they were normally propagated on the cultural medium supplemented with 2 mM rutin for those pathogenic bacteria. The enhanced resistance was associated with primed expression of several pathogenesis-related genes. We also demonstrated that the rutin-mediated priming resistance was attenuated in npr1, eds1, eds5, pad4-1, ndr1 mutants, and NahG transgenic Arabidopsis plant, while not in either snc1-11, ein2-5 or jar1 mutants. We concluded that the rutin-priming defense signal was modulated by the salicylic acid (SA)-dependent pathway from an early stage upstream of NDR1 and EDS1. PMID:26751786

  19. Rutin-Mediated Priming of Plant Resistance to Three Bacterial Pathogens Initiating the Early SA Signal Pathway.

    PubMed

    Yang, Wei; Xu, Xiaonan; Li, Yang; Wang, Yingzi; Li, Ming; Wang, Yong; Ding, Xinhua; Chu, Zhaohui

    2016-01-01

    Flavonoids are ubiquitous in the plant kingdom and have many diverse functions, including UV protection, auxin transport inhibition, allelopathy, flower coloring and insect resistance. Here we show that rutin, a proud member of the flavonoid family, could be functional as an activator to improve plant disease resistances. Three plant species pretreated with 2 mM rutin were found to enhance resistance to Xanthomonas oryzae pv. oryzae, Ralstonia solanacearum, and Pseudomonas syringae pv. tomato strain DC3000 in rice, tobacco and Arabidopsis thaliana respectively. While they were normally propagated on the cultural medium supplemented with 2 mM rutin for those pathogenic bacteria. The enhanced resistance was associated with primed expression of several pathogenesis-related genes. We also demonstrated that the rutin-mediated priming resistance was attenuated in npr1, eds1, eds5, pad4-1, ndr1 mutants, and NahG transgenic Arabidopsis plant, while not in either snc1-11, ein2-5 or jar1 mutants. We concluded that the rutin-priming defense signal was modulated by the salicylic acid (SA)-dependent pathway from an early stage upstream of NDR1 and EDS1. PMID:26751786

  20. Rutin-Mediated Priming of Plant Resistance to Three Bacterial Pathogens Initiating the Early SA Signal Pathway.

    PubMed

    Yang, Wei; Xu, Xiaonan; Li, Yang; Wang, Yingzi; Li, Ming; Wang, Yong; Ding, Xinhua; Chu, Zhaohui

    2016-01-01

    Flavonoids are ubiquitous in the plant kingdom and have many diverse functions, including UV protection, auxin transport inhibition, allelopathy, flower coloring and insect resistance. Here we show that rutin, a proud member of the flavonoid family, could be functional as an activator to improve plant disease resistances. Three plant species pretreated with 2 mM rutin were found to enhance resistance to Xanthomonas oryzae pv. oryzae, Ralstonia solanacearum, and Pseudomonas syringae pv. tomato strain DC3000 in rice, tobacco and Arabidopsis thaliana respectively. While they were normally propagated on the cultural medium supplemented with 2 mM rutin for those pathogenic bacteria. The enhanced resistance was associated with primed expression of several pathogenesis-related genes. We also demonstrated that the rutin-mediated priming resistance was attenuated in npr1, eds1, eds5, pad4-1, ndr1 mutants, and NahG transgenic Arabidopsis plant, while not in either snc1-11, ein2-5 or jar1 mutants. We concluded that the rutin-priming defense signal was modulated by the salicylic acid (SA)-dependent pathway from an early stage upstream of NDR1 and EDS1.

  1. Enhancements to INO's broadband SWIR/MWIR spectroscopic lidar

    NASA Astrophysics Data System (ADS)

    Lambert-Girard, Simon; Babin, François; Allard, Martin; Piché, Michel

    2013-09-01

    Recent advances in the INO broadband SWIR/MWIR spectroscopic lidar will be presented. The system is designed for the detection of gaseous pollutants via active infrared differential optical absorption spectroscopy (DOAS). Two distinctive features are a sub-nanosecond PPMgO:LN OPG capable of generating broadband (10 to <100 nm FWHM) and tunable (1.5 to 3.8 μm) SWIR/MWIR light, and an in-house gated MCT-APD focal plane array used in the output plane of a grating spectrograph. The operation consists in closely gating the returns from back-scattering off topographic features, and is thus, for now, a path integrated measurement. All wavelengths are emitted and received simultaneously, for low concentration measurements and DOAS fitting methods are then applied. The OPG approach enables the generation of moderate FWHM continua with high spectral energy density and tunable to absorption features of many molecules. Recent measurements demonstrating a minimum sensitivity of 10 ppm-m for methane around 3.3 μm with ˜ 2 mW average power in less than 10 seconds will be described. Results of enhancements to the laser source using small or large bandwidth seeds constructed from telecom off-the-shelf components indicate that the OPG output spectral energy density can have controllable spectral widths and shapes. It also has a slightly more stable spectral shape from pulse to pulse than without the seed (25 % enhancement). Most importantly, the stabilized output spectra will allow more sensitive measurements.

  2. Mulberry leaf extract mediated synthesis of gold nanoparticles and its anti-bacterial activity against human pathogens

    NASA Astrophysics Data System (ADS)

    Adavallan, K.; Krishnakumar, N.

    2014-06-01

    Gold nanoparticles (Au-NPs) were synthesized at room temperature using Morus alba (mulberry) leaf extract as reducing and stabilizing agent. The development of plant mediated synthesis of nanoparticles is gaining importance due to its simplicity, low cost, non-toxicity, eco-friendliness, long term stability and reproducible aqueous synthesis method to obtain a self-assembly of nearly monodispersed Au-NPs. The formation and morphology of biosynthesized nanoparticles are investigated with the help of UV-Vis spectroscopy, dynamic light scattering (DLS), transmission electron microscopy (TEM), atomic force microscopy (AFM), x-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FT-IR) techniques. Au-NPs formation was screened by UV-Vis spectroscopy through color conversion due to surface plasmon resonance band at 538 nm for Au-NPs. DLS studies revealed that the average size of Au-NPs was 50 nm. TEM studies showed the particles to be nearly spherical with few irregular shapes and particle size ranges 15-53 nm. The AFM image clearly shows the surface morphology of the well-dispersed Au-NPs with less than 50 nm. The high crystallinity of nanoparticles is evident from bright circular spots in the selected area electron diffraction (SAED) pattern. X-ray diffraction pattern showed high purity and face-centered cubic structure of Au-NPs. The FT-IR results indicate the presence of different functional groups present in the biomolecule capping the nanoparticles. Further, biosynthesized Au-NPs show strong zone of inhibition against Vibrio cholera (gram-negative) and Staphylococcus aureus (gram-positive) whereas, chemically synthesized Au-NPs and mulberry leaf extract exhibit a fair zone of inhibition.

  3. Role of the DmpR-Mediated Regulatory Circuit in Bacterial Biodegradation Properties in Methylphenol-Amended Soils

    PubMed Central

    Sarand, Inga; Skärfstad, Eleonore; Forsman, Mats; Romantschuk, Martin; Shingler, Victoria

    2001-01-01

    Pathway substrates and some structural analogues directly activate the regulatory protein DmpR to promote transcription of the dmp operon genes encoding the (methyl)phenol degradative pathway of Pseudomonas sp. strain CF600. While a wide range of phenols can activate DmpR, the location and nature of substituents on the basic phenolic ring can limit the level of activation and thus utilization of some compounds as assessed by growth on plates. Here we address the role of the aromatic effector response of DmpR in determining degradative properties in two soil matrices that provide different nutritional conditions. Using the wild-type system and an isogenic counterpart containing a DmpR mutant with enhanced ability to respond to para-substituted phenols, we demonstrate (i) that the enhanced in vitro biodegradative capacity of the regulator mutant strain is manifested in the two different soil types and (ii) that exposure of the wild-type strain to 4-methylphenol-contaminated soil led to rapid selection of a subpopulation exhibiting enhanced capacities to degrade the compound. Genetic and functional analyses of 10 of these derivatives demonstrated that all harbored a single mutation in the sensory domain of DmpR that mediated the phenotype in each case. These findings establish a dominating role for the aromatic effector response of DmpR in determining degradation properties. Moreover, the results indicate that the ability to rapidly adapt regulator properties to different profiles of polluting compounds may underlie the evolutionary success of DmpR-like regulators in controlling aromatic catabolic pathways. PMID:11133441

  4. The Bacterial Fermentation Product Butyrate Influences Epithelial Signaling via Reactive Oxygen Species-Mediated Changes in Cullin-1 Neddylation1

    PubMed Central

    Kumar, Amrita; Wu, Huixia; Collier-Hyams, Lauren S.; Kwon, Young-Man; Hanson, Jason M.; Neish, Andrew S.

    2010-01-01

    The human enteric flora plays a significant role in intestinal health and disease. Populations of enteric bacteria can inhibit the NF-κB pathway by blockade of IκB-α ubiquitination, a process catalyzed by the E3-SCFβ-TrCP ubiquitin ligase. The activity of this ubiquitin ligase is regulated via covalent modification of the Cullin-1 subunit by the ubiquitin-like protein NEDD8. We previously reported that interaction of viable commensal bacteria with mammalian intestinal epithelial cells resulted in a rapid and reversible generation of reactive oxygen species (ROS) that modulated neddylation of Cullin-1 and resulted in suppressive effects on the NF-κB pathway. Herein, we demonstrate that butyrate and other short chain fatty acids supplemented to model human intestinal epithelia in vitro and human tissue ex vivo results in loss of neddylated Cul-1 and show that physiological concentrations of butyrate modulate the ubiquitination and degradation of a target of the E3-SCFβ-TrCP ubiquitin ligase, the NF-κB inhibitor IκB-α. Mechanistically, we show that physiological concentrations of butyrate induces reactive oxygen species that transiently alters the intracellular redox balance and results in inactivation of the NEDD8-conjugating enzyme Ubc12 in a manner similar to effects mediated by viable bacteria. Because the normal flora produces significant amounts of butyrate and other short chain fatty acids, these data provide a functional link between a natural product of the intestinal normal flora and important epithelial inflammatory and proliferative signaling pathways. PMID:19109186

  5. Lithium and valproate decrease inositol mass and increase expression of the yeast INO1 and INO2 genes for inositol biosynthesis.

    PubMed

    Vaden, D L; Ding, D; Peterson, B; Greenberg, M L

    2001-05-01

    Bipolar affective disorder (manic-depressive illness) is a chronic, severe, debilitating illness affecting 1-2% of the population. The Food and Drug Administration-approved drugs lithium and valproate are not completely effective in the treatment of this disorder, and the mechanisms underlying their therapeutic effects have not been established. We are employing genetic and molecular approaches to identify common targets of lithium and valproate in the yeast Saccharomyces cerevisiae. We show that both drugs affect molecular targets in the inositol metabolic pathway. Lithium and valproate cause a decrease in intracellular myo-inositol mass and an increase in expression of both a structural (INO1) and a regulatory (INO2) gene required for inositol biosynthesis. The opi1 mutant, which exhibits constitutive expression of INO1, is more resistant to inhibition of growth by lithium but not by valproate, suggesting that valproate may inhibit the Ino1p-catalyzed synthesis of inositol 1-phosphate. Consistent with this possibility, growth in valproate leads to decreased synthesis of inositol monophosphate. Thus, both lithium and valproate perturb regulation of the inositol biosynthetic pathway, albeit via different mechanisms. This is the first demonstration of increased expression of genes in the inositol biosynthetic pathway by both lithium and valproate. Because inositol is a key regulator of many cellular processes, the effects of lithium and valproate on inositol synthesis have far-reaching implications for predicting genetic determinants of responsiveness and resistance to these agents. PMID:11278273

  6. Green synthesis of bacterial mediated anti-proliferative gold nanoparticles: inducing mitotic arrest (G2/M phase) and apoptosis (intrinsic pathway).

    PubMed

    Kumar, C Ganesh; Poornachandra, Y; Chandrasekhar, Cheemalamarri

    2015-11-28

    The physiochemical and biological properties of microbial derived gold nanoparticles have potential applications in various biomedical domains as well as in cancer therapy. We have fabricated anti-proliferative bacterial mediated gold nanoparticles (b-Au NPs) using a culture supernatant of Streptomyces clavuligerus and later characterized them by UV-visible, TEM, DLS, XRD and FT-IR spectroscopic techniques. The capping agent responsible for the nanoparticle formation was characterized based on SDS-PAGE and MALDI-TOF-MS analyses. They were tested for anticancer activity in A549, HeLa and DU145 cell lines. The biocompatibility and non-toxic nature of the nanoparticles were tested on normal human lung cell line (MRC-5). The b-Au NPs induced the cell cycle arrest in G2/M phase and also inhibited the microtubule assembly in DU145 cells. Mechanistic studies, such as ROS, MMP, Cyt-c, GSH, caspases 9, 8 and 3 activation and the Annexin V-FITC staining, along with the above parameters tested provided sufficient evidence that the b-Au NPs induced apoptosis through the intrinsic pathway. The results supported the use of b-Au NPs for future therapeutic application in cancer therapy and other biomedical applications. PMID:26503300

  7. Enzyme-mediated bacterial biodegradation of an azo dye (C.I. Acid blue 113): reuse of treated dye wastewater in post-tanning operations.

    PubMed

    Senthilvelan, T; Kanagaraj, J; Panda, R C

    2014-11-01

    "Dyeing" is a common practice used to color the hides during the post-tanning operations in leather processing generating plenty of wastewater. The waste stream containing dye as pollutant is severely harmful to living beings. An azo dye (C.I. Acid Blue 113) has been biodegraded effectively by bacterial culture mediated with azoreductase enzyme to reduce the pollution load in the present investigation. The maximum rate of dye degradation was found to be 96 ± 4 and 92 ± 4 % for the initial concentrations of 100 and 200 mg/l, respectively. The enzyme activity was measured using NADH as a substrate. Fourier transform infrared spectroscopy (FT-IR) analysis was confirmed that the transformation of azo linkage could be transformed into N2 or NH3 or incorporated into complete biomass. Breaking down of dye molecules to various metabolites (such as aniline, naphthalene-1,4-diamine, 3-aminobenzenesulfonic acid, naphthalene-1-sulfonic acid, 8-aminonaphthalene-1-sulfonic acid, 5,8-diaminonaphthalene-1-sulfonic acid) was confirmed by gas chromatography and mass spectra (GC-MS) and mass (electrospray ionization (ESI)) spectra analysis. The treated wastewater could be reused for dyeing operation in the leather processing, and the properties of produced leather were evaluated by conventional methods that revealed to have improved dye penetration into the grain layer of experimental leather sample and resulted in high levelness of dyeing, which helps to obtain the desired smoothness and soft leather properties.

  8. Development of a visual loop-mediated isothermal amplification method for rapid detection of the bacterial pathogen Pseudomonas putida of the large yellow croaker (Pseudosciaena crocea).

    PubMed

    Mao, Zhijuan; Qiu, Yangyu; Zheng, Lei; Chen, Jigang; Yang, Jifang

    2012-06-01

    In recent years, the large yellow croaker (Pseudosciaena crocea), an important marine fish farmed in the coastal areas of Zhejiang province, east China, has become severely endangered as a result of the bacterial pathogen Pseudomonas putida. This paper reports the development of a visual loop-mediated isothermal amplification (LAMP) assay for rapid detection of the pathogen. Four primers, F3, B3, FIP and BIP, were designed on the basis of DNA sequence of the rpoN gene of P. putida. After optimization of the reaction conditions, the detection limit of LAMP assay was 4.8cfu per reaction, 10-fold higher than that of conventional PCR. The assay showed high specificity to discriminate all P. putida isolates from nine other Gram-negative bacteria. The assay also successfully detected the pathogen DNA in the tissues of infected fish. For visual LAMP without cross-contamination, SYBR Green I was embedded in a microcrystalline wax capsule and preset in the reaction tubes; after the reaction the wax was melted at 85°C to release the dye and allow intercalation with the amplicons. The simple, highly sensitive, highly specific and cost-effective characteristics of visual LAMP may encourage its application in the rapid diagnosis of this pathogen.

  9. Bacterial cell surface hydrophobicity properties in the mediation of in vitro adhesion by the rabbit enteric pathogen Escherichia coli strain RDEC-1.

    PubMed Central

    Drumm, B; Neumann, A W; Policova, Z; Sherman, P M

    1989-01-01

    The role of hydrophobicity in the attachment of enteropathogens to gastrointestinal mucosa is controversial. In vitro binding of Escherichia coli RDEC-1 to rabbit intestine is dependent on the expression of pili. We examined in vitro adherence of piliated RDEC-1 after altering either the hydrophobicity of the organisms, the hydrophobicity of the substrate for attachment, or the surface tension of the suspending liquid. Hydrophobicity of RDEC-1 was determined using four complementary methods. In each assay piliated RDEC-1 demonstrated relatively more hydrophobic properties compared with both organisms grown to suppress pilus expression and a mutant that cannot express mannose-resistant pili. When piliated RDEC-1 were pretreated with tetramethyl urea to disrupt hydrophobic bonds surface hydrophobicity decreased. Concurrently, bacterial adherence to rabbit ileal microvillus membranes, mucus and mucin was reduced. Binding of piliated organisms to hydrophobic surfaces was significantly higher compared to both nonpiliated bacteria and the adherence of piliated RDEC-1 to relatively hydrophilic surfaces. Addition of propanol reduced the surface tension of the suspending liquid, and decreased adhesion of piliated RDEC-1 to polystyrene by 80%. Conversely, adherence of piliated organisms to a hydrophilic surface increased 12-fold after lowering the surface tension of the suspending liquid. We conclude that hydrophobic properties have a role in mediating in vitro adherence of this E. coli enteric pathogen. Images PMID:2572606

  10. Arabidopsis wat1 (walls are thin1)-mediated resistance to the bacterial vascular pathogen, Ralstonia solanacearum, is accompanied by cross-regulation of salicylic acid and tryptophan metabolism.

    PubMed

    Denancé, Nicolas; Ranocha, Philippe; Oria, Nicolas; Barlet, Xavier; Rivière, Marie-Pierre; Yadeta, Koste A; Hoffmann, Laurent; Perreau, François; Clément, Gilles; Maia-Grondard, Alessandra; van den Berg, Grardy C M; Savelli, Bruno; Fournier, Sylvie; Aubert, Yann; Pelletier, Sandra; Thomma, Bart P H J; Molina, Antonio; Jouanin, Lise; Marco, Yves; Goffner, Deborah

    2013-01-01

    Inactivation of Arabidopsis WAT1 (Walls Are Thin1), a gene required for secondary cell-wall deposition, conferred broad-spectrum resistance to vascular pathogens, including the bacteria Ralstonia solanacearum and Xanthomonas campestris pv. campestris, and the fungi Verticillium dahliae and Verticillium albo-atrum. Introduction of NahG, the bacterial salicylic acid (SA)-degrading salicylate hydroxylase gene, into the wat1 mutant restored full susceptibility to both R. solanacearum and X. campestris pv. campestris. Moreover, SA content was constitutively higher in wat1 roots, further supporting a role for SA in wat1-mediated resistance to vascular pathogens. By combining transcriptomic and metabolomic data, we demonstrated a general repression of indole metabolism in wat1-1 roots as shown by constitutive down-regulation of several genes encoding proteins of the indole glucosinolate biosynthetic pathway and reduced amounts of tryptophan (Trp), indole-3-acetic acid and neoglucobrassicin, the major form of indole glucosinolate in roots. Furthermore, the susceptibility of the wat1 mutant to R. solanacearum was partially restored when crossed with either the trp5 mutant, an over-accumulator of Trp, or Pro35S:AFB1-myc, in which indole-3-acetic acid signaling is constitutively activated. Our original hypothesis placed cell-wall modifications at the heart of the wat1 resistance phenotype. However, the results presented here suggest a mechanism involving root-localized metabolic channeling away from indole metabolites to SA as a central feature of wat1 resistance to R. solanacearum.

  11. Control of TMEM16A by INO-4995 and other inositolphosphates

    PubMed Central

    Tian, Yuemin; Schreiber, Rainer; Wanitchakool, Podchanart; Kongsuphol, Patthara; Sousa, Marisa; Uliyakina, Inna; Palma, Marta; Faria, Diana; Traynor-Kaplan, Alexis E; Fragata, José I; Amaral, Margarida D; Kunzelmann, Karl

    2013-01-01

    Background And Purpose Ca2+-dependent Cl− secretion (CaCC) in airways and other tissues is due to activation of the Cl− channel TMEM16A (anoctamin 1). Earlier studies suggested that Ca2+-activated Cl− channels are regulated by membrane lipid inositol phosphates, and that 1-O-octyl-2-O-butyryl-myo-inositol 3,4,5,6-tetrakisphosphate octakis(propionoxymethyl) ester (INO-4995) augments CaCC. Here we examined whether TMEM16A is the target for INO-4995 and if the channel is regulated by inositol phosphates. Experimental Approach The effects of INO-4995 on CaCC were examined in overexpressing HEK293, colonic and primary airway epithelial cells as well as Xenopus oocytes. We used patch clamping, double electrode voltage clamp and Ussing chamber techniques. Key Results We found that INO-4995 directly activates a TMEM16A whole cell conductance of 6.1 ± 0.9 nS pF–1 in overexpressing cells. The tetrakisphosphates Ins(3,4,5,6)P4 or Ins(1,3,4,5)P4 and enzymes controlling levels of InsP4 or PIP2 and PIP3 had no effects on the magnitude or kinetics of TMEM16A currents. In contrast in Xenopus oocytes, human airways and colonic cells, which all express TMEM16A endogenously, Cl− currents were not acutely activated by INO-4995. However incubation with INO-4995 augmented 1.6- to 4-fold TMEM16A-dependent Cl− currents activated by ionomycin or ATP, while intracellular Ca2+ signals were not affected. The potentiating effect of INO-4995 on transient ATP-activated TMEM16A-currents in cystic fibrosis (CF) airways was twice of that observed in non-CF airways. Conclusions And Implications These data indicate that TMEM16A is the target for INO-4995, although the mode of action appears different for overexpressed and endogenous channels. INO-4995 may be useful for the treatment of CF lung disease. PMID:22946960

  12. iNOS Activity Modulates Inflammation, Angiogenesis, and Tissue Fibrosis in Polyether-Polyurethane Synthetic Implants

    PubMed Central

    Cassini-Vieira, Puebla; Araújo, Fernanda Assis; da Costa Dias, Filipi Leles; Russo, Remo Castro; Andrade, Silvia Passos; Teixeira, Mauro Martins; Barcelos, Luciola Silva

    2015-01-01

    There is considerable interest in implantation techniques and scaffolds for tissue engineering and, for safety and biocompatibility reasons, inflammation, angiogenesis, and fibrosis need to be determined. The contribution of inducible nitric oxide synthase (iNOS) in the regulation of the foreign body reaction induced by subcutaneous implantation of a synthetic matrix was never investigated. Here, we examined the role of iNOS in angiogenesis, inflammation, and collagen deposition induced by polyether-polyurethane synthetic implants, using mice with targeted disruption of the iNOS gene (iNOS−/−) and wild-type (WT) mice. The hemoglobin content and number of vessels were decreased in the implants of iNOS−/− mice compared to WT mice 14 days after implantation. VEGF levels were also reduced in the implants of iNOS−/− mice. In contrast, the iNOS−/− implants exhibited an increased neutrophil and macrophage infiltration. However, no alterations were observed in levels of CXCL1 and CCL2, chemokines related to neutrophil and macrophage migration, respectively. Furthermore, the implants of iNOS−/− mice showed boosted collagen deposition. These data suggest that iNOS activity controls inflammation, angiogenesis, and fibrogenesis in polyether-polyurethane synthetic implants and that lack of iNOS expression increases foreign body reaction to implants in mice. PMID:26106257

  13. The Complex Role of iNOS in Acutely-Rejecting Cardiac Transplants

    PubMed Central

    Pieper, Galen M.; Roza, Allan M.

    2008-01-01

    This review summarizes the evidence for a detrimental role of nitric oxide (NO) derived from inducible NO synthase (iNOS) and/or reactive nitrogen species such as peroxynitrite in acutely-rejecting cardiac transplants. In chronic cardiac transplant rejection, iNOS may have an opposing beneficial component. The purpose of this review is primarily to address issues related to acute rejection which is a recognized risk factor for chronic rejection. The evidence for a detrimental role is based upon strategies involving non-selective NOS inhibitors, NO neutralizers, selective iNOS inhibitors and iNOS gene deletion in rodent models of cardiac rejection. The review is discussed in the context of the impact on various components including graft survival, histological rejection and cardiac function which may contribute in toto to the process of graft rejection. Possible limitations of each strategy are discussed in order to understand better the variance in published findings including issues related to the potential importance of cell localization of iNOS expression. Finally, the concept of a dual role of NO and its down-stream product, peroxynitrite, in rejection vs. immune regulation is discussed. PMID:18291116

  14. Anti-Inflammatory Effects of Flavonoids: Genistein, Kaempferol, Quercetin, and Daidzein Inhibit STAT-1 and NF-κB Activations, Whereas Flavone, Isorhamnetin, Naringenin, and Pelargonidin Inhibit only NF-κB Activation along with Their Inhibitory Effect on iNOS Expression and NO Production in Activated Macrophages

    PubMed Central

    Hämäläinen, Mari; Nieminen, Riina; Vuorela, Pia; Heinonen, Marina; Moilanen, Eeva

    2007-01-01

    In inflammation, bacterial products and proinflammatory cytokines induce the formation of large amounts of nitric oxide (NO) by inducible nitric oxide synthase (iNOS), and compounds that inhibit NO production have anti-inflammatory effects. In the present study, we systematically investigated the effects of 36 naturally occurring flavonoids and related compounds on NO production in macrophages exposed to an inflammatory stimulus (lipopolysaccharide, LPS), and evaluated the mechanisms of action of the effective compounds. Flavone, the isoflavones daidzein and genistein, the flavonols isorhamnetin, kaempferol and quercetin, the flavanone naringenin, and the anthocyanin pelargonidin inhibited iNOS protein and mRNA expression and also NO production in a dose-dependent manner. All eight active compounds inhibited the activation of nuclear factor-κB (NF-κB), which is a significant transcription factor for iNOS. Genistein, kaempferol, quercetin, and daidzein also inhibited the activation of the signal transducer and activator of transcription 1 (STAT-1), another important transcription factor for iNOS. The present study characterises the effects and mechanisms of naturally occurring phenolic compounds on iNOS expression and NO production in activated macrophages. The results partially explain the pharmacological efficacy of flavonoids as anti-inflammatory compounds. PMID:18274639

  15. Cytokine-mediated induction of cyclo-oxygenase-2 by activation of tyrosine kinase in bovine endothelial cells stimulated by bacterial lipopolysaccharide.

    PubMed Central

    Akarasereenont, P.; Bakhle, Y. S.; Thiemermann, C.; Vane, J. R.

    1995-01-01

    1. The induction of cyclo-oxygenase-2 (COX-2) afforded by bacterial lipopolysaccharide (LPS, endotoxin) in bovine aortic endothelial cells (BAEC) is mediated by tyrosine kinase. LPS also causes the generation of several cytokines including interleukin-1 beta (IL-1 beta), tumour necrosis factor-alpha (TNF-alpha), epidermal growth factor (EGF) and platelet-derived growth factor (PDGF). This study investigates whether endogenous IL-1 beta, TNF-alpha, EGF or PDGF contribute to the induction of COX-2 elicited by LPS in BAEC and if their action is due to activation of tyrosine kinase. Furthermore, we have studied the induction of COX-2 by exogenous cytokines. 2. Accumulation of 6-oxo-prostaglandin (PG) F1 alpha in cultures of BAEC was measured by radioimmunoassay at 24 h after addition of either LPS (1 microgram ml-1) alone or LPS together with a polyclonal antibody to one of the various cytokines. In experiments designed to measure 'COX activity', 6-oxo-PGF1 alpha generated by BAEC activated with recombinant human IL-1 beta, TNF-alpha, EGF or PDGF for 12 h was measured after incubation of washed cells with exogenous arachidonic acid (30 microM for 15 min). Western blot analysis determined the expression of COX-2 protein in BAEC. 3. The accumulation of 6-oxo-PGF1 alpha caused by LPS in BAEC was attenuated by co-incubation with one of the polyclonal antibodies, anti-IL-1 beta, anti-TNF-alpha, anti-EGF, anti-PDGF or with the IL-1 receptor antagonist, in a dose-dependent manner. Exogenous IL-1 beta, TNF-alpha or EGF also caused an increase in COX activity, while PDGF was ineffective.(ABSTRACT TRUNCATED AT 250 WORDS) Images Figure 4 PMID:7582449

  16. Agrobacterium-mediated transformation of Eucalyptus globulus using explants with shoot apex with introduction of bacterial choline oxidase gene to enhance salt tolerance.

    PubMed

    Matsunaga, Etsuko; Nanto, Kazuya; Oishi, Masatoshi; Ebinuma, Hiroyasu; Morishita, Yoshihiko; Sakurai, Nozomu; Suzuki, Hideyuki; Shibata, Daisuke; Shimada, Teruhisa

    2012-01-01

    Eucalyptus globulus is one of the most economically important plantation hardwoods for paper making. However, its low transformation frequency has prevented genetic engineering of this species with useful genes. We found the hypocotyl section with a shoot apex has the highest regeneration ability among another hypocotyl sections, and have developed an efficient Agrobacterium-mediated transformation method using these materials. We then introduced a salt tolerance gene, namely a bacterial choline oxidase gene (codA) with a GUS reporter gene, into E. globulus. The highest frequency of transgenic shoot regeneration from hypocotyls with shoot apex was 7.4% and the average frequency in four experiments was 4.0%, 12-fold higher than that from hypocotyls without shoot apex. Using about 10,000 explants, over 250 regenerated buds were confirmed as transformants by GUS analysis. Southern blot analysis of 100 elongated shoots confirmed successful generation of stable transformants. Accumulation of glycinebetaine was investigated in 44 selected transgenic lines, which showed 1- to 12-fold higher glycinebetaine levels than non-transgenic controls. Rooting of 16 transgenic lines was successful using a photoautotrophic method under enrichment with 1,000 ppm CO(2). The transgenic whole plantlets were transplanted into potting soil and grown normally in a growth room. They showed salt tolerance to 300 mM NaCl. The points of our system are using explants with shoot apex as materials, inhibiting the elongation of the apex on the selection medium, and regenerating transgenic buds from the side opposite to the apex. This approach may also solve transformation problems in other important plants.

  17. UVB exposure enhanced benzanthrone-induced inflammatory responses in SKH-1 mouse skin by activating the expression of COX-2 and iNOS through MAP kinases/NF-κB/AP-1 signalling pathways.

    PubMed

    Abbas, Sabiya; Alam, Shamshad; Pal, Anu; Kumar, Mahadeo; Singh, Dhirendra; Ansari, Kausar Mahmood

    2016-10-01

    This study was conducted to explore the role of UVB on benzanthrone (BA)-induced skin inflammation and its mechanism/s. SKH-1 hairless mice were topically exposed with BA (25 and 50 mg/kg b.wt) either alone or along with UVB (50 mJ/cm(2)) for 24 h and estimation of ROS, histopathological analysis, myeloperoxidase (MPO) activity, mast cell staining, immunohistochemistry for COX-2 and iNOS as well as western blotting for MAPKs, p-NF-κB, c-jun, c-fos COX-2 and iNOS were carried out. Enhanced ROS generation, increased epidermal thickness, mast cell number, MPO activity, enhanced expression of COX-2 and iNOS, MAPKs, c-jun, c-fos, NF-κB were found in BA either alone or when followed by UVB treatment, compared to the control groups. Expression of COX-2, iNOS and phosphorylation of ERK1/2 were found to be more enhanced in BA and UVB- exposed group compared to BA and UVB only group, while phosphorylation of JNK1/2, p38, NF-κB and expression of c-jun and c-fos were comparable with BA and UVB only groups. In summary, we suggest that UVB exposure enhanced BA-induced SKH-1 skin inflammation possibly via oxidative stress-mediated activation of MAPKs-NF-κB/AP-1 signalling, which subsequently increased the expression of COX-2 and iNOS and led to inflammation in SKH-1 mouse skin.

  18. Association of INOS, TRAIL, TGF-β2, TGF-β3, and IgL genes with response to Salmonella enteritidis in poultry

    PubMed Central

    2003-01-01

    Several candidate genes were selected, based on their critical roles in the host's response to intracellular bacteria, to study the genetic control of the chicken response to Salmonella enteritidis (SE). The candidate genes were: inducible nitric oxide synthase (INOS), tumor necrosis factor related apoptosis inducing ligand (TRAIL), transforming growth factor β2 (TGF-β2), transforming growth factor β3 (TGF-β3), and immunoglobulin G light chain (IgL). Responses to pathogenic SE colonization or to SE vaccination were measured in the Iowa Salmonella response resource population (ISRRP). Outbred broiler sires and three diverse, highly inbred dam lines produced 508 F1 progeny, which were evaluated as young chicks for either bacterial load isolated from spleen or cecum contents after pathogenic SE inoculation, or the circulating antibody level after SE vaccination. Fragments of each gene were sequenced from the founder lines of the resource population to identify genomic sequence variation. Single nucleotide polymorphisms (SNP) were identified, then PCR-RFLP techniques were developed to genotype the F1 resource population. Linear mixed models were used for statistical analyses. Because the inbred dam lines always contributed one copy of the same allele, the heterozygous sire allele effects could be assessed in the F1 generation. Association analyses revealed significant effects of the sire allele of TRAIL-StyI on the spleen (P < 0.07) and cecum (P < 0.0002) SE bacterial load. Significant effects (P < 0.04) were found on the cecum bacterial load for TGF-β3-BsrI. Varied and moderate association was found for SE vaccine antibody response for all genes. This is the first reported study on the association of SNP in INOS, TRAIL, TGF-β2, TGF-β3, and IgL with the chicken response to SE. Identification of candidate genes to improve the immune response may be useful for marker-assisted selection to enhance disease resistance. PMID:12927083

  19. Development of Antisense Therapeutic and Imaging Agents to Detect and Suppress Inducible Nitric Oxide Synthase (iNOS) Expression in Acute Lung Injury (ALI)

    NASA Astrophysics Data System (ADS)

    Shen, Yuefei

    This dissertation focuses on the development and investigation of antisense imaging and therapeutic agents, combined with nanotechnology, to detect and suppress inducible nitric oxide synthase (iNOS) expression for the diagnosis and treatment of acute lung injury (ALI). To achieve this goal, several efforts were made. The first effort was the identification and characterization of high binding affinity antisense peptide nucleic acids (PNAs) and shell-crosslinked knedel-like nanoparticle (SCK)-PNA conjugates to the iNOS mRNA. Antisense binding sites on the iNOS mRNA were first mapped by a procedure for rapidly generating a library of antisense accessible sites on native mRNAs (MASL) which involves reverse transcription of whole cell mRNA extracts with a random oligodeoxynucleotide primer followed by mRNA-specific PCR. Antisense PNAs against the antisense accessible sites were accordingly synthesized and characterized. The second effort was the investigation of cationic shell crosslinked knedel-like nanoparticle (cSCK)-mediated siRNA delivery to suppress iNOS expression for the treatment of ALI. siRNA with its unique gene-specific properties could serve as a promising therapeutic agent, however success in this area has been challenged by a lack of efficient biocompatible transfection agents. cSCK with its nanometer size and positive charge previously showed efficient cellular delivery of phosphorothioate ODNs (oligodeoxynucleotides), plasmid DNA and PNA. Herein, cSCK showed good siRNA binding and facilitated efficient siRNA transfection in HeLa, a mouse macrophage cell line and other human cell lines. cSCK led to greater silencing efficiency than Lipofectamine 2000 in HeLa cells as determined by the viability following transfection with cytotoxic and non-cytotoxic siRNAs, as well in 293T and HEK cells, and was comparable in BEAS-2B and MCF10a cells. The third effort was the preparation of an iNOS imaging probe through electrostatic complexation between a radiolabeled

  20. Glutathione S-transferase P1 suppresses iNOS protein stability in RAW264.7 macrophage-like cells after LPS stimulation.

    PubMed

    Cao, Xiang; Kong, Xiuqin; Zhou, Yi; Lan, Lei; Luo, Lan; Yin, Zhimin

    2015-01-01

    Glutathione S-transferase P1 (GSTP1) is a ubiquitous expressed protein which plays an important role in the detoxification and xenobiotics metabolism. Previous studies showed that GSTP1 was upregulated by the LPS stimulation in RAW264.7 macrophage-like cells and GSTP1 overexpression downregulated lipopolysaccharide (LPS) induced inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression. Here we show that GSTP1 physically associates with the oxygenase domain of iNOS by the G-site domain and decreases the protein level of iNOS dimer. Both overexpression and RNA interference (RNAi) experiments indicate that GSTP1 downregulates iNOS protein level and increases S-nitrosylation and ubiquitination of iNOS. The Y7F mutant type of GSTP1 physically associates with iNOS, but shows no effect on iNOS protein content, iNOS S-nitrosylation, and changes in iNOS from dimer to monomer, suggesting the importance of enzyme activity of GSTP1 in regulating iNOS S-nitrosylation and stability. GSTM1, another member of GSTs shows no significant effect on regulation of iNOS. In conclusion, our study reveals the novel role of GSTP1 in regulation of iNOS by affecting S-nitrosylation, dimerization, and stability, which provides a new insight for analyzing the regulation of iNOS and the anti-inflammatory effects of GSTP1. PMID:26361746

  1. A Polypodium leucotomos extract inhibits solar-simulated radiation-induced TNF-alpha and iNOS expression, transcriptional activation and apoptosis.

    PubMed

    Jańczyk, Agnieska; Garcia-Lopez, M Angeles; Fernandez-Peñas, Pablo; Alonso-Lebrero, Jose Luis; Benedicto, Ignacio; López-Cabrera, Manuel; Gonzalez, Salvador

    2007-10-01

    In this report, we have examined the molecular basis of the photoprotective effect of a hydrophilic extract of the fern Polypodium leucotomos (PL) in vitro, using a solar simulator as the source of UV radiation (SSR). We found that pretreatment of human keratinocytes with PL inhibited SSR-mediated increase of tumor necrosis factor (TNF)-alpha and also abrogated nitric oxide (NO) production. Consistent with this, PL blocked the induction of inducible nitric oxide synthase (iNOS) elicited by SSR. In addition, PL inhibited the SSR-mediated transcriptional activation of NF-kappaB and AP1. Finally, we demonstrated that pretreatment with PL exerted a cytoprotective effect against SSR-induced damage, resulting in increased cell survival. Together, these data postulate a multifactor mechanism of protection not exclusively reliant on the antioxidant capability of PL, and strengthen the basic knowledge on the photoprotective effect of this botanical agent.

  2. Prodigiosin inhibits gp91(phox) and iNOS expression to protect mice against the oxidative/nitrosative brain injury induced by hypoxia-ischemia.

    PubMed

    Chang, Chia-Che; Wang, Yea-Hwey; Chern, Chang-Ming; Liou, Kuo-Tong; Hou, Yu-Chang; Peng, Yu-Ta; Shen, Yuh-Chiang

    2011-11-15

    This study aimed to explore the mechanisms by which prodigiosin protects against hypoxia-induced oxidative/nitrosative brain injury induced by middle cerebral artery occlusion/reperfusion (MCAo/r) injury in mice. Hypoxia in vitro was modeled using oxygen-glucose deprivation (OGD) followed by reoxygenation of BV-2 microglial cells. Our results showed that treatment of mice that have undergone MCAo/r injury with prodigiosin (10 and 100μg/kg, i.v.) at 1h after hypoxia ameliorated MCAo/r-induced oxidative/nitrosative stress, brain infarction, and neurological deficits in the mice, and enhanced their survival rate. MCAo/r induced a remarkable production in the mouse brains of reactive oxygen species (ROS) and a significant increase in protein nitrosylation; this primarily resulted from enhanced expression of NADPH oxidase 2 (gp91(phox)), inducible nitric oxide synthase (iNOS), and the infiltration of CD11b leukocytes due to breakdown of blood-brain barrier (BBB) by activation of nuclear factor-kappa B (NF-κB). All these changes were significantly diminished by prodigiosin. In BV-2 cells, OGD induced ROS and nitric oxide production by up-regulating gp91(phox) and iNOS via activation of the NF-κB pathway, and these changes were suppressed by prodigiosin. In conclusion, our results indicate that prodigiosin reduces gp91(phox) and iNOS expression possibly by impairing NF-κB activation. This compromises the activation of microglial and/or inflammatory cells, which then, in turn, mediates prodigiosin's protective effect in the MCAo/r mice.

  3. Tumor necrosis factor-alpha and nerve growth factor synergistically induce iNOS in pheochromocytoma cells.

    PubMed

    Macdonald, N J; Taglialatela, G

    2000-11-01

    Inducible nitric oxide synthase (iNOS) has been reported in tangle-bearing neurons of patients with Alzheimer's disease (AD), and can be induced by tumor necrosis factor-alpha (TNFalpha). High CNS levels of TNFalpha are associated with neurodegenerative diseases such as AD, where neurons dependent on neurotrophins such as nerve growth factor (NGF) are particularly affected. In this study we determined the effect of TNFalpha on iNOS in NGF-responsive pheochromocytoma (PC12) cells. We found that while TNFalpha and NGF alone were unable to induce iNOS, their simultaneous addition resulted in iNOS induction and the release of nitric oxide. Our results suggest that synergistic iNOS induction by TNFalpha and NGF may occur in selective population of NGF-responsive neurons in the presence of elevated CNS levels of TNFalpha.

  4. Expression of the calcium-independent cytokine-inducible (iNOS) isoform of nitric oxide synthase in rat placenta.

    PubMed Central

    Casado, M; D-iaz-Guerra, M J; Rodrigo, J; Fernández, A P; Boscá, L; Martín-Sanz, P

    1997-01-01

    The presence of the calcium-independent cytokine-inducible nitric oxide synthase (iNOS) has been investigated in rat placenta from day 19 of gestation till delivery. iNOS has been detected at the mRNA, enzyme activity and protein levels in complete placenta. Immunocytochemical detection of iNOS was heterogeneously distributed in control placenta. Intraperitoneal injection of pregnant rats at 21 days of gestation with lipopolysaccharide (LPS) increased the iNOS immunoreactivity in the decidua basalis of the placenta, and, when the mRNA levels and enzyme activity were measured in total tissue, a moderate increase (approx. 160%) was observed. A constitutive nuclear factor kappaB activity was observed in placenta from both control and LPS-treated animals. These results indicate constitutive expression of iNOS in rat placenta. PMID:9164857

  5. I. Development of Metal-Mediated SPOT-Synthesis Methods for the Efficient Construction of Small-Molecule Macroarrays. II. Design and Synthesis of Novel Bacterial Biofilm Inhibitors

    NASA Astrophysics Data System (ADS)

    Frei, Reto

    I. The use of small molecule probes to explore biological phenomena has become a valuable tool in chemical biology. As a result, methods that permit the rapid synthesis and biological evaluation of such compounds are highly sought-after. The small molecule macroarray represents one such approach for the synthesis and identification of novel bioactive agents. Macroarrays are readily constructed via the SPOT-synthesis technique on planar cellulose membranes, yielding spatially addressed libraries of ˜10-1000 unique compounds. We sought to expand the arsenal of chemical reactions compatible with this solid-phase platform, and developed highly efficient SPOT-synthesis protocols for the Mizoroki-Heck, Suzuki-Miyaura, and copper-catalyzed azide-alkyne cycloaddition reaction. We demonstrated that these metal-mediated reactions can be implemented, either individually or sequentially, for the efficient construction of small molecules in high purity on rapid time scales. Utilizing these powerful C-C and C-N bond forming coupling reactions, we constructed a series of macroarrays based on novel stilbene, phenyl-naphthalene, and triazole scaliblds. Subsequent biological testing of the stilbene and phenyl-naphthalene libraries revealed several potent antagonists and agonists, respectively, of the quorum sensing (QS) receptor LuxR in Vibrio fischeri. II. Bacteria living within biofilms are notorious for their resistance to known antibiotic agents, and constitute a major human health threat. Methods to attenuate biofilm growth would have a significant impact on the management of bacterial infections. Despite intense research efforts, small molecules capable of either inhibiting or dispersing biolilms remain scarce. We utilized natural products with purported anti-biofilm or QS inhibitory activity as sources of structural insight to guide the synthesis of novel biofilm modulators with improved activities. These studies revealed 2-aminobenzimidazole derivatives as highly potent

  6. Synergy and antagonism in regulation of recombinant human INO80 chromatin remodeling complex

    PubMed Central

    Willhoft, Oliver; Bythell-Douglas, Rohan; McCormack, Elizabeth A.; Wigley, Dale B.

    2016-01-01

    We have purified a minimal core human Ino80 complex from recombinant protein expressed in insect cells. The complex comprises one subunit each of an N-terminally truncated Ino80, actin, Arp4, Arp5, Arp8, Ies2 and Ies6, together with a single heterohexamer of the Tip49a and Tip49b proteins. This core complex has nucleosome sliding activity that is similar to that of endogenous human and yeast Ino80 complexes and is also inhibited by inositol hexaphosphate (IP6). We show that IP6 is a non-competitive inhibitor that acts by blocking the stimulatory effect of nucleosomes on the ATPase activity. The IP6 binding site is located within the C-terminal region of the Ino80 subunit. We have also prepared complexes lacking combinations of Ies2 and Arp5/Ies6 subunits that reveal regulation imposed by each of them individually and synergistically that couples ATP hydrolysis to nucleosome sliding. This coupling between Ies2 and Arp5/Ies6 can be overcome in a bypass mutation of the Arp5 subunit that is active in the absence of Ies2. These studies reveal several underlying mechanisms for regulation of ATPase activity involving a complex interplay between these protein subunits and IP6 that in turn controls nucleosome sliding. PMID:27257055

  7. Structural analyses of the chromatin remodeling enzymes INO80-C and SWR-C

    PubMed Central

    Watanabe, Shinya; Tan, Dongyan; Lakshminarasimhan, Mahadevan; Washburn, Michael P.; Hong, Eun-Jin Erica; Walz, Thomas; Peterson, Craig L.

    2015-01-01

    INO80-C and SWR-C are conserved members of a subfamily of ATP-dependent chromatin remodeling enzymes that function in transcription and genome-maintenance pathways. A crucial role for these enzymes is to control chromosomal distribution of the H2A.Z histone variant. Here we use electron microscopy (EM) and two-dimensional (2D) class averaging to demonstrate that these remodeling enzymes have similar overall architectures. Each enzyme is characterized by a dynamic ‘tail’ domain and a compact ‘head’ that contains Rvb1/Rvb2 subunits organized as hexameric rings. EM class averages and mass spectrometry support the existence of single heterohexameric rings in both SWR-C and INO80-C. EM studies define the position of the Arp8/Arp4/Act1 module within INO80-C, and we find that this module enhances nucleosome binding affinity but is largely dispensable for remodeling activities. In contrast, the Ies6/Arp5 module is essential for INO80-C remodeling, and furthermore this module controls conformational changes that may couple nucleosome binding to remodeling. PMID:25964121

  8. Antioxidative effects of cinnamomi cortex: A potential role of iNOS and COX-II

    PubMed Central

    Chung, Jin-Won; Kim, Jeong-Jun; Kim, Sung-Jin

    2011-01-01

    Background: Cinnamomi cortex has wide varieties of pharmacological actions such as anti-inflammatory action, anti-platelet aggregation, and improving blood circulation. In this study, we tested to determine whether the Cinnamomi cortex extract has antioxidant activities. Materials and Methods: Antioxidative actions were explored by measuring free radical scavenging activity, NO levels, and reducing power. The mechanism of antioxidative action of Cinnamomi cortex was determined by measuring iNOS and COX-II expression in lipopolysaccharide (LPS) stimulated Raw cells. Results: Seventy percent methanolic extract of Cinnamomi cortex exerted significant 1,1-diphenyl--2--picrylhydrazyl (DPPH) free radicals and NO scavenging activities in a dose-dependent manner. More strikingly, the Cinnamomi cortex extract exerted dramatic reducing power activity (13-fold over control). Production of iNOS induced by LPS was significantly inhibited by the Cinnamomi cortex extract, suggesting that it inhibits NO production by suppressing iNOS expression. Additionally, COX-2 induced by LPS was dramatically inhibited by the Cinnamomi cortex extract. Conclusion: These results suggest that 70% methanolic extract of Cinnamomi cortex exerts significant antioxidant activity via inhibiting iNOS and COX-II induction. PMID:22262934

  9. Targeting NOX, INOS and COX-2 in inflammatory cells: chemoprevention using food phytochemicals.

    PubMed

    Murakami, Akira; Ohigashi, Hajime

    2007-12-01

    Biological, biochemical and physical stimuli activate inflammatory leukocytes, such as macrophages, resulting in induction and synthesis of proinflammatory proteins and enzymes, together with free radicals, as innate immune responses. On the other hand, chronic and dysregulated activation of some inducible enzymes, including NADPH oxidase (NOX), inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2, have been shown to play pivotal roles in the development of certain inflammatory diseases such as oncogenesis. While the use of synthetic agents, especially those targeting molecules, is an attractive and reasonable approach to prevent carcinogenesis, it should be noted that traditional herbs and spices also exist along with their active constituents, which have been demonstrated to disrupt inflammatory signal transduction pathways. In this mini-review, the molecular mechanisms of activation or induction of NOX, iNOS and COX-2, as well as some food phytochemicals with marked potential to regulate those key inflammatory molecules, are highlighted. For example, 1'-acetoxychavicol acetate, which occurs in the rhizomes of the subtropical Zingiberaceae plant, has been shown to attenuate NOX-derived superoxide generation in macrophages, as well as lipopolysaccharide-induced nitric oxide and prostaglandin E(2) production through the suppression of iNOS and COX-2 synthesis, respectively. Notably, this phytochemical has exhibited a wide range of cancer prevention activities in several rodent models of inflammation-associated carcinogenesis. Herein, the cancer preventive potentials of several food phytochemicals targeting the induction of NOX, iNOS and COX-2 are described.

  10. Role of iNOS in Bystander Signaling Between Macrophages and Lymphoma Cells

    SciTech Connect

    Ghosh, Somnath; Maurya, Dharmendra Kumar; Krishna, Malini

    2008-12-01

    Purpose: The present report describes the bystander effects of radiation between similar and dissimilar cells and the role of iNOS in such communication. Materials and Methods: EL-4 and RAW 264.7 cells were exposed to 5 Gy {gamma}-irradiation. The medium from irradiated cells was transferred to unirradiated cells. Results: Irradiated EL-4 cells as well as those cultured in the presence of medium from {gamma}-irradiated EL-4 cells showed an upregulation of NF-{kappa}B, iNOS, p53, and p21/waf1 genes. The directly irradiated and the bystander EL-4 cells showed an increase in DNA damage, apoptosis, and NO production. Bystander signaling was also found to exist between RAW 264.7 (macrophage) and EL-4 (lymphoma) cells. Unstimulated or irradiated RAW 264.7 cells did not induce bystander effect in unirradiated EL-4 cells, but LPS stimulated and irradiated RAW 264.7 cells induced an upregulation of NF-{kappa}B and iNOS genes and increased the DNA damage in bystander EL-4 cells. Treatment of EL-4 or RAW 264.7 cells with L-NAME significantly reduced the induction of gene expression and DNA damage in the bystander EL-4 cells, whereas treatment with cPTIO only partially reduced the induction of gene expression and DNA damage in the bystander EL-4 cells. Conclusions: It was concluded that active iNOS in the irradiated cells was essential for bystander response.

  11. Transcriptional regulation of the human iNOS gene by IL-1beta in endothelial cells.

    PubMed Central

    Kolyada, A. Y.; Madias, N. E.

    2001-01-01

    BACKGROUND: Vascular endothelium participates in the control of vascular tone and function via the release of nitric oxide (NO) by the endothelial-type NO synthase (eNOS). Inducible NO synthase (iNOS) expression in endothelial cells occurs in many clinical conditions following induction by lipopolysaccharide or cytokines and generates large quantities of NO that result in endothelial cell activation and dysfunction. No information exists on the transcriptional regulation of the human iNOS gene (or that of other species) in endothelial cells. MATERIALS AND METHODS: We examined the transcriptional regulation of the human iNOS gene by interleukin-1beta (IL-1beta) in rat pulmonary microvascular endothelial cells (PVEC) by transient cotransfections of different iNOS-promoter constructs and cDNA of different transcription factors and regulatory proteins. RESULTS: The -1034/+88 bp iNOS promoter was strongly induced by IL-1beta, the regulatory elements for such induction being localized downstream of -205 bp. Cotransfection experiments with NF-kappaB isoforms, IkappaB isoforms, and IKK mutants suggested that the NF-kappaB site at -115/-106 bp is important, but not sufficient, for induction of iNOS promoter and that the role of NF-kappaB is partially independent of its binding site. C/EBP sites within the -205/+88 bp region were shown to be responsible, along with NF-kappaB site, for induction of iNOS promoter by IL-1beta. Overexpression of C/EBPalpha, C/EBPdelta, and liver-enriched activator protein (LAP) activated the promoter, whereas overexpression of liver-enriched inhibitory protein (LIP) strongly suppressed it. C/EBPbeta (LAP and LIP isoforms) was constitutively present in PVEC and was induced (approximately 2-fold) by IL-1beta, whereas C/EBPdelta was not constitutively expressed but was strongly induced by IL-1beta. Both C/EBPbeta and C/EBPdelta participated in DNA-protein complex formation. CONCLUSION: Both NF-kappaB and C/EBP pathways are important for the

  12. Upregulated iNOS and oxidative damage to the cochlear stria vascularis due to noise stress.

    PubMed

    Shi, Xiaorui; Nuttall, Alfred L

    2003-03-28

    Our previous work has revealed increased nitric oxide (NO) production in the cochlear perilymph following noise stress. However, it is not clear if the increase of NO is related to iNOS and whether NO-related oxidative stress can cause vascular tissue damage. In this study, iNOS immunoreactivity, NO production, and reactive oxygen species (ROS) in the lateral wall were examined in normal mice and compared with similar animals exposed to 120 dBA broadband noise, 3 h/day, for 2 consecutive days. In the normal animals, iNOS expression was not observed in the vascular endothelium of the stria vascularis and only weak iNOS immunoactivity was detected in the marginal cells. However, expression of iNOS in the wall of the blood vessels of stria vascularis and marginal cells was observed after loud sound stress (LSS). Relatively low levels of NO production and low ROS activity were detected in the stria vascularis in the unstimulated condition. In contrast, NO production was increased and ROS activity was elevated in the stria vascularis after LSS. These changes were attenuated by the iNOS inhibitor, GW 274150. To explore whether noise induces apoptotic processes in the stria vascularis, we examined morphological changes in endothelial- and marginal-cells. In vitro, annexin-V phosphatidylserine (PS) (to label and detect early evidence of apoptosis) was combined with propidium iodide (PI) (to probe plasma membrane integrity). PI alone was used in fixed tissues to detect later stage apoptotic cells by morphology of the nuclei. Following LSS, PS was expressed on cell surfaces of endothelial cells of blood vessels and marginal cells of the stria vascularis. Later stage apoptosis, characterized by irregular nuclei and condensation of nuclei, was also observed in these cells. The data indicate that increased iNOS expression and production of both NO and ROS following noise stress may lead to marginal cell pathology, and the dysfunction of cochlear microcirculation by inducing

  13. Nitrosyl Iodide, Ino: Millimeter-Wave Spectroscopy Guided by AB Initio Quantum Chemical Computation

    NASA Astrophysics Data System (ADS)

    Bailleux, Stephane; Duflot, Denis; Aiba, Shohei; Ozeki, Hiroyuki

    2015-06-01

    In the series of the nitrosyl halides, XNO (where X = {F, Cl, Br, I}), the millimeter-wave spectrum of INO remains so far unknown. We report our investigation on the first high-resolution rotational spectroscopy of nitrosyl iodide, INO. One of the motivation for this work comes from the growing need in developing a more complete understanding of atmospheric chemistry, especially halogen and nitrogen oxides chemistry that adversely impacts ozone levels. In the family of the nitrogen oxyhalides such as nitrosyl (XNO), nitryl (XNO), nitrite (XONO), and nitrate (XON0_2) halides, those with X = {F, Cl, Br} have been well studied, both theoretically and experimentally. However, relatively little is known about the iodine-containing analogues, although they also are of potential importance in tropospheric chemistry. In 1991, the Fourier-transform IR spectroscopic detection of INO, INO_2 and IONO_2 in the gas phase has been reported The INO molecule was generated by in situ mixing continuously I_2 and NO in a 50-cm long reaction glass tube whose outlet was connected to the absorption cell using a teflon tube. At the time of writing this abstract, 68 μ_a-type transitions (K_a = 0-10), all weak, have been successfully assigned. The hyperfine structures due to both I and N nuclei will also be presented. S.B. and D.D. acknowledge support from the Laboratoire d'Excellence CaPPA (Chemical and Physical Properties of the Atmosphere) through contract ANR-10-LABX-005 of the Programme d'Investissement d'Avenir. I. Barnes, K. H. Becker and J. Starcke, J. Phys. Chem. 1991, 95, 9736-9740.

  14. Prodigiosin inhibits gp91{sup phox} and iNOS expression to protect mice against the oxidative/nitrosative brain injury induced by hypoxia-ischemia

    SciTech Connect

    Chang, Chia-Che; Wang, Yea-Hwey; Chern, Chang-Ming; Liou, Kuo-Tong; Hou, Yu-Chang; Peng, Yu-Ta; Shen, Yuh-Chiang

    2011-11-15

    This study aimed to explore the mechanisms by which prodigiosin protects against hypoxia-induced oxidative/nitrosative brain injury induced by middle cerebral artery occlusion/reperfusion (MCAo/r) injury in mice. Hypoxia in vitro was modeled using oxygen-glucose deprivation (OGD) followed by reoxygenation of BV-2 microglial cells. Our results showed that treatment of mice that have undergone MCAo/r injury with prodigiosin (10 and 100 {mu}g/kg, i.v.) at 1 h after hypoxia ameliorated MCAo/r-induced oxidative/nitrosative stress, brain infarction, and neurological deficits in the mice, and enhanced their survival rate. MCAo/r induced a remarkable production in the mouse brains of reactive oxygen species (ROS) and a significant increase in protein nitrosylation; this primarily resulted from enhanced expression of NADPH oxidase 2 (gp91{sup phox}), inducible nitric oxide synthase (iNOS), and the infiltration of CD11b leukocytes due to breakdown of blood-brain barrier (BBB) by activation of nuclear factor-kappa B (NF-{kappa}B). All these changes were significantly diminished by prodigiosin. In BV-2 cells, OGD induced ROS and nitric oxide production by up-regulating gp91{sup phox} and iNOS via activation of the NF-{kappa}B pathway, and these changes were suppressed by prodigiosin. In conclusion, our results indicate that prodigiosin reduces gp91{sup phox} and iNOS expression possibly by impairing NF-{kappa}B activation. This compromises the activation of microglial and/or inflammatory cells, which then, in turn, mediates prodigiosin's protective effect in the MCAo/r mice. -- Highlights: Black-Right-Pointing-Pointer Prodigiosin ameliorated brain infarction and deficits. Black-Right-Pointing-Pointer Prodigiosin protected against hypoxia/reperfusion-induced brain injury. Black-Right-Pointing-Pointer Prodigiosin diminished oxidative/nitrosativestress and leukocytes infiltration. Black-Right-Pointing-Pointer Prodigiosin reduced BBB breakdown. Black

  15. Arctigenin inhibits lipopolysaccharide-induced iNOS expression in RAW264.7 cells through suppressing JAK-STAT signal pathway.

    PubMed

    Kou, Xianjuan; Qi, Shimei; Dai, Wuxing; Luo, Lan; Yin, Zhimin

    2011-08-01

    Arctigenin has been demonstrated to have an anti-inflammatory function, but the precise mechanisms of its action remain to be fully defined. In the present study, we determined the effects of arctigenin on lipopolysaccharide (LPS)-induced production of proinflammatory mediators and the underlying mechanisms involved in RAW264.7 cells. Our results indicated that arctigenin exerted its anti-inflammatory effect by inhibiting ROS-dependent STAT signaling through its antioxidant activity. Arctigenin also significantly reduced the phosphorylation of STAT1 and STAT 3 as well as JAK2 in LPS-stimulated RAW264.7 cells. The inhibitions of STAT1 and STAT 3 by arctigenin prevented their translocation to the nucleus and consequently inhibited expression of iNOS, thereby suppressing the expression of inflammation-associated genes, such as IL-1β, IL-6 and MCP-1, whose promoters contain STAT-binding elements. However, COX-2 expression was slightly inhibited at higher drug concentrations (50 μM). Our data demonstrate that arctigenin inhibits iNOS expression via suppressing JAK-STAT signaling pathway in macrophages.

  16. Attenuation of smoke induced neuronal and physiological changes by bacoside rich extract in Wistar rats via down regulation of HO-1 and iNOS.

    PubMed

    Pandareesh, M D; Anand, T

    2014-01-01

    Bacopa monniera is well known herbal medicine for its neuropharmacological effects. It alleviates variety of disorders including neuronal and physiological changes. Crackers smoke is a potent risk factor that leads to free radical mediated oxidative stress in vivo. The aim of the current study is to evaluate the protective efficacy of B. monniera extract (BME) against crackers smoke induced neuronal and physiological changes via modulating inducible nitric oxide synthase (iNOS) and hemeoxygenase-1 (HO-1) expression in rats. Rats were exposed to smoke for 1h for a period of 3 weeks and consecutively treated with BME at three different dosages (i.e., 10, 20 and 40 mg/kg b.wt.). Our results elucidate that BME treatment ameliorates histopathalogical changes, reactive oxygen species levels, lipid peroxidation, acetylcholine esterase activity and brain neurotransmitter levels to normal. BME supplementation efficiently inhibited HO-1 expression and nitric oxide generation by down-regulating iNOS expression. Smoke induced depletion of antioxidant enzyme status, monoamine oxidase activity was also replenished by BME supplementation. Thus the present study indicates that BME ameliorates various impairments associated with neuronal and physiological changes in rats exposed to crackers smoke by its potent neuromodulatory, antioxidant and adaptogenic propensity.

  17. Protection of Tong-Sai-Mai Decoction against Apoptosis Induced by H2O2 in PC12 Cells: Mechanisms via Bcl-2-Mitochondria-ROS-INOS Pathway

    PubMed Central

    Lee, Maxwell Kim Kit; Lu, Yin; Di, Liu-qing; Xu, Hui-qin

    2014-01-01

    Tong-Sai-Mai decoction (TSM) is a Chinese materia medica polyherbal formulation that has been applied in treating brain ischemia for hundreds of years. Because it could repress the oxidative stress in in vivo studies, now we focus on the in vitro studies to investigate the mechanism by targeting the oxidative stress dependent signaling. The relation between the neurogenesis and the reactive oxygen species (ROS) production remains largely unexamined. PC12 cells are excitable cell types widely used as in vitro model for neuronal cells. Most marker genes that are related to neurotoxicity, apoptosis, and cell cycles are expressed at high levels in these cells. The aim of the present study is to explore the cytoprotection of TSM against hydrogen peroxide- (H2O2-) induced apoptosis and the molecular mechanisms underlying PC12 cells. Our findings revealed that TSM cotreatment with H2O2 restores the expression of bcl-2, inducible nitric oxide synthase (INOS), and mitochondria membrane potential. Meanwhile, it reduces intracellular [Ca2+] concentration, lactate dehydrogenase (LDH) release, and the expression of caspase-3 and bax. The results of the present study suggested that the cytoprotective effects of the TSM might be mediated, at least in part, by the bcl-2-mitochondria-ROS-INOS pathway. Due to its nontoxic characteristics, TSM could be further developed to treat the neurodegenerative diseases which are closely associated with the oxidative stress. PMID:25404948

  18. Chromatin Remodeling Factors Isw2 and Ino80 Regulate Checkpoint Activity and Chromatin Structure in S Phase

    PubMed Central

    Lee, Laura; Rodriguez, Jairo; Tsukiyama, Toshio

    2015-01-01

    When cells undergo replication stress, proper checkpoint activation and deactivation are critical for genomic stability and cell survival and therefore must be highly regulated. Although mechanisms of checkpoint activation are well studied, mechanisms of checkpoint deactivation are far less understood. Previously, we reported that chromatin remodeling factors Isw2 and Ino80 attenuate the S-phase checkpoint activity in Saccharomyces cerevisiae, especially during recovery from hydroxyurea. In this study, we found that Isw2 and Ino80 have a more pronounced role in attenuating checkpoint activity during late S phase in the presence of methyl methanesulfonate (MMS). We therefore screened for checkpoint factors required for Isw2 and Ino80 checkpoint attenuation in the presence of MMS. Here we demonstrate that Isw2 and Ino80 antagonize checkpoint activators and attenuate checkpoint activity in S phase in MMS either through a currently unknown pathway or through RPA. Unexpectedly, we found that Isw2 and Ino80 increase chromatin accessibility around replicating regions in the presence of MMS through a novel mechanism. Furthermore, through growth assays, we provide additional evidence that Isw2 and Ino80 partially counteract checkpoint activators specifically in the presence of MMS. Based on these results, we propose that Isw2 and Ino80 attenuate S-phase checkpoint activity through a novel mechanism. PMID:25701287

  19. An inherited immunoglobulin class-switch recombination deficiency associated with a defect in the INO80 chromatin remodeling complex

    PubMed Central

    Kracker, Sven; Di Virgilio, Michela; Schwartzentruber, Jeremy; Cuenin, Cyrille; Forveille, Monique; Deau, Marie-Céline; McBride, Kevin M.; Majewski, Jacek; Gazumyan, Anna; Seneviratne, Suranjith; Grimbacher, Bodo; Kutukculer, Necil; Herceg, Zdenko; Cavazzana, Marina; Jabado, Nada; Nussenzweig, Michel C.; Fischer, Alain; Durandy, Anne

    2015-01-01

    Background Immunoglobulin class-switch recombination defects (CSR-D) are rare primary immunodeficiencies characterized by impaired production of switched immunoglobulin isotypes and normal or elevated IgM levels. They are caused by impaired T:B cooperation or intrinsic B cell defects. However, many immunoglobulin CSR-Ds are still undefined at the molecular level. Objective This study's objective was to delineate new causes of immunoglobulin CSR-Ds and thus gain further insights into the process of immunoglobulin class-switch recombination (CSR). Methods Exome sequencing in 2 immunoglobulin CSR-D patients identified variations in the INO80 gene. Functional experiments were performed to assess the function of INO80 on immunoglobulin CSR. Results We identified recessive, nonsynonymous coding variations in the INO80 gene in 2 patients affected by defective immunoglobulin CSR. Expression of wild-type INO80 in patients' fibroblastic cells corrected their hypersensitivity to high doses of γ-irradiation. In murine CH12-F3 cells, the INO80 complex accumulates at Sα and Eμ regions of the IgH locus, and downregulation of INO80 as well as its partners Reptin and Pontin impaired CSR. In addition, Reptin and Pontin were shown to interact with activation-induced cytidine deaminase. Finally, an abnormal separation of sister chromatids was observed upon INO80 downregulation in CH12-F3 cells, pinpointing its role in cohesin activity. Conclusion INO80 deficiency appears to be associated with defective immunoglobulin CSR. We propose that the INO80 complex modulates cohesin function that may be required during immunoglobulin switch region synapsis. PMID:25312759

  20. Do calcium-mediated cellular signalling pathways, prostaglandin E2 (PGE2), estrogen or progesterone receptor antagonists, or bacterial endotoxins affect bovine placental function in vitro?

    PubMed

    Weems, Y S; Randel, R D; Carstens, G E; Welsh, T H; Weems, C W

    2004-04-01

    media treated with RU-486 increased (P < or = 0.05) at 4 and 8 h compared to vehicle controls and was not affected by other treatments (P > or = 0.05). Concentrations of PGE2 in media at 4 and 8 h were lower (P < or = 0.05) when compared to controls except treatment with PGE2 at 4 and 8h and RU-486 at 8h (P > or = 0.05). PGF2alpha was increased (P < or = 0.05) by RU-486 at 8h and no other treatment affected PGF2alpha at 4 or 8 h (P < or = 0.05). In conclusion, modulators of cellular calcium signalling pathways given alone do not affect bovine placental progesterone secretion at the days studied and progesterone receptor-mediated events appear to suppress placental progesterone, PGF2alpha, and PGE2 secretion in cattle. In addition, PGE2 does not appear to regulate bovine placental progesterone secretion when the corpus luteum is functional and bacterial endotoxin does not appear to affect bovine placental secretion of PGF2alpha or PGE2. PMID:15287156

  1. The effect of aspirin nanoemulsion on TNFα and iNOS in gastric tissue in comparison with conventional aspirin

    PubMed Central

    Mahmoud, Fatma Abd Elhalim; Hashem, Khalid S; Hussein Elkelawy, Asmaa Mohammed M

    2015-01-01

    aspirin, indicated by significant decreases in TNFα, iNOS, prostaglandin E2, and malondialdehyde levels, and also significant increases in glutathione, glutathione reductase, glutathione peroxidase, catalase, and superoxide dismutase. The biochemical results were confirmed by histopathological studies. Conclusion Aspirin nanoemulsion has less toxic effect on the gastric mucosa compared to ordinary aspirin. This can be indicated by the increase of the antioxidant activity and the decrease of the inflammatory mediators in the gastric tissue. PMID:26345150

  2. Orostachys japonicus Inhibits Expression of the TLR4, NOD2, iNOS, and COX-2 Genes in LPS-Stimulated Human PMA-Differentiated THP-1 Cells by Inhibiting NF-κB and MAPK Activation

    PubMed Central

    Woo, Hong-Jung; Kim, Youngchul

    2015-01-01

    Orostachys japonicus is traditionally used as an inflammatory agent. In this report, we investigated the effects of O. japonicus extract on the expression of genes encoding pathogen-recognition receptors (TLR2, TLR4, NOD1, and NOD2) and proinflammatory factors (iNOS, COX-2, and cytokines) in LPS-stimulated PMA-differentiated THP-1 cells and the NF-κB and MAPK pathways. O. japonicus induced toxicity at high concentrations but had no effect at concentrations lower than 25 μg/mL. O. japonicus inhibited LPS-induced TLR4 and NOD2 mRNA levels, suppressed LPS-induced iNOS and COX-2 transcription and translocation, and downregulated LPS-induced proinflammatory cytokine (IL-1β, IL-6, IL-8, and TNF-α) mRNA levels. In addition, O. japonicus inhibited LPS-induced NF-κB activation and IκBα degradation and suppressed LPS-induced JNK, p38 MAPK, and ERK phosphorylation. Overall, our results demonstrate that the anti-inflammatory effects of O. japonicus are mediated by suppression of NF-κB and MAPK signaling, resulting in reduced TLR4, NOD2, iNOS, and COX-2 expression and inhibition of inflammatory cytokine expression. PMID:25810745

  3. Development and commissioning of the HARDROC based readout for the INO-ICAL experiment

    NASA Astrophysics Data System (ADS)

    Kumar, Ashok; Gaur, Ankit; Phogat, Aman; Rafik, Md.; Naimuddin, Md.

    2016-10-01

    Glass based Resistive Plate Chambers (RPCs) are going to be used as an active element in the Iron Calorimeter (ICAL) experiment at the India based Neutrino Observatory (INO), which is being constructed to study atmospheric neutrinos. Though the RPC detector operational parameters are more or less finalized, the readout electronics is being developed using various technologies. The ICAL experiment will consist of about 29,000 RPC detectors of 2 m × 2 m in size with each detector having 64 readout channels both in the X and Y directions. The present study focusses on multi-channel electronics based on SiGe 350 nm technology as an option for the INO-ICAL RPC detectors. The study includes commissioning and usage of frontend application specific integrated circuit (ASIC) HARDROC chip in which 64 channels are handled independently to perform zero suppression. We present first testbench results using the HARDROC chip with the aim to use it finally in the ICAL experiment.

  4. Two G-protein-coupled-receptor candidates, Cand2 and Cand7, are involved in Arabidopsis root growth mediated by the bacterial quorum-sensing signals N-acyl-homoserine lactones.

    PubMed

    Jin, Guoping; Liu, Fang; Ma, Hong; Hao, Shaoyan; Zhao, Qian; Bian, Zirui; Jia, Zhenhua; Song, Shuishan

    2012-01-20

    Many Gram-negative bacteria use N-acyl-homoserine lactones (AHLs) as quorum sensing (QS) signaling molecules to coordinate their group behavior. Recently, it was shown that plants can perceive and respond to these bacterial AHLs. However, little is known about the molecular mechanism underlying the response of plants to bacterial QS signals. In this study, we show that the promotion of root elongation in wild type Arabidopsis thaliana induced by the AHLs N-3-oxo-hexanoyl-homoserine lactone (3OC6-HSL) or N-3-oxo-octanoyl-homoserine lactone (3OC8-HSL) was completely abolished in plants with loss-of-function mutations in two candidate G-protein Coupled Receptors (GPCRs), Cand2 and Cand7. Furthermore, real-time PCR analysis revealed that the expression levels of Cand2 and Cand7 were elevated in plants treated with 3OC6-HSL or 3OC8-HSL. These results suggest that Cand2 and Cand7 are involved in the regulation of root growth by bacterial AHLs and that GPCRs play a role in mediating interactions between plants and microbes.

  5. THE ROLE OF MIR-212 AND INOS IN ALCOHOL-INDUCED INTESTINAL BARRIER DYSFUNCTION AND STEATOHEPATITIS

    PubMed Central

    Tang, Yueming; Zhang, Lijuan; Forsyth, Christopher B.; Shaikh, Maliha; Song, Shiwen; Keshavarzian, Ali

    2015-01-01

    Background Alcoholic liver disease (ALD) is commonly associated with intestinal barrier dysfunction. Alcohol-induced dysregulation of intestinal tight junction (TJ) proteins, such as Zonula Occludens-1 (ZO-1), plays an important role in alcohol-induced gut leakiness. However, the mechanism of alcohol-induced disruption of TJ proteins is not well established. The goal of this study was to elucidate this mechanism by studying the role of MicroRNA 212 (miR-212) and inducible nitric oxide synthase (iNOS) in alcohol-induced gut leakiness. Methods The permeability of the Caco-2 monolayer was assessed by transepithelial electrical resistance (TER) and flux of fluorescein sulfonic acid (FSA). miR-212 was measured by real time PCR. The wild type, iNOS knockout, and miR-212 knockdown mice were fed with alcohol diet (29% of total calories, 4.5% v/v) for 8 weeks. The LNA-anti-miR-212 was used to inhibit miR-212 expression in mice. The alcohol-induced intestinal permeability, miR-212 expression and liver injuries in mice were measured. Results Our in vitro monolayer and in vivo mice studies showed that: (1) alcohol-induced over-expression of the intestinal miR-212 and intestinal hyperpermeability is prevented by using miR-212 knock-down techniques; and (2). iNOS is upregulated in the intestine by alcohol and that iNOS signaling is required for alcohol-induced miR-212 over-expression, ZO-1 disruption, gut leakiness and steatohepatis. Conclusions These studies thus support a novel miR-212 mechanism for alcohol-induced gut leakiness and a potential target that could be exploited for therapeutic intervention to prevent leaky gut and liver injury in alcoholics. PMID:26207424

  6. Bacterial Sialidase

    NASA Technical Reports Server (NTRS)

    2004-01-01

    Data shows that elevated sialidase in bacterial vaginosis patients correlates to premature births in women. Bacterial sialidase also plays a significant role in the unusual colonization of Pseudomonas aeruginosa in cystic fibrosis patients. Crystals of Salmonella sialidase have been reproduced and are used for studying the inhibitor-enzyme complexes. These inhibitors may also be used to inhibit a trans-sialidase of Trypanosome cruzi, a very similar enzyme to bacterial sialidase, therefore preventing T. cruzi infection, the causitive agent of Chagas' disease. The Center for Macromolecular Crystallography suggests that inhibitors of bacterial sialidases can be used as prophylactic drugs to prevent bacterial infections in these critical cases.

  7. Edaravone attenuates hippocampal damage in an infant mouse model of pneumococcal meningitis by reducing HMGB1 and iNOS expression via the Nrf2/HO-1 pathway

    PubMed Central

    Li, Zheng; Ma, Qian-qian; Yan, Yan; Xu, Feng-dan; Zhang, Xiao-ying; Zhou, Wei-qin; Feng, Zhi-chun

    2016-01-01

    ), nor it affected the protein levels of HMGB1 and iNOS in the hippocampus of the mice with mild meningitis. Conclusion: Edaravone produces neuroprotective actions in a mouse model of pneumococcal meningitis by reducing neuronal apoptosis and HMGB1 and iNOS expression in the hippocampus via the Nrf2/HO-1 pathway. Thus, edaravone may be a promising agent for the treatment of bacterial meningitis. PMID:27569388

  8. [The role of bacterial endotoxins, receptors and cytokines in the pathogenesis of septic (endotoxin) shock].

    PubMed

    Lazarov, S; Balutsov, M; Ianev, E

    2000-01-01

    Sepsis, resistant to therapy, results in the development of septic (endotoxin) shock. The latter is caused by the endotoxins of different Gram-negative bacteria. Endotoxin (bacterial lipopdisacharide--LPS) interacts with cells through specific membrane or plasma soluble endotoxin receptors (sCD14, mlD14, LBP, CD13/CD14, CD16, CD116/CD18, L-selectin, etc.). Endotoxin interaction with the mCD14 receptor of the monocytes, macrophages and the neutrophils results in the production of a number of proinflammatory cytokines--tumor necrosis factor alpha (TNF alpha), interleukines 1 and 6 (IL-1 and IL-6, etc), antiinflammatory cytokines--interleukines 10 and 12 (IL-10 and IL-12), cell adhesion molecules (P-selectin, E-selectin, ICAM-1, VCAM-1, etc.) and inducible enzymes: inducible NO synthase (iNOS), inducible phospholipase A2 (cPL-A2), inducible cyclooxygenase (COX-2). All pathologic processes in the structure and function of human body during endotoxin shock are a result of the disbalance of a number of mediators with a proinflammatory and antiinflammatory effects.

  9. Delineation and mutational analysis of the Yersinia pseudotuberculosis YopE domains which mediate translocation across bacterial and eukaryotic cellular membranes.

    PubMed Central

    Schesser, K; Frithz-Lindsten, E; Wolf-Watz, H

    1996-01-01

    Pathogenic yersiniae deliver a number of different effector molecules, which are referred to as Yops, into the cytosol of eukaryotic cells via a type III secretion system. To identify the regions of YopE from Yersinia pseudotuberculosis that are necessary for its translocation across the bacterial and eukaryotic cellular membranes, we constructed a series of hybrid genes which consisted of various amounts of yopE fused to the adenylate cyclase-encoding domain of the cyclolysin gene (cyaA) of Bordetella pertussis. By assaying intact cells for adenylate cyclase activity, we show that a YopE-Cya protein containing just the 11 amino-terminal residues of YopE is efficiently exported to the exterior surface of the bacterial cell. Single amino acid replacements of the first seven YopE residues significantly decreased the amount of reporter protein detected on the cell surface, suggesting that the extreme amino-terminal region of YopE is recognized by the secretion machinery. As has recently been shown for the Y. enterocolitica YopE protein (M.-P. Sory, A. Boland, I. Lambermont, and G. R. Cornelis, Proc. Natl. Acad. Sci. USA 92:11998-12002, 1995), we found that export to the cell surface was not sufficient for YopE-Cya proteins to be delivered into the eukaryotic cytoplasm. For traversing the HeLa cell membrane, at least 49 yopE-encoded residues were required. Replacement of leucine 43 of YopE with glycine severely affected the delivery of the reporter protein into HeLa cells. Surprisingly, export from the bacterial cell was also not sufficient for YopE-Cya proteins to be released from the bacterial cell surface into the culture supernatant. At least 75 residues of YopE were required to detect activity of the corresponding reporter protein in the culture supernatant, suggesting that a release domain exists in this region of YopE. We also show that the chaperone-like protein YerA required at least 75 YopE residues to form a stable complex in vitro with YopE-Cya proteins and

  10. Bacterial Proteasomes

    PubMed Central

    Jastrab, Jordan B.; Darwin, K. Heran

    2015-01-01

    Interest in bacterial proteasomes was sparked by the discovery that proteasomal degradation is required for the pathogenesis of Mycobacterium tuberculosis, one of the world's deadliest pathogens. Although bacterial proteasomes are structurally similar to their eukaryotic and archaeal homologs, there are key differences in their mechanisms of assembly, activation, and substrate targeting for degradation. In this article, we compare and contrast bacterial proteasomes with their archaeal and eukaryotic counterparts, and we discuss recent advances in our understanding of how bacterial proteasomes function to influence microbial physiology. PMID:26488274

  11. Intestinal expression of Fas and Fas ligand is upregulated by bacterial signaling through TLR4 and TLR5, with activation of Fas modulating intestinal TLR-mediated inflammation.

    PubMed

    Fernandes, Philana; O'Donnell, Charlotte; Lyons, Caitriona; Keane, Jonathan; Regan, Tim; O'Brien, Stephen; Fallon, Padraic; Brint, Elizabeth; Houston, Aileen

    2014-12-15

    TLRs play an important role in mediating intestinal inflammation and homeostasis. Fas is best studied in terms of its function in apoptosis, but recent studies demonstrate that Fas signaling may mediate additional functions such as inflammation. The role of Fas, and the Fas ligand (FasL), in the intestine is poorly understood. The aim of this study was to evaluate potential cross-talk between TLRs and Fas/FasL system in intestinal epithelial cells (IECs). IECs were stimulated with TLR ligands, and expression of Fas and FasL was investigated. Treatment with TLR4 and TLR5 ligands, but not TLR2 and 9 ligands, increased expression of Fas and FasL in IECs in vitro. Consistent with this finding, expression of intestinal Fas and FasL was reduced in vivo in the epithelium of TLR4 knockout (KO), 5KO, and germ-free mice, but not in TLR2KO mice. Modulating Fas signaling using agonistic anti-Fas augmented TLR4- and TLR5-mediated TNF-α and IL-8 production by IECs. In addition, suppression of Fas in IECs reduced the ability of TLR4 and TLR5 ligands and the intestinal pathogens Salmonella typhimurium and Listeria monocytogenes to induce the expression of IL-8. In conclusion, this study demonstrates that extensive cross-talk in IECs occurs between the Fas and TLR signaling pathways, with the FasL/Fas system playing a role in TLR-mediated inflammatory responses in the intestine.

  12. Evaluation of a loop-mediated isothermal amplification method for rapid detection of channel catfish Ictalurus punctatus important bacterial pathogen Edwardsiella ictaluri.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Channel catfish Ictalurus punctatus infected with Edwardsiella ictaluri results in $40 - 50 million annual losses in profits to catfish producers. Early detection of this pathogen is necessary for disease control and reduction of economic loss. In this communication, the loop-mediated isothermal a...

  13. Hypoxia regulates iNOS expression in human normal peritoneal and adhesion fibroblasts through NF-κB activation mechanism

    PubMed Central

    Jiang, Zhong L.; Fletcher, Nicole M.; Diamond, Michael P.; Abu-Soud, Husam M.; Saed, Ghassan M.

    2009-01-01

    Objective To determine the mechanism by which hypoxia increases expression of iNOS in human normal peritoneal and adhesion fibroblasts. Design Prospective experimental study. Setting University medical center. Patient(s) Primary cultures of fibroblasts from normal peritoneum and adhesion tissues. Intervention(s) Hypoxia treated cells. Main Outcome Measure(s) We utilized real-time RT-PCR to quantify mRNA levels of iNOS and NF-κB. Western blots were used to determine iNOS, NF-κB, IκB-α and phospho-IκB expression levels in normal peritoneal and adhesion fibroblasts in response to hypoxia. Result(s) Hypoxia resulted in a significant increase in iNOS and NF-κB expression in normal and adhesion fibroblasts. Furthermore, both cell types manifested lower levels of NF-κB, cytoplasmic phospho-IκB-α, and iNOS proteins. In contrast, they manifested higher levels of cytoplasmic IκB-α and IκB-α/NF-κB ratios as well as phosphorylated-IκB-α/NF-κB ratio. Under hypoxic conditions, both cell types exhibited significantly decreased cytoplasmic NF-κB, IκB-α levels, and significantly increased cytoplasmic phospho-IκB-α, iNOS, and NF-κB protein levels. Conclusions Hypoxia increases iNOS expression by a mechanism involving activation of NF-κB. The ratio of IκB-α/NF-κB or IκB-α/p-IκB-α can be used to monitor activation. PMID:18281043

  14. A Pilot Study of Bacterial Genes with Disrupted ORFs Reveals a Surprising Profusion of Protein Sequence Recoding Mediated by Ribosomal Frameshifting and Transcriptional Realignment

    PubMed Central

    Sharma, Virag; Firth, Andrew E.; Antonov, Ivan; Fayet, Olivier; Atkins, John F.; Borodovsky, Mark; Baranov, Pavel V.

    2011-01-01

    Bacterial genome annotations contain a number of coding sequences (CDSs) that, in spite of reading frame disruptions, encode a single continuous polypeptide. Such disruptions have different origins: sequencing errors, frameshift, or stop codon mutations, as well as instances of utilization of nontriplet decoding. We have extracted over 1,000 CDSs with annotated disruptions and found that about 75% of them can be clustered into 64 groups based on sequence similarity. Analysis of the clusters revealed deep phylogenetic conservation of open reading frame organization as well as the presence of conserved sequence patterns that indicate likely utilization of the nonstandard decoding mechanisms: programmed ribosomal frameshifting (PRF) and programmed transcriptional realignment (PTR). Further enrichment of these clusters with additional homologous nucleotide sequences revealed over 6,000 candidate genes utilizing PRF or PTR. Analysis of the patterns of conservation apparently associated with nontriplet decoding revealed the presence of both previously characterized frameshift-prone sequences and a few novel ones. Since the starting point of our analysis was a set of genes with already annotated disruptions, it is highly plausible that in this study, we have identified only a fraction of all bacterial genes that utilize PRF or PTR. In addition to the identification of a large number of recoded genes, a surprising observation is that nearly half of them are expressed via PTR—a mechanism that, in contrast to PRF, has not yet received substantial attention. PMID:21673094

  15. Temperature-mediated differences in bacterial kidney disease expression and survival in Renibacterium salmoninarum-challenged bull trout and other salmonids

    USGS Publications Warehouse

    Jones, D.T.; Moffitt, C.M.; Peters, K.K.

    2007-01-01

    Resource managers considering restoration and reconnection of watersheds to protect and enhance threatened populations of bull trout Salvelinus confluentus have little information about the consequences of bacterial kidney disease (BKD) caused by Renibacterium salmoninarum. To better understand the response of bull trout to R. salmoninarum challenge, we conducted several laboratory experiments at two water temperatures. The extent, severity, and lethality of BKD in bull trout were compared with those of similarly challenged lake trout S. namaycush, Arctic char S. alpinus, Chinook salmon Oncorhynchus tshawytscha, and rainbow trout O. mykiss. The lethal dose of bacterial cells necessary to induce 50% mortality (LD50) was 10-fold lower at the 15??C challenge than at the 9??C challenge. Of the species tested, bull trout were relatively resistant to BKD, Arctic char were the most susceptible among Salvelinus species, and Chinook salmon were the most susceptible among Oncorhynchus species tested. Mean time to death was more rapid for all fish tested at 15??C than for fish challenged at 9??C. These results suggest that infection of bull trout with BKD likely poses a low risk to successful restoration of threatened populations. ?? Copyright by the American Fisheries Society 2007.

  16. The human Ino80 binds to microtubule via the E-hook of tubulin: Implications for the role in spindle assembly

    SciTech Connect

    Park, Eun-Jung; Hur, Shin-Kyoung; Lee, Han-Sae; Lee, Shin-Ai; Kwon, Jongbum

    2011-12-16

    Highlights: Black-Right-Pointing-Pointer The N-terminal domain of hIno80 is important for binding to the spindle. Black-Right-Pointing-Pointer The hIno80 N-terminal domain binds to tubulin and microtubule in vitro. Black-Right-Pointing-Pointer The E-hook of tubulin is critical for hIno80 binding to tubulin and microtubule. Black-Right-Pointing-Pointer Tip49a does not bind to microtubule and dispensable for spindle formation. -- Abstract: The human INO80 chromatin remodeling complex, comprising the Ino80 ATPase (hIno80) and the associated proteins such as Tip49a, has been implicated in a variety of nuclear processes other than transcription. We previously have found that hIno80 interacts with tubulin and co-localizes with the mitotic spindle and is required for spindle formation. To better understand the role of hIno80 in spindle formation, we further investigated the interaction between hIno80 and microtubule. Here, we show that the N-terminal domain, dispensable for the nucleosome remodeling activity, is important for hIno80 to interact with tubulin and co-localize with the spindle. The hIno80 N-terminal domain binds to monomeric tubulin and polymerized microtubule in vitro, and the E-hook of tubulin, involved in the polymerization of microtubule, is critical for this binding. Tip49a, which has been reported to associate with the spindle, does not bind to microtubule in vitro and dispensable for spindle formation in vivo. These results suggest that hIno80 can play a direct role in the spindle assembly independent of its chromatin remodeling activity.

  17. Bioactive diterpenoids from Trigonostemon chinensis: Structures, NO inhibitory activities, and interactions with iNOS.

    PubMed

    Xu, Jing; Peng, Maoqin; Sun, Xiaocong; Liu, Xingyu; Tong, Ling; Su, Guochen; Ohizumi, Yasushi; Lee, Dongho; Guo, Yuanqiang

    2016-10-01

    A phytochemical investigation to obtain new NO inhibitors led to the isolation of two new (1 and 2) and four known (3-6) diterpenoids from Trigonostemon chinensis. Their structures were elucidated on the basis of extensive 1D and 2D NMR spectroscopic data analyses, and the absolute configurations of new compounds were established by experimental and calculated ECD spectra. The inhibitory activities on lipopolysaccharide-induced NO production in murine microglial BV-2 cells of these diterpenoids were evaluated, and all of the compounds showed inhibitory effects. The interactions of bioactive compounds with iNOS protein were also studied by molecular docking. PMID:27570243

  18. Performance of the prototype gas recirculation system with built-in RGA for INO RPC system

    NASA Astrophysics Data System (ADS)

    Bhuyan, M.; Datar, V. M.; Joshi, A.; Kalmani, S. D.; Mondal, N. K.; Rahman, M. A.; Satyanarayana, B.; Verma, P.

    2012-01-01

    An open loop gas recovery and recirculation system has been developed for the INO RPC system. The gas mixture coming from RPC exhaust is first desiccated by passing through molecular sieve (3 Å+4 Å). Subsequent scrubbing over basic active alumina removes toxic and acidic contaminants. The Isobutane and Freon are then separated by diffusion and liquefied by fractional condensation by cooling up to -26C. A Residual Gas Analyser (RGA) is being used in the loop to study the performance of the recirculation system. The results of the RGA analysis will be discussed.

  19. Melatonin Enhances the Anti-Tumor Effect of Fisetin by Inhibiting COX-2/iNOS and NF-κB/p300 Signaling Pathways

    PubMed Central

    Yu, Zhenlong; Xiao, Yao; Wang, Jingshu; Qiu, Huijuan; Yu, Wendan; Tang, Ranran; Yuan, Yuhui; Guo, Wei; Deng, Wuguo

    2014-01-01

    Melatonin is a hormone identified in plants and pineal glands of mammals and possesses diverse physiological functions. Fisetin is a bio-flavonoid widely found in plants and exerts antitumor activity in several types of human cancers. However, the combinational effect of melatonin and fisetin on antitumor activity, especially in melanoma treatment, remains unclear. Here, we tested the hypothesis that melatonin could enhance the antitumor activity of fisetin in melanoma cells and identified the underlying molecular mechanisms. The combinational treatment of melanoma cells with fisetin and melatonin significantly enhanced the inhibitions of cell viability, cell migration and clone formation, and the induction of apoptosis when compared with the treatment of fisetin alone. Moreover, such enhancement of antitumor effect by melatonin was found to be mediated through the modulation of the multiply signaling pathways in melanoma cells. The combinational treatment of fisetin with melatonin increased the cleavage of PARP proteins, triggered more release of cytochrome-c from the mitochondrial inter-membrane, enhanced the inhibition of COX-2 and iNOS expression, repressed the nuclear localization of p300 and NF-κB proteins, and abrogated the binding of NF-κB on COX-2 promoter. Thus, these results demonstrated that melatonin potentiated the anti-tumor effect of fisetin in melanoma cells by activating cytochrome-c-dependent apoptotic pathway and inhibiting COX-2/iNOS and NF-κB/p300 signaling pathways, and our study suggests the potential of such a combinational treatment of natural products in melanoma therapy. PMID:25000190

  20. Melatonin enhances the anti-tumor effect of fisetin by inhibiting COX-2/iNOS and NF-κB/p300 signaling pathways.

    PubMed

    Yi, Canhui; Zhang, Yong; Yu, Zhenlong; Xiao, Yao; Wang, Jingshu; Qiu, Huijuan; Yu, Wendan; Tang, Ranran; Yuan, Yuhui; Guo, Wei; Deng, Wuguo

    2014-01-01

    Melatonin is a hormone identified in plants and pineal glands of mammals and possesses diverse physiological functions. Fisetin is a bio-flavonoid widely found in plants and exerts antitumor activity in several types of human cancers. However, the combinational effect of melatonin and fisetin on antitumor activity, especially in melanoma treatment, remains unclear. Here, we tested the hypothesis that melatonin could enhance the antitumor activity of fisetin in melanoma cells and identified the underlying molecular mechanisms. The combinational treatment of melanoma cells with fisetin and melatonin significantly enhanced the inhibitions of cell viability, cell migration and clone formation, and the induction of apoptosis when compared with the treatment of fisetin alone. Moreover, such enhancement of antitumor effect by melatonin was found to be mediated through the modulation of the multiply signaling pathways in melanoma cells. The combinational treatment of fisetin with melatonin increased the cleavage of PARP proteins, triggered more release of cytochrome-c from the mitochondrial inter-membrane, enhanced the inhibition of COX-2 and iNOS expression, repressed the nuclear localization of p300 and NF-κB proteins, and abrogated the binding of NF-κB on COX-2 promoter. Thus, these results demonstrated that melatonin potentiated the anti-tumor effect of fisetin in melanoma cells by activating cytochrome-c-dependent apoptotic pathway and inhibiting COX-2/iNOS and NF-κB/p300 signaling pathways, and our study suggests the potential of such a combinational treatment of natural products in melanoma therapy.

  1. The SPRY domain–containing SOCS box protein SPSB2 targets iNOS for proteasomal degradation

    PubMed Central

    Kuang, Zhihe; Lewis, Rowena S.; Curtis, Joan M.; Zhan, Yifan; Saunders, Bernadette M.; Babon, Jeffrey J.; Kolesnik, Tatiana B.; Low, Andrew; Masters, Seth L.; Willson, Tracy A.; Kedzierski, Lukasz; Yao, Shenggen; Handman, Emanuela

    2010-01-01

    Inducible nitric oxide (NO) synthase (iNOS; NOS2) produces NO and related reactive nitrogen species, which are critical effectors of the innate host response and are required for the intracellular killing of pathogens such as Mycobacterium tuberculosis and Leishmania major. We have identified SPRY domain–containing SOCS (suppressor of cytokine signaling) box protein 2 (SPSB2) as a novel negative regulator that recruits an E3 ubiquitin ligase complex to polyubiquitinate iNOS, resulting in its proteasomal degradation. SPSB2 interacts with the N-terminal region of iNOS via a binding interface on SPSB2 that has been mapped by nuclear magnetic resonance spectroscopy and mutational analyses. SPSB2-deficient macrophages showed prolonged iNOS expression, resulting in a corresponding increase in NO production and enhanced killing of L. major parasites. These results lay the foundation for the development of small molecule inhibitors that could disrupt the SPSB–iNOS interaction and thus prolong the intracellular lifetime of iNOS, which may be beneficial in chronic and persistent infections. PMID:20603330

  2. Purification and characterisation of aquamarine blue pigment from the shells of abalone (Haliotis discus hannai Ino).

    PubMed

    Cai, Zhixing; Wu, Jiulin; Chen, Li; Guo, Wei; Li, Jianhua; Wang, Jiabin; Zhang, Qiqing

    2011-09-01

    Aquamarine blue pigment (ABP) from the shells of abalone (Haliotis discus hannai Ino) was extracted using a precipitation adsorption method and further purified via semi-preparative HPLC. The ABP with molecular weight of 582.8 was identified as a polyenic compound by NMR. The colour value of ABP was E1cm(1%)612nm=534.3. ABP can dissolve in water, ethanol, methanol, acetic acid and DMSO but was scarcely soluble in chloroform, aether, acetone, petroleum ether and cyclohexane. ABP was relatively stable between 25 and 100°C, from pH 2 to pH 12, under UV-light and indoor natural light. However, it was bleached by H2O2 and Na2SO3 and even unstable under sunlight. The stability of ABP was slightly influenced by metal ions (Ca(2+), Cu(2+), Fe(2+) and so on) and food addictives (sodium chloride, sugar, starch and so on). This is the first report on the characterisation of pigment obtained from Haliotis discus hannai Ino.

  3. Low fucose containing bacterial polysaccharide facilitate mitochondria-dependent ROS-induced apoptosis of human lung epithelial carcinoma via controlled regulation of MAPKs-mediated Nrf2/Keap1 homeostasis signaling.

    PubMed

    Chowdhury, Sougata Roy; Sengupta, Suman; Biswas, Subir; Sen, Ramkrishna; Sinha, Tridib Kumar; Basak, Ratan Kumar; Adhikari, Basudam; Bhattacharyya, Arindam

    2015-12-01

    Reactive oxygen species (ROS), the key mediators of cellular oxidative stress and redox dysregulation involved in cancer initiation and progression, have recently emerged as promising targets for anticancer drug discovery. Continuous free radical assault upsets homeostasis in cellular redox system and regulates the associated signaling pathways to mediate stress-induced cell death. This study investigates the dose-specific pro-oxidative behavior of a bacterial fucose polysaccharide, which attenuated proliferation of different cancer cells. In the fermentation process, Bacillus megaterium RB-05 [GenBank Accession Number HM371417] was found to biosynthesize a polysaccharide with low-fucose content (4.9%), which conferred the maximum anti-proliferative activity (750 µg/mL) against human lung cancer epithelial cells (A549) during preliminary screening. Structural elucidation and morphological characterization of the duly purified polysaccharide was done using HPLC, GC-MS, (1)H/(13)C NMR, and microscopy. The polysaccharide exhibited concentration- and time-dependent anti-proliferative effects against A549 cells by inducing intracellular ROS level and regulating the mitochondrial membrane-permeability following the apoptotic pathway. This process encompasses activation of caspase-8/9/3/7, increase in the ratio of Bax/Bcl2 ratio, translocation of Bcl2-associated X protein (Bax) and cytochrome c, decrease in expression of anti-apoptotic members of Bcl2 family, and phosphorylation of mitogen activated protein kinases (MAPKs). Apoptosis was attenuated upon pretreatment with specific caspase-inhibitors. Simultaneously, during apoptosis, the ROS-mediated stress as well as activated MAPKs triggered nuclear translocation of transcription factors like nuclear factor (erythroid-derived)-like 2 (Nrf2) and promoted further transcription of downstream cytoprotective genes, which somehow perturbed the chemotherapeutic efficacy of the polysaccharide, although using CuPP, a chemical

  4. Supernatural supersymmetry: Phenomenological implications of anomaly-mediated supersymmetry breaking

    NASA Astrophysics Data System (ADS)

    Feng, Jonathan L.; Moroi, Takeo

    2000-05-01

    We discuss the phenomenology of supersymmetric models in which supersymmetry breaking terms are induced by the super-Weyl anomaly. Such a scenario is envisioned to arise when supersymmetry breaking takes place in another world, i.e., on another brane. We review the anomaly-mediated framework and study in detail the minimal anomaly-mediated model parametrized by only 3+1 parameters: Maux, m0, tan β, and sgn(μ). The renormalization group equations exhibit a novel ``focus point'' (as opposed to fixed point) behavior, which allows squark and slepton masses far above their usual naturalness bounds. We present the superparticle spectrum and highlight several implications for high energy colliders. Three lightest supersymmetric particle (LSP) candidates exist: the W-ino, the stau, and the tau sneutrino. For the W-ino LSP scenario, light W-ino triplets with the smallest possible mass splittings are preferred; such W-inos are within reach of run II Fermilab Tevatron searches. Finally, we study a variety of sensitive low energy probes, including b-->sγ, the anomalous magnetic moment of the muon, and the electric dipole moments of the electron and neutron.

  5. Supernatural supersymmetry: Phenomenological implications of anomaly-mediated supersymmetry breaking

    SciTech Connect

    Feng, Jonathan L.; Moroi, Takeo

    2000-05-01

    We discuss the phenomenology of supersymmetric models in which supersymmetry breaking terms are induced by the super-Weyl anomaly. Such a scenario is envisioned to arise when supersymmetry breaking takes place in another world, i.e., on another brane. We review the anomaly-mediated framework and study in detail the minimal anomaly-mediated model parametrized by only 3+1 parameters: M{sub aux}, m{sub 0}, tan {beta}, and sgn({mu}). The renormalization group equations exhibit a novel ''focus point'' (as opposed to fixed point) behavior, which allows squark and slepton masses far above their usual naturalness bounds. We present the superparticle spectrum and highlight several implications for high energy colliders. Three lightest supersymmetric particle (LSP) candidates exist: the W-ino, the stau, and the tau sneutrino. For the W-ino LSP scenario, light W-ino triplets with the smallest possible mass splittings are preferred; such W-inos are within reach of run II Fermilab Tevatron searches. Finally, we study a variety of sensitive low energy probes, including b{yields}s{gamma}, the anomalous magnetic moment of the muon, and the electric dipole moments of the electron and neutron. (c) 2000 The American Physical Society.

  6. Bacterial Keratitis

    MedlinePlus

    ... very quickly, and if left untreated, can cause blindness. The bacteria usually responsible for this type of ... to intense ultraviolet radiation exposure, e.g. snow blindness or welder's arc eye). Next Bacterial Keratitis Symptoms ...

  7. Bioactive products of arginine in sepsis: tissue and plasma composition after LPS and iNOS blockade.

    PubMed

    Lortie, M J; Ishizuka, S; Schwartz, D; Blantz, R C

    2000-06-01

    Blockade or gene deletion of inducible nitric oxide synthase (iNOS) fails to fully abrogate all the sequelae leading to the high morbidity of septicemia. An increase in substrate uptake may be necessary for the increased production of nitric oxide (NO), but arginine is also a precursor for other bioactive products. Herein, we demonstrate an increase in alternate arginine products via arginine and ornithine decarboxylase in rats given lipopolysaccharide (LPS). The expression of iNOS mRNA in renal tissue was evident 60 but not 30 min post-LPS, yet a rapid decrease in blood pressure was obtained within 30 min that was completely inhibited by selective iNOS blockade. Plasma levels of arginine and ornithine decreased by at least 30% within 60 min of LPS administration, an effect not inhibited by the iNOS blocker L-N(6)(1-iminoethyl)lysine (L-NIL). Significant increases in plasma nitrates and citrulline occurred only 3-4 h post-LPS, an effect blocked by L-NIL pretreatment. The intracellular composition of organs harvested 6 h post-LPS reflected tissue-specific profiles of arginine and related metabolites. Tissue arginine concentration, normally an order of magnitude higher than in plasma, did not decrease after LPS. Pretreatment with L-NIL had a significant impact on the disposition of tissue arginine that was organ specific. These data demonstrate changes in arginine metabolism before and after de novo iNOS activity. Selective blockade of iNOS did not prevent uptake and can deregulate the production of other bioactive arginine metabolites.

  8. Interferon-gamma increases hPepT1-mediated uptake of di-tripeptides including the bacterial tripeptide fMLP in polarized intestinal epithelia.

    PubMed

    Buyse, Marion; Charrier, Laetitia; Sitaraman, Shanthi; Gewirtz, Andrew; Merlin, Didier

    2003-11-01

    Interferon-gamma causes a global phenotypic switch in intestinal epithelial function, in which enterocytes become immune accessory cells. The phenotypic switch is characterized by a down-regulation of membrane transporters and up-regulation of immune accessory molecules in intestinal epithelial cells. However, the effect of interferon-gamma on the intestinal epithelia di-tripeptide hPepT1 transporter has not been investigated. In this study we demonstrate that 1) interferon-gamma increases di-tripeptide uptake in dose- and time-dependent manner in model intestinal epithelia (Caco-2 BBE cell monolayers), 2) the increase in di-tripeptides induced by interferon-gamma is hPepT1 mediated, 3) interferon-gamma does not affect the hPept1 expression at the mRNA and protein levels 4) interferon-gamma increases the intracellular pH and consequently enhances the H+-electrochemical gradient across apical plasma membrane in model intestinal epithelia (Caco2-BBE monolayers). We suggest that interferon-gamma could increase the hPepT1 mediated di-tripeptides uptake in inflamed epithelial cells. Under these conditions, interferon-gamma will increase the intracellular amount of such diverse prokaryotic and eucaryotic small di-tripeptides in inflamed epithelial cells. The intracellular accumulation of such di-tripeptides may be important in enterocytes becoming immune accessory cells.

  9. Quorum-sensing agr mediates bacterial oxidation response via an intramolecular disulfide redox switch in the response regulator AgrA

    PubMed Central

    Sun, Fei; Liang, Haihua; Kong, Xiangqian; Xie, Sherrie; Cho, Hoonsik; Deng, Xin; Ji, Quanjiang; Zhang, Haiyan; Alvarez, Sophie; Hicks, Leslie M.; Bae, Taeok; Luo, Cheng; Jiang, Hualiang; He, Chuan

    2012-01-01

    Oxidation sensing and quorum sensing significantly affect bacterial physiology and host–pathogen interactions. However, little attention has been paid to the cross-talk between these two seemingly orthogonal signaling pathways. Here we show that the quorum-sensing agr system has a built-in oxidation-sensing mechanism through an intramolecular disulfide switch possessed by the DNA-binding domain of the response regulator AgrA. Biochemical and mass spectrometric analysis revealed that oxidation induces the intracellular disulfide bond formation between Cys-199 and Cys-228, thus leading to dissociation of AgrA from DNA. Molecular dynamics (MD) simulations suggest that the disulfide bond formation generates a steric clash responsible for the abolished DNA binding of the oxidized AgrA. Mutagenesis studies further established that Cys-199 is crucial for oxidation sensing. The oxidation-sensing role of Cys-199 is further supported by the observation that the mutant Staphylococcus aureus strain expressing AgrAC199S is more susceptible to H2O2 owing to repression of the antioxidant bsaA gene under oxidative stress. Together, our results show that oxidation sensing is a component of the quorum-sensing agr signaling system, which serves as an intrinsic checkpoint to ameliorate the oxidation burden caused by intense metabolic activity and potential host immune response. PMID:22586129

  10. Spatiotemporally Resolved Tracking of Bacterial Responses to ROS-Mediated Damage at the Single-Cell Level with Quantitative Functional Microscopy.

    PubMed

    Barroso, Álvaro; Grüner, Malte; Forbes, Taylor; Denz, Cornelia; Strassert, Cristian A

    2016-06-22

    Herein we report on the implementation of photofunctional microparticles in combination with optical tweezers for the investigation of bacterial responses to oxidative stress by means of quantitative functional microscopy. A combination of a strongly hydrophobic axially substituted Si(IV) phthalocyanine adsorbed onto silica microparticles was developed, and the structural and photophysical characterization was carried out. The microparticles are able to produce reactive oxygen species under the fluorescence microscope upon irradiation with red light, and the behavior of individual bacteria can be consequently investigated in situ and in real time at the single cell level. For this purpose, a methodology was introduced to monitor phototriggered changes with spatiotemporal resolution. The defined distance between the photoactive particles and individual bacteria can be fixed under the microscope before the photosensitization process is started, and the photoinduced damage can be monitored by tracing the time-dependent fluorescence turn-on of a suitable marker. The results showed a distance-dependent photoinduced death time, defined as the onset of the incorporation of propidium iodide. Our methodology constitutes a new tool for the in vitro design and evaluation of photosensitizers for the treatment of cancer and infectious diseases with the aid of functional optical microscopy, as it enables a quantitative response evaluation of living systems toward oxidative stress. More generally, it provides a way to understand the response of an ensemble of living entities to reactive oxygen species by analyzing the behavior of a set of individual organisms. PMID:27227509

  11. Leptospira Interrogans Induces Fibrosis in the Mouse Kidney through Inos-Dependent, TLR- and NLR-Independent Signaling Pathways

    PubMed Central

    Fanton d'Andon, Martine; Quellard, Nathalie; Fernandez, Béatrice; Ratet, Gwenn; Lacroix-Lamandé, Sonia; Vandewalle, Alain; Boneca, Ivo G.; Goujon, Jean-Michel; Werts, Catherine

    2014-01-01

    Background Leptospira (L.) interrogans are bacteria responsible for a worldwide reemerging zoonosis. Rodents carry L. interrogans asymptomatically in their kidneys and excrete bacteria in the urine, contaminating the environment. Humans get infected through skin contact and develop a mild or severe leptospirosis that may lead to renal failure and fibrosis. L. interrogans provoke an interstitial nephritis, but the induction of fibrosis caused by L. interrogans has not been studied in murine models. Innate immune receptors from the TLR and NLR families have recently been shown to play a role in the development and progression of tissue fibrosis in the lung, liver and kidneys under different pathophysiological situations. We recently showed that TLR2, TLR4, and NLRP3 receptors were crucial in the defense against leptospirosis. Moreover, infection of a human cell line with L. interrogans was shown to induce TLR2-dependent production of fibronectin, a component of the extracellular matrix. Therefore, we thought to assess the presence of renal fibrosis in L. interrogans infected mice and to analyze the contribution of some innate immune pathways in this process. Methodology/principal findings Here, we characterized by immunohistochemical studies and quantitative real-time PCR, a model of Leptospira-infected C57BL/6J mice, with chronic carriage of L. interrogans inducing mild renal fibrosis. Using various strains of transgenic mice, we determined that the renal infiltrates of T cells and, unexpectedly, TLR and NLR receptors, are not required to generate Leptospira-induced renal fibrosis. We also show that the iNOS enzyme, known to play a role in Leptospira-induced interstitial nephritis, also plays a role in the induction of renal fibrosis. Conclusion/significance To our knowledge, this work provides the first experimental murine model of sustained renal fibrosis induced by a chronic bacterial infection that may be peculiar, since it does not rely on TLR or NLR receptors

  12. Bacterial Fucose-Rich Polysaccharide Stabilizes MAPK-Mediated Nrf2/Keap1 Signaling by Directly Scavenging Reactive Oxygen Species during Hydrogen Peroxide-Induced Apoptosis of Human Lung Fibroblast Cells

    PubMed Central

    Roy Chowdhury, Sougata; Sinha, Tridib Kumar; Sen, Ramkrishna; Basak, Ratan Kumar; Adhikari, Basudam; Bhattacharyya, Arindam

    2014-01-01

    Continuous free radical assault upsets cellular homeostasis and dysregulates associated signaling pathways to promote stress-induced cell death. In spite of the continuous development and implementation of effective therapeutic strategies, limitations in treatments for stress-induced toxicities remain. The purpose of the present study was to determine the potential therapeutic efficacy of bacterial fucose polysaccharides against hydrogen peroxide (H2O2)-induced stress in human lung fibroblast (WI38) cells and to understand the associated molecular mechanisms. In two different fermentation processes, Bacillus megaterium RB-05 biosynthesized two non-identical fucose polysaccharides; of these, the polysaccharide having a high-fucose content (∼42%) conferred the maximum free radical scavenging efficiency in vitro. Structural characterizations of the purified polysaccharides were performed using HPLC, GC-MS, and 1H/13C/2D-COSY NMR. H2O2 (300 µM) insult to WI38 cells showed anti-proliferative effects by inducing intracellular reactive oxygen species (ROS) and by disrupting mitochondrial membrane permeability, followed by apoptosis. The polysaccharide (250 µg/mL) attenuated the cell death process by directly scavenging intracellular ROS rather than activating endogenous antioxidant enzymes. This process encompasses inhibition of caspase-9/3/7, a decrease in the ratio of Bax/Bcl2, relocalization of translocated Bax and cytochrome c, upregulation of anti-apoptotic members of the Bcl2 family and a decrease in the phosphorylation of MAPKs (mitogen activated protein kinases). Furthermore, cellular homeostasis was re-established via stabilization of MAPK-mediated Nrf2/Keap1 signaling and transcription of downstream cytoprotective genes. This molecular study uniquely introduces a fucose-rich bacterial polysaccharide as a potential inhibitor of H2O2-induced stress and toxicities. PMID:25412177

  13. Bacterial fucose-rich polysaccharide stabilizes MAPK-mediated Nrf2/Keap1 signaling by directly scavenging reactive oxygen species during hydrogen peroxide-induced apoptosis of human lung fibroblast cells.

    PubMed

    Roy Chowdhury, Sougata; Sengupta, Suman; Biswas, Subir; Sinha, Tridib Kumar; Sen, Ramkrishna; Basak, Ratan Kumar; Adhikari, Basudam; Bhattacharyya, Arindam

    2014-01-01

    Continuous free radical assault upsets cellular homeostasis and dysregulates associated signaling pathways to promote stress-induced cell death. In spite of the continuous development and implementation of effective therapeutic strategies, limitations in treatments for stress-induced toxicities remain. The purpose of the present study was to determine the potential therapeutic efficacy of bacterial fucose polysaccharides against hydrogen peroxide (H2O2)-induced stress in human lung fibroblast (WI38) cells and to understand the associated molecular mechanisms. In two different fermentation processes, Bacillus megaterium RB-05 biosynthesized two non-identical fucose polysaccharides; of these, the polysaccharide having a high-fucose content (∼ 42%) conferred the maximum free radical scavenging efficiency in vitro. Structural characterizations of the purified polysaccharides were performed using HPLC, GC-MS, and (1)H/(13)C/2D-COSY NMR. H2O2 (300 µM) insult to WI38 cells showed anti-proliferative effects by inducing intracellular reactive oxygen species (ROS) and by disrupting mitochondrial membrane permeability, followed by apoptosis. The polysaccharide (250 µg/mL) attenuated the cell death process by directly scavenging intracellular ROS rather than activating endogenous antioxidant enzymes. This process encompasses inhibition of caspase-9/3/7, a decrease in the ratio of Bax/Bcl2, relocalization of translocated Bax and cytochrome c, upregulation of anti-apoptotic members of the Bcl2 family and a decrease in the phosphorylation of MAPKs (mitogen activated protein kinases). Furthermore, cellular homeostasis was re-established via stabilization of MAPK-mediated Nrf2/Keap1 signaling and transcription of downstream cytoprotective genes. This molecular study uniquely introduces a fucose-rich bacterial polysaccharide as a potential inhibitor of H2O2-induced stress and toxicities.

  14. Central nervous action of interleukin-1 mediates activation of limbic structures and behavioural depression in response to peripheral administration of bacterial lipopolysaccharide.

    PubMed

    Konsman, J P; Veeneman, J; Combe, C; Poole, S; Luheshi, G N; Dantzer, R

    2008-12-01

    Although receptors for the pro-inflammatory cytokine interleukin-1 have long been known to be expressed in the brain, their role in fever and behavioural depression observed during the acute phase response (APR) to tissue infection remains unclear. This may in part be due to the fact that interleukin-1 in the brain is bioactive only several hours after peripheral administration of bacterial lipopolysaccharide (LPS). To study the role of cerebral interleukin-1 action in temperature and behavioural changes, and activation of brain structures during the APR, interleukin-1 receptor antagonist (IL-1ra; 100 microg) was infused into the lateral brain ventricle 4 h after intraperitoneal (i.p.) LPS injection (250 microg/kg) in rats. I.p. LPS administration induced interleukin-1beta (IL-1beta) production in systemic circulation as well as in brain circumventricular organs and the choroid plexus. Intracerebroventricular (i.c.v.) infusion of IL-1ra 4 h after i.p. LPS injection attenuated the reduction in social interaction, a cardinal sign of behavioural depression during sickness, and c-Fos expression in the amygdala and bed nucleus of the stria terminalis. However, LPS-induced fever, rises in plasma corticosterone, body weight loss and c-Fos expression in the hypothalamus and caudal brainstem were not altered by i.c.v. infusion of IL-1ra. These findings, together with our previous observations showing that i.c.v. infused IL-1ra diffuses throughout perivascular spaces, where macrophages express interleukin-1 receptors, can be interpreted to suggest that circulating or locally produced brain IL-1beta acts on these cells to bring about behavioural depression and activation of limbic structures during the APR after peripheral LPS administration.

  15. Central nervous action of interleukin-1 mediates activation of limbic structures and behavioural depression in response to peripheral administration of bacterial lipopolysaccharide.

    PubMed

    Konsman, J P; Veeneman, J; Combe, C; Poole, S; Luheshi, G N; Dantzer, R

    2008-12-01

    Although receptors for the pro-inflammatory cytokine interleukin-1 have long been known to be expressed in the brain, their role in fever and behavioural depression observed during the acute phase response (APR) to tissue infection remains unclear. This may in part be due to the fact that interleukin-1 in the brain is bioactive only several hours after peripheral administration of bacterial lipopolysaccharide (LPS). To study the role of cerebral interleukin-1 action in temperature and behavioural changes, and activation of brain structures during the APR, interleukin-1 receptor antagonist (IL-1ra; 100 microg) was infused into the lateral brain ventricle 4 h after intraperitoneal (i.p.) LPS injection (250 microg/kg) in rats. I.p. LPS administration induced interleukin-1beta (IL-1beta) production in systemic circulation as well as in brain circumventricular organs and the choroid plexus. Intracerebroventricular (i.c.v.) infusion of IL-1ra 4 h after i.p. LPS injection attenuated the reduction in social interaction, a cardinal sign of behavioural depression during sickness, and c-Fos expression in the amygdala and bed nucleus of the stria terminalis. However, LPS-induced fever, rises in plasma corticosterone, body weight loss and c-Fos expression in the hypothalamus and caudal brainstem were not altered by i.c.v. infusion of IL-1ra. These findings, together with our previous observations showing that i.c.v. infused IL-1ra diffuses throughout perivascular spaces, where macrophages express interleukin-1 receptors, can be interpreted to suggest that circulating or locally produced brain IL-1beta acts on these cells to bring about behavioural depression and activation of limbic structures during the APR after peripheral LPS administration. PMID:19087175

  16. Bacterial rheotaxis

    PubMed Central

    Marcos; Fu, Henry C.; Powers, Thomas R.; Stocker, Roman

    2012-01-01

    The motility of organisms is often directed in response to environmental stimuli. Rheotaxis is the directed movement resulting from fluid velocity gradients, long studied in fish, aquatic invertebrates, and spermatozoa. Using carefully controlled microfluidic flows, we show that rheotaxis also occurs in bacteria. Excellent quantitative agreement between experiments with Bacillus subtilis and a mathematical model reveals that bacterial rheotaxis is a purely physical phenomenon, in contrast to fish rheotaxis but in the same way as sperm rheotaxis. This previously unrecognized bacterial taxis results from a subtle interplay between velocity gradients and the helical shape of flagella, which together generate a torque that alters a bacterium's swimming direction. Because this torque is independent of the presence of a nearby surface, bacterial rheotaxis is not limited to the immediate neighborhood of liquid–solid interfaces, but also takes place in the bulk fluid. We predict that rheotaxis occurs in a wide range of bacterial habitats, from the natural environment to the human body, and can interfere with chemotaxis, suggesting that the fitness benefit conferred by bacterial motility may be sharply reduced in some hydrodynamic conditions. PMID:22411815

  17. Bacterial Cell Wall Components

    NASA Astrophysics Data System (ADS)

    Ginsberg, Cynthia; Brown, Stephanie; Walker, Suzanne

    Bacterial cell-surface polysaccharides cells are surrounded by a variety of cell-surface structures that allow them to thrive in extreme environments. Components of the cell envelope and extracellular matrix are responsible for providing the cells with structural support, mediating intercellular communication, allowing the cells to move or to adhere to surfaces, protecting the cells from attack by antibiotics or the immune system, and facilitating the uptake of nutrients. Some of the most important cell wall components are polysaccharide structures. This review discusses the occurrence, structure, function, and biosynthesis of the most prevalent bacterial cell surface polysaccharides: peptidoglycan, lipopolysaccharide, arabinogalactan, and lipoarabinomannan, and capsular and extracellular polysaccharides. The roles of these polysaccharides in medicine, both as drug targets and as therapeutic agents, are also described.

  18. Bacterial chemoreceptors and chemoeffectors.

    PubMed

    Bi, Shuangyu; Lai, Luhua

    2015-02-01

    Bacteria use chemotaxis signaling pathways to sense environmental changes. Escherichia coli chemotaxis system represents an ideal model that illustrates fundamental principles of biological signaling processes. Chemoreceptors are crucial signaling proteins that mediate taxis toward a wide range of chemoeffectors. Recently, in deep study of the biochemical and structural features of chemoreceptors, the organization of higher-order clusters in native cells, and the signal transduction mechanisms related to the on-off signal output provides us with general insights to understand how chemotaxis performs high sensitivity, precise adaptation, signal amplification, and wide dynamic range. Along with the increasing knowledge, bacterial chemoreceptors can be engineered to sense novel chemoeffectors, which has extensive applications in therapeutics and industry. Here we mainly review recent advances in the E. coli chemotaxis system involving structure and organization of chemoreceptors, discovery, design, and characterization of chemoeffectors, and signal recognition and transduction mechanisms. Possible strategies for changing the specificity of bacterial chemoreceptors to sense novel chemoeffectors are also discussed.

  19. Bacterial Genome Instability

    PubMed Central

    Darmon, Elise

    2014-01-01

    SUMMARY Bacterial genomes are remarkably stable from one generation to the next but are plastic on an evolutionary time scale, substantially shaped by horizontal gene transfer, genome rearrangement, and the activities of mobile DNA elements. This implies the existence of a delicate balance between the maintenance of genome stability and the tolerance of genome instability. In this review, we describe the specialized genetic elements and the endogenous processes that contribute to genome instability. We then discuss the consequences of genome instability at the physiological level, where cells have harnessed instability to mediate phase and antigenic variation, and at the evolutionary level, where horizontal gene transfer has played an important role. Indeed, this ability to share DNA sequences has played a major part in the evolution of life on Earth. The evolutionary plasticity of bacterial genomes, coupled with the vast numbers of bacteria on the planet, substantially limits our ability to control disease. PMID:24600039

  20. Luteolin and luteolin-7-O-glucoside from dandelion flower suppress iNOS and COX-2 in RAW264.7 cells.

    PubMed

    Hu, Chun; Kitts, David D

    2004-10-01

    Both reactive oxygen- and nitrogen-derived reactive species play important roles in physiological and pathophysiological conditions. Flavones, luteolin and luteolin-7-O-glucoside along with a rich plant source of both flavones, namely dandelion (Taraxacum officinale) flower extract were studied for antioxidant activity in different in vitro model systems. In this current study, luteolin and luteolin-7-O-glucoside at concentrations lower than 20 microM, significantly (p < 0.05) suppressed the productions of nitric oxide and prostaglandin E2 (PGE2) in bacterial lipopolysaccharide activated-mouse macrophage RAW264.7 cells without introducing cytotoxicity. The inhibitory effects were further attributed to the suppression of both inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein expression, and not reduced enzymatic activity. Similar suppression for both inducible enzymes was also found with the presence of dandelion flower extract, specifically, the ethyl acetate fraction of dandelion flower extract which contained 10% luteolin and luteolin-7-O-glucoside. PMID:15543940

  1. New players in the same old game: a system level in silico study to predict type III secretion system and effector proteins in bacterial genomes reveals common themes in T3SS mediated pathogenesis

    PubMed Central

    2013-01-01

    Background Type III secretion system (T3SS) plays an important role in virulence or symbiosis of many pathogenic or symbiotic bacteria [CHM 2:291–294, 2007; Physiology (Bethesda) 20:326–339, 2005]. T3SS acts like a tunnel between a bacterium and its host through which the bacterium injects ‘effector’ proteins into the latter [Nature 444:567–573, 2006; COSB 18:258–266, 2008]. The effectors spatially and temporally modify the host signalling pathways [FEMS Microbiol Rev 35:1100–1125, 2011; Cell Host Microbe5:571–579, 2009]. In spite its crucial role in host-pathogen interaction, the study of T3SS and the associated effectors has been limited to a few bacteria [Cell Microbiol 13:1858–1869, 2011; Nat Rev Microbiol 6:11–16, 2008; Mol Microbiol 80:1420–1438, 2011]. Before one set out to perform systematic experimental studies on an unknown set of bacteria it would be beneficial to identify the potential candidates by developing an in silico screening algorithm. A system level study would also be advantageous over traditional laboratory methods to extract an overriding theme for host-pathogen interaction, if any, from the vast resources of data generated by sequencing multiple bacterial genomes. Results We have developed an in silico protocol in which the most conserved set of T3SS proteins was used as the query against the entire bacterial database with increasingly stringent search parameters. It enabled us to identify several uncharacterized T3SS positive bacteria. We adopted a similar strategy to predict the presence of the already known effectors in the newly identified T3SS positive bacteria. The huge resources of biochemical data [FEMS Microbiol Rev 35:1100–1125, 2011; Cell Host Microbe 5:571–579, 2009; BMC Bioinformatics 7(11):S4, 2010] on the T3SS effectors enabled us to search for the common theme in T3SS mediated pathogenesis. We identified few cellular signalling networks in the host, which are manipulated by most of the T3SS

  2. Negative regulation of inducible nitric-oxide synthase expression mediated through transforming growth factor-beta-dependent modulation of transcription factor TCF11.

    PubMed

    Berg, David T; Gupta, Akanksha; Richardson, Mark A; O'Brien, Lee A; Calnek, David; Grinnell, Brian W

    2007-12-21

    Inducible nitric-oxide synthase (iNOS) plays a central role in the regulation of vascular function and response to injury. A central mediator controlling iNOS expression is transforming growth factor-beta (TGF-beta), which represses its expression through a mechanism that is poorly understood. We have identified a binding site in the iNOS promoter that interacts with the nuclear heterodimer TCF11/MafG using chromatin immunoprecipitation and mutation analyses. We demonstrate that binding at this site acts to repress the induction of iNOS gene expression by cytokines. We show that this repressor is induced by TGF-beta1 and by Smad6-short, which enhances TGF-beta signaling. In contrast, the up-regulation of TCF11/MafG binding could be suppressed by overexpression of the TGF-beta inhibitor Smad7, and a small interfering RNA to TCF11 blocked the suppression of iNOS by TGF-beta. The binding of TCF11/MafG to the iNOS promoter could be enhanced by phorbol 12-myristate 13-acetate and suppressed by the protein kinase C inhibitor staurosporine. Moreover, the induction of TCF11/MafG binding by TGF-beta and Smad6-short could be blocked by staurosporine, and the effect of TGF-beta was blocked by the selective protein kinase C inhibitor calphostin C. Consistent with the in vitro data, we found suppression of TCF11 coincident with iNOS up-regulation in a rat model of endotoxemia, and we observed a highly significant negative correlation between TCF11 and nitric oxide production. Furthermore, treatment with activated protein C, a serine protease effective in septic shock, blocked the down-regulation of TCF11 and suppressed endotoxin-induced iNOS. Overall, our results demonstrate a novel mechanism by which iNOS expression is regulated in the context of inflammatory activation.

  3. Development of 2 m×2 m size glass RPCs for INO

    NASA Astrophysics Data System (ADS)

    Bhuyan, M.; Datar, V. M.; Kalmani, S. D.; Lahamge, S. M.; Mohammed, S.; Mondal, N. K.; Nagaraj, P.; Redij, A.; Samuel, D.; Saraf, M. N.; Satyanarayana, B.; Shinde, R. R.; Verma, P.

    2012-01-01

    The India-based Neutrino Observatory (INO) collaboration is planning to build a massive 50 kt magnetised iron calorimeter (ICAL) detector to study atmospheric neutrinos and to precisely determine the neutrino oscillation parameters. About 30,000 2 m×2 m size glass Resistive Plate Chambers (RPCs) will be used as active detector elements in ICAL. Starting with a small 30 cm ×30 cm size RPCs, we have now succeeded in developing full size 2 m ×2 m RPCs. The fabrication procedures used for these large size RPCs as well as their performance will be discussed in this paper. We will also present some preliminary results from our studies on SF6 based gas mixtures on the RPC operation and characteristics.

  4. Isolation, identification, and antioxidant activity of polysaccharides from the shell of abalone (Haliotis discus hannai Ino).

    PubMed

    Wang, Z L; Liang, H B; Guo, W; Peng, Z F; Chen, J D; Zhang, Q Q

    2014-07-04

    In this study, two antioxidative substances, a homogeneous polysaccharide [abalone shell polysaccharide (ASP-1), corresponding to the first peak by size exclusion chromatography] and a non-polysaccharide compound [abalone shell compound (ACS-2), corresponding to the second peak by size exclusion chromatography], were extracted from the abalone (Haliotis discus hannai Ino) shell. We primarily focused on the investigation of ASP-1. As a heteropolysaccharide, ASP-1 is comprised of 9.3% uronic acid and 86.4% saccharide, the latter including mannose, ribose, rhamnose, glucose, galactose, arabinose, and two unknown monosaccharides, NO1 and NO2, with a mass ratio of 9.5:10.1:2.2:18.2:21.8:5.5:16.5:16.2. The antioxidant activity assays indicated that 5.0 mg/mL ASP-1 has significant scavenging effects on superoxide radicals (86.2%) compared to the positive control of ascorbic acid (95.6%).

  5. Effects of dietary menadione on the activity of antioxidant enzymes in abalone, Haliotis discus hannai Ino

    NASA Astrophysics Data System (ADS)

    Fu, Jinghua; Xu, Wei; Mai, Kangsen; Zhang, Wenbing; Feng, Xiuni; Liufu, Zhiguo

    2012-01-01

    A 240-day growth experiment in a re-circulating water system was conducted to investigate the effects of dietary menadione on the growth and antioxidant responses of abalone Haliotis discus hannai Ino. Triplicate groups of juvenile abalone (initial weight: 1.19 ± 0.01 g; shell length: 19.23 ± 0.01 mm) were fed to satiation with 3 semi-purified diets containing 0, 10, and 1 000 mg menadione sodium bisulfite (MSB)/kg, respectively. Results show that there were no significant differences in the rate of weight gain or in the daily increment in shell length of abalone among different treatments. Activities of superoxide dismutase (SOD), glutathione peroxidase (GPX), glutathione S-transferase (GST) and glutathione reductase (GR) in viscera were significantly decreased with dietary menadione. However, activities of these enzymes except for GPX in muscle were increased. Therefore, antioxidant responses of abalone were increased in muscle and decreased in viscera by dietary menadione.

  6. Enteropathogenic Escherichia coli outer membrane proteins induce iNOS by activation of NF-kappaB and MAP kinases.

    PubMed

    Malladi, Vasantha; Puthenedam, Manjula; Williams, Peter H; Balakrishnan, Arun

    2004-12-01

    Enteropathogenic Escherichia coli (EPEC) infects the human intestinal epithelium and is a major cause of infantile diarrhea in developing countries. Nitric oxide (NO) is an important modulator of intestinal inflammatory response. The aim of the present study was to investigate whether EPEC outer membrane proteins (OMPs) up regulate epithelial cell expression of inducible nitric oxide synthase (iNOS) and to examine the role of NF-kappaB and MAP kinases (MAPK) on nitrite production. iNOS mRNA expression was assessed by RT-PCR. Nitrite levels were measured by Griess reaction. NF-kappaB activation by OMPs was evaluated by EMSA and immunoblotting was done to detect MAPK activation. EPEC OMP up regulated iNOS, induced nitrite production and NF-kappaB and MAPK were activated in caco-2 cells. The nitrite levels decreased when NF-kappaB and MAPK inhibitors were used. Thus, EPEC OMPs induce iNOS expression and NO production through activation of NF-kappaB and MAPK.

  7. Luteolin, a bioflavonoid inhibits azoxymethane-induced colon carcinogenesis: Involvement of iNOS and COX-2

    PubMed Central

    Pandurangan, Ashok Kumar; Kumar, Suresh Ananda Sadagopan; Dharmalingam, Prakash; Ganapasam, Sudhandiran

    2014-01-01

    Colon cancer (CRC) is a serious health problem through worldwide. Development of novel drug without side effect for this cancer was crucial. Luteolin (LUT), a bioflavonoid has many beneficial effects such as antioxidant, anti-inflammatory, anti-proliferative properties. Azoxymethane (AOM), a derivative of 1, 2-Dimethyl hydrazine (DMH) was used for the induction of CRC in Balb/C mice. CRC was induced by intraperitoneal injection of AOM to mice at the dose of 15 mg/body kg weight for 3 weeks. Mouse was treated with LUT at the dose of 1.2 mg/body kg weight orally until end of the experiment. The expression of inducible nitric oxide synthase (iNOS) and cyclooxygense (COX)-2 were analyzed by RT-PCR and immunohistochemistry. The expressions of iNOS and COX-2 were increased in the case of AOM induction. Administration of LUT effectively reduced the expressions of iNOS and COX-2. The present study revealed that, LUT suppresses both iNOS and COX-2 expressions and act as an anti-inflammatory role against CRC. PMID:24991108

  8. The Effect of Garlic Extract on Expression of INFγ And Inos Genes in Macrophages Infected with Leishmania major

    PubMed Central

    Gharavi, MJ; Nobakht, M; Khademvatan, SH; Bandani, E; Bakhshayesh, M; Roozbehani, M

    2011-01-01

    Background The study was aimed to show the effect of molecular mechanism of Aqueous Garlic Extract (AGE) on expression of IFNγ and iNOS genes in Leishmania major. Methods Leishmania major promastigotes (MRHO/IR/75/ER) were added to the in-vitro cultured J774 cell line, the cells were incubated for 72 hours. Various concentrations of garlic extract (9.25, 18.5, 37, 74, 148 mg/ml) were added to the infected cells. MTT assay was applied for cellular proliferation. After 72 hours of incubation, supernatants were collected and total RNA was extracted from the infected cells. The express of IFNγ and iNOS genes were studied by RT-PCR method. Results The colorimetric MTT assay after 3 days of incubation showed cytotoxic effect of garlic extract with an IC50 of 37 mg/ml. In addition, IFNγ and iNOS genes expression by RT-PCR indicated that garlic extract lead to over expression of these genes in J774 cell line infected with L. major. Conclusion Garlic extract exerts cytotoxic effect on infected J774 cell line. In addition, the hypothesis that garlic can improve cellular immunity with raising the expression of IFNγ and of iNOS genes confirmed. PMID:22347300

  9. Aloe vera toxic effects: expression of inducible nitric oxide synthase (iNOS) in testis of Wistar rat

    PubMed Central

    Asgharzade, Samira; Rafieian-kopaei, Mahmoud; Mirzaeian, Amin; Reiisi, Somaye; Salimzadeh, Loghman

    2015-01-01

    Objective(s): Nitric oxide (NO), a product of inducible nitric oxide synthase (iNOS), contributes in germ cell apoptosis. This study was aimed to evaluate the effects of Aloe vera gel (AVG) on male Wistar rat reproductive organ, serum NO level, and expression of iNOS gene in leydig cells. Materials and Methods: Adult male Wistar rats (n=36) were used for experiments in three groups. The experimental groups were orally administered with the AVG extract solution once-daily as follow: 150 mg.kg-1; group A, 300 mg.kg-1; group B, and only normal saline; group C (control group). They were mated with untreated females and the reproductive and chemical parameters were assessed for each group, including semen quality, serum testosterone, sperm fertility, gonad and body weight, serum NO concentration (by the Griess method), and iNOS gene expression (using RT-PCR). Results: The testes weight, serum testosterone, as well as sperm count and fertility of the AVG treated groups were significantly reduced when compared to the control (P<0.001). Concentration of serum NO was significantly increased (37.1±4.63 µM) in the administrated group with higher AVG concentration, compared to the control group (P<0.001; 10.19±0.87 µM); however, iNOS mRNA expression was increased in the treated animals (P<0.001). Conclusion: iNOS may play a functional role in spermatogenesis via apoptosis, reducing sperm count, but further studies are needed to illustrate the mechanisms by which AVG exerts its negative effects on spermatogenesis and sperm quality. PMID:26730330

  10. Rate and severity of HIV-associated dementia (HAD): correlations with Gp41 and iNOS.

    PubMed Central

    Adamson, D. C.; McArthur, J. C.; Dawson, T. M.; Dawson, V. L.

    1999-01-01

    BACKGROUND: Fifteen to thirty percent of AIDS patients develop some type of neurologic disorder during the course of their illness and the vast majority of these neurologic disorders will be HIV-associated dementia (HAD). These patients can exhibit varying degrees of severity and rates of progression of HAD. Neuropathologic variables that are associated with the rate of progression of HAD are not known. MATERIALS AND METHODS: Tissue was collected at autopsy from the Johns Hopkins University HIV Neurology Program. Seventy-one AIDS patients of this prospectively characterized population were followed until death to obtain information on dementia severity and the rate of neurological progression. Immunoblot analysis of immunological nitric oxide synthase (iNOS), HAM56, gp41, p24, gp120, and beta-tubulin was performed and the levels of iNOS, HAM56, gp41, and p24 were normalized to beta-tubulin and analyzed for significance by means of the Kruskal-Wallis test for multiple groups. RESULTS: We have identified unique groups within this spectrum and designated them slow, moderate, and rapid progressors. Slow and moderate progressors' neurological progression occurs over a course of months to years, whereas the rapid progressors' disease shows rapid increases in severity over weeks to months. In the present study we demonstrate that the severity and rate of progression of HAD correlates significantly with levels of the HIV-1 coat protein, gp41, iNOS, and HAM56, a marker of microglial/macrophage activation. CONCLUSION: The severity and rate of progression of HAD correlates with indices of immune activation as well as levels of iNOS and gp41. There appears to be a threshold effect in which high levels of gp41, iNOS, and immune activation are particularly associated with severe (Memorial Sloan-Kettering score 3 to 4) and rapidly progressive HAD. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:10203575

  11. Role of Snail Activation in Alcohol-induced iNOS-mediated Disruption of Intestinal Epithelial Cell Permeability

    PubMed Central

    Forsyth, Christopher B.; Tang, Yueming; Shaikh, Maliha; Zhang, Lijuan; Keshavarzian, Ali

    2013-01-01

    Background Chronic alcohol use results in many pathological effects including alcoholic liver disease (ALD). ALD pathogenesis requires endotoxemia. Our previous studies showed that increased intestinal permeability is the major cause of endotoxemia and that this gut leakiness is dependent on alcohol stimulation of inducible nitric oxide synthase (iNOS) in both alcoholic subjects and rodent models of alcoholic steatohepatitis (ASH). The mechanism of the alcohol-induced, iNOS-mediated disruption of the intestinal barrier function is not known. We have recently shown that alcohol stimulates activation of the transcription factor Snail and biomarkers of epithelial mesenchymal transition. Since activated Snail disrupts tight junctional proteins , we hypothesized that activation of Snail by iNOS might be one of the key signaling pathways mediating alcohol stimulated intestinal epithelial cell hyperpermeability. Methods We measured intestinal permeability in alcohol-fed C57BL/6 control and iNOS KO mice and measured Snail protein expression in the intestines of these mice. We then examined intestinal epithelial permeability using the Caco-2 cell model of the intestinal barrier ± siRNA inhibition of Snail. We assessed Snail activation by alcohol in Caco-2 cells ± inhibition of iNOS with L-NIL or siRNA. Finally, we assessed Snail activation by alcohol ± inhibition with siRNA for p21-activated kinase (PAK1). Results Our data show that chronic alcohol feeding promotes intestinal hyperpermeability in wild type BL/6 but not in iNOS KO mice. Snail protein expression was increased in the intestines of alcohol-treated wild type mice but not in iNOS KO mice. SiRNA inhibition of Snail significantly inhibited alcohol-induced hyperpermeability in Caco-2 cell monolayers. Alcohol stimulation of SnailpS246 activation was blocked by inhibition of iNOS with L-NIL or with siRNA. SiRNA inhibition of PAK1 significantly inhibited alcohol-mediated activation of Snail in Caco-2 cells

  12. [Bacterial vaginosis].

    PubMed

    Romero Herrero, Daniel; Andreu Domingo, Antonia

    2016-07-01

    Bacterial vaginosis (BV) is the main cause of vaginal dysbacteriosis in the women during the reproductive age. It is an entity in which many studies have focused for years and which is still open for discussion topics. This is due to the diversity of microorganisms that cause it and therefore, its difficult treatment. Bacterial vaginosis is probably the result of vaginal colonization by complex bacterial communities, many of them non-cultivable and with interdependent metabolism where anaerobic populations most likely play an important role in its pathogenesis. The main symptoms are an increase of vaginal discharge and the unpleasant smell of it. It can lead to serious consequences for women, such as an increased risk of contracting sexually transmitted infections including human immunodeficiency virus and upper genital tract and pregnancy complications. Gram stain is the gold standard for microbiological diagnosis of BV, but can also be diagnosed using the Amsel clinical criteria. It should not be considered a sexually transmitted disease but it is highly related to sex. Recurrence is the main problem of medical treatment. Apart from BV, there are other dysbacteriosis less characterized like aerobic vaginitis of which further studies are coming slowly but are achieving more attention and consensus among specialists. PMID:27474242

  13. Discovery of inducible nitric oxide synthase (iNOS) inhibitor development candidate KD7332, part 1: Identification of a novel, potent, and selective series of quinolinone iNOS dimerization inhibitors that are orally active in rodent pain models.

    PubMed

    Bonnefous, Céline; Payne, Joseph E; Roppe, Jeffrey; Zhuang, Hui; Chen, Xiaohong; Symons, Kent T; Nguyen, Phan M; Sablad, Marciano; Rozenkrants, Natasha; Zhang, Yan; Wang, Li; Severance, Daniel; Walsh, John P; Yazdani, Nahid; Shiau, Andrew K; Noble, Stewart A; Rix, Peter; Rao, Tadimeti S; Hassig, Christian A; Smith, Nicholas D

    2009-05-14

    There are three isoforms of dimeric nitric oxide synthases (NOS) that convert arginine to citrulline and nitric oxide. Inducible NOS is implicated in numerous inflammatory diseases and, more recently, in neuropathic pain states. The majority of existing NOS inhibitors are either based on the structure of arginine or are substrate competitive. We describe the identification from an ultra high-throughput screen of a novel series of quinolinone small molecule, nonarginine iNOS dimerization inhibitors. SAR studies on the screening hit, coupled with an in vivo lipopolysaccharide (LPS) challenge assay measuring plasma nitrates and drug levels, rapidly led to the identification of compounds 12 and 42--potent inhibitors of the human and mouse iNOS enzyme that were highly selective over endothelial NOS (eNOS). Following oral dosing, compounds 12 and 42 gave a statistical reduction in pain behaviors in the mouse formalin model, while 12 also statistically reduced neuropathic pain behaviors in the chronic constriction injury (Bennett) model.

  14. Altered responses to bacterial infection and endotoxic shock in mice lacking inducible nitric oxide synthase.

    PubMed

    MacMicking, J D; Nathan, C; Hom, G; Chartrain, N; Fletcher, D S; Trumbauer, M; Stevens, K; Xie, Q W; Sokol, K; Hutchinson, N

    1995-05-19

    Mice deficient in inducible nitric oxide synthase (iNOS) were generated to test the idea that iNOS defends the host against infectious agents and tumor cells at the risk of contributing to tissue damage and shock. iNOS-/-mice failed to restrain the replication of Listeria monocytogenes in vivo or lymphoma cells in vitro. Bacterial endotoxic lipopolysaccharide (LPS) caused shock and death in anesthetized wild-type mice, but in iNOS-/-mice, the fall in central arterial blood pressure was markedly attenuated and early death averted. However, unanesthetized iNOS-/-mice suffered as much LPS-induced liver damage as wild type, and when primed with Propionobacterium acnes and challenged with LPS, they succumbed at the same rate as wild type. Thus, there exist both iNOS-dependent and iNOS-independent routes to LPS-induced hypotension and death.

  15. Evaluation of cell-mediated immune responses and bacterial clearance in 6-10 months old water buffalo (Bubalus bubalis) experimentally vaccinated with four dosages of commercial Brucella abortus strain RB51 vaccine.

    PubMed

    Diptee, M D; Adesiyun, A A; Asgarali, Z; Campbell, M; Fosgate, G T

    2005-07-15

    Thirty water buffalo, obtained from a brucellosis-free farm, were used to evaluate cell-mediated immune responses and bacterial clearance in response to vaccination with Brucella abortus strain RB51 (RB51) in a dose-response study. The animals were randomly divided into five treatment groups. Groups I--V received the recommended dose (RD) of RB51 vaccine once, RD twice 4 weeks apart, double RD once, double RD twice 4 weeks apart and saline once, respectively. Cell-mediated immune response to RB51 was assessed by the histological examination of haematoxylin and eosin (H&E) stained sections of lymph nodes draining the sites of inoculation and by comparison of stimulation indices (SI) derived from gamma interferon (IFN-gamma) assay. A mixture of cytoplasmic proteins from B. melitensis B115 (brucellergene) was used as a specific antigenic stimulus to peripheral blood mononuclear cells (PBMC) and lymph node mononuclear cells (LNMC) up to 22 post-initial-inoculation week (PIW). Supernatants harvested at 18-24h after the in vitro antigenic stimulus were assayed for their IFN-gamma content by using a commercial sandwich enzyme-linked immunosorbent assay (ELISA) kit. Clearance of RB51 was assessed by the sequential immunohistochemical examination of sections of draining lymph nodes post-inoculation. There was no observable expansion of the deep cortex of lymph nodes on H&E sections indicating poor T-cell stimulation. All group V (control) water buffalo PBMC ELISA values were negative (SI<2.2) at all PIW sampling intervals. Overall PBMC IFN-gamma assay detected vaccinates from treatment groups' I--IV 67% (4/6), 83% (5/6), 33% (2/6) and 67% (4/6), respectively. LNMC IFN-gamma assay was unimpressive and there was a negative correlation (--.08) between the results of PBMC and LNMC of IFN-gamma assay. Clearance of RB51 occurred between 4 and 6 PIW in treatment groups I and III and between 6 and 12 PIW in groups II and IV. RB51 was not detected in any of the control animals at

  16. Inhibitory effects of Euterpe oleracea Mart. on nitric oxide production and iNOS expression.

    PubMed

    Matheus, Maria Eline; de Oliveira Fernandes, Sidnei Bessa; Silveira, Cristiane Silva; Rodrigues, Verônica Pinto; de Sousa Menezes, Fabio; Fernandes, Patricia Dias

    2006-09-19

    The palm Euterpe oleracea is a plant of great economic value in Brazil. Although the heart of palm extracted from its trunk is considered a delicacy the world over, its fruits are popular only among Brazilians. In some poor regions of Brazil, there are reports on the popular use of its juice in the treatment of several disorders, mainly those of oxidative onset as cardiovascular ones. Because of its wide utilization; because there are very few scientific studies of this species, and to discover if its use in folk medicine for problems related with oxidation is in fact justifiable, we decided, in this study, to evaluate the effects of Euterpe oleracea flowers, fruits and spikes fractions on: nitric oxide (NO) production, NO scavenger capacity, and on the expression of inducible nitric oxide synthase enzyme, as well. Results showed that the fractions obtained from fruits were the most potent in inhibiting NO production, followed by those from flowers and spikes. Only in high doses, did some fractions reduce cell viability. Reduction on NO production was not due to NO scavenger activity. These results were accompanied by inhibition of iNOS expression. The more pronounced effect was observed in the fractions in which the concentration of cyanidin-3-O-glucoside and cyanidin-3-O-rhamnoside were higher. To sum up, our results indicate that fractions from Euterpe oleracea inhibits NO production by reducing the levels of inducible nitric oxide synthase expression. PMID:16635558

  17. Anticoagulant Activity and Structural Characterization of Polysaccharide from Abalone (Haliotis discus hannai Ino) Gonad.

    PubMed

    Zhao, Jun; Yang, Jingfeng; Song, Shuang; Zhou, Dayong; Qiao, Weizhou; Zhu, Ce; Liu, Shuyin; Zhu, Beiwei

    2016-01-01

    In this study, we aimed at characterizing the structure and the anticoagulant activity of a polysaccharide fraction (AGP33) isolated from the gonads of Haliotis discus hannai Ino. AGP33 was extracted by enzymatic hydrolysis and purified by ion-exchange and gel-filtration chromatography. The backbone fraction of AGP33 (BAGP33), which appeared to contain of mannose, glucose and galactose, was prepared by partial acid hydrolysis. According to methylation and nuclear magnetic resonance (NMR) spectroscopy, the backbone of AGP33 was identified as mainly consisting of 1→3-linked, 1→4-linked, and 1→6-linked monosaccharides. AGP33 is a sulfated polysaccharide with sulfates occur at 3-O- and 4-O-positions. It prolonged thromboplastin time (APTT), thrombin time (TT) and prothrombin time (PT) compared to a saline control solution in a dosage-dependent manner. AGP33 exhibited an extension (p < 0.01) of APTT compared to the saline group at concentrations higher than 5 μg/mL. AGP33 exhibited higher anticoagulant activity than its desulfated product (AGP33-des) and BAGP33. The results showed that polysaccharide with higher molecular weight and sulfate content demonstrated greater anticoagulant activity. PMID:27338320

  18. Anticoagulant Activity and Structural Characterization of Polysaccharide from Abalone (Haliotis discus hannai Ino) Gonad.

    PubMed

    Zhao, Jun; Yang, Jingfeng; Song, Shuang; Zhou, Dayong; Qiao, Weizhou; Zhu, Ce; Liu, Shuyin; Zhu, Beiwei

    2016-06-08

    In this study, we aimed at characterizing the structure and the anticoagulant activity of a polysaccharide fraction (AGP33) isolated from the gonads of Haliotis discus hannai Ino. AGP33 was extracted by enzymatic hydrolysis and purified by ion-exchange and gel-filtration chromatography. The backbone fraction of AGP33 (BAGP33), which appeared to contain of mannose, glucose and galactose, was prepared by partial acid hydrolysis. According to methylation and nuclear magnetic resonance (NMR) spectroscopy, the backbone of AGP33 was identified as mainly consisting of 1→3-linked, 1→4-linked, and 1→6-linked monosaccharides. AGP33 is a sulfated polysaccharide with sulfates occur at 3-O- and 4-O-positions. It prolonged thromboplastin time (APTT), thrombin time (TT) and prothrombin time (PT) compared to a saline control solution in a dosage-dependent manner. AGP33 exhibited an extension (p < 0.01) of APTT compared to the saline group at concentrations higher than 5 μg/mL. AGP33 exhibited higher anticoagulant activity than its desulfated product (AGP33-des) and BAGP33. The results showed that polysaccharide with higher molecular weight and sulfate content demonstrated greater anticoagulant activity.

  19. Effects of polysaccharides from abalone (Haliotis discus hannai Ino) on HepG2 cell proliferation.

    PubMed

    Wang, Yu-Ming; Wu, Feng-Juan; Du, Lei; Li, Guo-Yun; Takahashi, Koretaro; Xue, Yong; Xue, Chang-Hu

    2014-05-01

    Three polysaccharides, AAP, AVAP I, and AVAP II, were isolated from abalone Haliotis discus hannai Ino. The polysaccharides' compositions were analysed, and their effects on HepG2 cell proliferation were assessed. AVAP I had a greater growth-stimulatory effect than AAP or AVAP II. The oligosaccharide of AVAP I (Oli-AVAP I) exhibited the same growth effects, but rhamnose, the primary monosaccharide of AVAP I and Oli-AVAP I, did not exhibit this activity. Moreover, AVAP I dramatically reduced the mRNA levels of CDK6 and Cyclin E1 but significantly increased Cyclin B1, CDK1 and Cyclin F. Interestingly, AVAP I remained able to induce cell proliferation in a low serum concentration medium. AVAP I could therefore promote HepG2 cell proliferation by regulating gene expression and accelerating the cell cycle process. AVAP I may be useful as a serum supplement for stimulating the proliferation of mammalian cells. Our results offer a comprehensive method for utilising the abalone viscera, which is usually discarded as waste.

  20. Clinical Implications of iNOS Levels in Triple-Negative Breast Cancer Responding to Neoadjuvant Chemotherapy

    PubMed Central

    Jiang, Nan; Zhang, Lei; Li, Yiming; Xu, Xiaoyin; Cai, Shouliang; Wei, Liang; Liu, Xuhong; Chen, Guanglei; Zhou, Yizhen; Liu, Cheng; Li, Zhan; Jin, Feng; Chen, Bo

    2015-01-01

    Triple-negative breast cancer is a high-risk breast cancer with poor survival rate. To date, there is a lack of targeted therapy for this type of cancer. One unique phenomenon is that inflammatory breast cancer is frequently triple negative. However, it is still ambiguous how inflammation influences triple-negative breast cancer growth and responding to chemotherapy. Herein, we investigated the levels of inflammation-associated enzyme, iNOS, in 20 triple-negative breast cancer patients’ tumors, and examined its correlation with patients’ responses to platinum-based neoadjuvant chemotherapy. Our studies showed that triple-negative breast cancer patients with attenuated iNOS levels in tumor cells after treatment showed better responses to platinum-based neoadjuvant chemotherapy than other triple-negative breast cancer patients. Our further in vitro studies confirmed that induction of proper levels of NO increased the resistance to cisplatin in triple-negative MDA-MB-231 cells. Our data suggest that aberrant high level of iNOS/NO are associated with less effectiveness of platinum-based neoadjuvant chemotherapy in triple-negative breast cancer. Therefore, we propose to monitor iNOS levels as a new predictor for triple-negative breast cancer patient’s response to platinum-based neoadjuvant chemotherapy. Moreover, iNOS/NO is considered as a potential target for combination therapy with platinum drugs for triple-negative breast cancer. PMID:26196284

  1. Bacterial Hydrodynamics

    NASA Astrophysics Data System (ADS)

    Lauga, Eric

    2016-01-01

    Bacteria predate plants and animals by billions of years. Today, they are the world's smallest cells, yet they represent the bulk of the world's biomass and the main reservoir of nutrients for higher organisms. Most bacteria can move on their own, and the majority of motile bacteria are able to swim in viscous fluids using slender helical appendages called flagella. Low-Reynolds number hydrodynamics is at the heart of the ability of flagella to generate propulsion at the micrometer scale. In fact, fluid dynamic forces impact many aspects of bacteriology, ranging from the ability of cells to reorient and search their surroundings to their interactions within mechanically and chemically complex environments. Using hydrodynamics as an organizing framework, I review the biomechanics of bacterial motility and look ahead to future challenges.

  2. Bacterial arthritis.

    PubMed

    Ho, G

    2001-07-01

    The septic arthritis literature of 2000 revisited several topics previously examined in some detail. These include septic arthritis in rheumatoid arthritis, rheumatic manifestations of bacterial endocarditis, and infectious complications of prosthetic joints. The trend in antibiotic prophylaxis to prevent late infections in total joint replacement is to narrow the targeted hosts to those most at risk, to define the procedures associated with the greatest risk of bacteremia, and to simplify the antibiotic regimen. The diagnoses of septic arthritis of the lumbar facet joint and septic arthritis caused by direct inoculation of bacteria by a foreign object penetrating the joint are facilitated by noninvasive imaging technologies. Septic arthritis caused by uncommon microorganisms and septic arthritis in immunocompromised hosts are other noteworthy topics in this year's literature. PMID:11555734

  3. Genomic insights into bacterial DMSP transformations.

    PubMed

    Moran, Mary Ann; Reisch, Chris R; Kiene, Ronald P; Whitman, William B

    2012-01-01

    Genomic and functional genomic methods applied to both model organisms and natural communities have rapidly advanced understanding of bacterial dimethylsulfoniopropionate (DMSP) degradation in the ocean. The genes for the two main pathways in bacterial degradation, routing DMSP to distinctly different biogeochemical fates, have recently been identified. The genes dmdA, -B, -C, and -D mediate the demethylation of DMSP and facilitate retention of carbon and sulfur in the marine microbial food web. The genes dddD, -L, -P, -Q, -W, and -Y mediate the cleavage of DMSP to dimethylsulfide (DMS), with important consequences for ocean-atmosphere sulfur flux. In ocean metagenomes, sufficient copies of these genes are present for approximately 60% of surface ocean bacterial cells to directly participate in DMSP degradation. The factors that regulate these two competing pathways remain elusive, but gene transcription analyses of natural bacterioplankton communities are making headway in unraveling the intricacies of bacterial DMSP processing in the ocean.

  4. Genomic Insights into Bacterial DMSP Transformations

    NASA Astrophysics Data System (ADS)

    Moran, Mary Ann; Reisch, Chris R.; Kiene, Ronald P.; Whitman, William B.

    2012-01-01

    Genomic and functional genomic methods applied to both model organisms and natural communities have rapidly advanced understanding of bacterial dimethylsulfoniopropionate (DMSP) degradation in the ocean. The genes for the two main pathways in bacterial degradation, routing DMSP to distinctly different biogeochemical fates, have recently been identified. The genes dmdA, -B, -C, and -D mediate the demethylation of DMSP and facilitate retention of carbon and sulfur in the marine microbial food web. The genes dddD, -L, -P, -Q, -W, and -Y mediate the cleavage of DMSP to dimethylsulfide (DMS), with important consequences for ocean-atmosphere sulfur flux. In ocean metagenomes, sufficient copies of these genes are present for ˜60% of surface ocean bacterial cells to directly participate in DMSP degradation. The factors that regulate these two competing pathways remain elusive, but gene transcription analyses of natural bacterioplankton communities are making headway in unraveling the intricacies of bacterial DMSP processing in the ocean.

  5. Effects of dietary carbohydrates sources on lipids compositions in abalone, Haliotis discus hannai Ino

    NASA Astrophysics Data System (ADS)

    Wang, Weifang; Mai, Kangsen; Zhang, Wenbing; Xu, Wei; Ai, Qinghui; Yao, Chunfeng; Li, Huitao

    2009-09-01

    A study was conducted to evaluate the effects of dietary carbohydrates on triglyceride, cholesterol and fatty acid concentrations in abalone, Haliotis discus hannai Ino. Six semi-purified diets with different carbohydrates (dextrin, heat-treated wheat starch, wheat starch, corn starch, tapioca starch and potato starch, respectively), all containing a carbohydrate level of 33.5%, were fed to abalone (initial shell length: 29.98 mm ± 0.09 mm; initial weight: 3.42 g ± 0.02 g) for 24 weeks in a recirculation system. The results indicate that serum triglyceride concentrations were significantly ( P < 0.05) higher in the abalone fed with dextrin, heat-treated wheat starch and wheat starch than those fed with corn starch, and serum cholesterol concentrations were significantly ( P < 0.05) higher in the abalone fed with dextrin, heat-treated wheat starch than those fed with corn starch. Fatty acid C20:4n-6 in the foot muscles were significantly ( P < 0.05) lower in the abalone fed with dextrin than those fed with wheat starch, corn starch, tapioca starch and potato starch. Fatty acid C20:4n-6 in hepatopancreas was significantly ( P < 0.05) lower in abalone fed with heat-treated wheat starch than those fed with corn starch, tapioca starch and potato starch. Fatty acid C22:6n-3 in the foot muscles were significantly ( P < 0.05) lower in the abalone fed with dextrin and heat-treated wheat starch than those fed with wheat starch and potato starch.

  6. Effect of ambient pressure variation on closed loop gas system for India based Neutrino Observatory (INO)

    NASA Astrophysics Data System (ADS)

    Satyanarayana, B.; Majumder, G.; Mondal, N. K.; Kalmani, S. D.; Shinde, R. R.; Joshi, A.

    2014-10-01

    Pilot unit of a closed loop gas mixing and distribution system for the INO project was designed and is being operated with 1.8meters × 1.9meters RPCs for about two years. A number of studies on controlling the flow and optimisation of the gas mixture through the RPC stack were carried out during this period. The gas system essentially measures and attempts to maintain absolute pressure inside the RPC gas volume. During typical Mumbai monsoon seasons, the barometric pressure changes rather rapidly, due to which the gas system fails to maintain the set differential pressure between the ambience and the RPC gas volume. As the safety bubblers on the RPC gas input lines are set to work on fixed pressure differentials, the ambient pressure changes lead to either venting out and thus wasting gas through safety bubblers or over pressuring the RPCs gas volume and thus degrading its performance. The above problem also leads to gas mixture contamination through minute leaks in gas gap. The problem stated above was solved by including the ambient barometric pressure as an input parameter in the closed loop. Using this, it is now possible to maintain any set differential pressure between the ambience and RPC gas volumes between 0 to 20mm of water column, thus always ensuring a positive pressure inside the RPC gas volume with respect to the ambience. This has resulted in improved performance of the gas system by maintaining the constant gas flow and reducing the gas toping up frequency. In this paper, we will highlight the design features and improvements of the closed loop gas system. We will present some of the performance studies and considerations for scaling up the system to be used with the engineering module and then followed by Iron Calorimeter detector (ICAL), which is designed to deploy about 30,000 RPCs of 1.8meters × 1.9 meters in area.

  7. Regulation of Inducible Nitric Oxide Synthase Expression by Viral A238L-Mediated Inhibition of p65/RelA Acetylation and p300 Transactivation

    PubMed Central

    Granja, Aitor G.; Sabina, Prado; Salas, María L.; Fresno, Manuel; Revilla, Yolanda

    2006-01-01

    Uncontrolled generation of nitric oxide (NO) by inducible nitric-oxide synthase (iNOS) can cause damage to host cells and inflammation, two undesirable events for virus spreading. African swine fever virus (ASFV) infection regulates iNOS-induced gene expression through the synthesis of the A238L virus protein. We here explored the role of A238L, an NF-κB and NFAT inhibitor, in the regulation of iNOS transcription in macrophages. NO production and iNOS mRNA and protein levels as well as iNOS promoter activity after lipopolysaccharide (LPS)-gamma interferon (IFN-γ) treatment were down-regulated both during ASFV infection and in Raw 264.7 cells stably expressing the viral protein. Overexpression of p300, but not of a histone acetyltransferase (HAT) defective mutant, reverted the A238L-mediated inhibition of both basal and LPS-IFN-γ-induced iNOS promoter activity. Following stimulation with LPS-IFN-γ, p65 and p300 interaction was abolished in Raw-A238L cells. Expression of A238L also inhibited p65/relA and p300 binding to the distal NF-κB sequence of the iNOS promoter together with p65 acetylation. Finally, A238L abrogated p300 transactivation mediated by a GAL4-p300 construction. These results provide evidence for an unique viral mechanism involved in transcriptional regulation of iNOS gene expression. PMID:17041221

  8. Airborne Bacterial Interactions: Functions Out of Thin Air?

    PubMed Central

    Audrain, Bianca; Létoffé, Sylvie; Ghigo, Jean-Marc

    2015-01-01

    Bacteria produce and release a large diversity of small molecules including organic and inorganic volatile compounds, hereafter referred to as bacterial volatile compounds (BVCs). Whereas BVCs were often only considered as wasted metabolic by-product sometimes perceived by animal olfactory systems, it is increasingly clear that they can also mediate cross-kingdom interactions with fungi, plants and animals. Recently, in vitro studies also reported the impact of BVCs on bacterial biology through modulation of antibiotic resistance, biofilm formation and virulence. Here, we review BVCs influence on bacterial adaptation to their environment and discuss the biological relevance of recently reported inter- and intra-species bacterial interactions mediated by BVCs. PMID:26733998

  9. Diverse Bacterial Microcompartment Organelles

    PubMed Central

    Chowdhury, Chiranjit; Sinha, Sharmistha; Chun, Sunny; Yeates, Todd O.

    2014-01-01

    SUMMARY Bacterial microcompartments (MCPs) are sophisticated protein-based organelles used to optimize metabolic pathways. They consist of metabolic enzymes encapsulated within a protein shell, which creates an ideal environment for catalysis and facilitates the channeling of toxic/volatile intermediates to downstream enzymes. The metabolic processes that require MCPs are diverse and widely distributed and play important roles in global carbon fixation and bacterial pathogenesis. The protein shells of MCPs are thought to selectively control the movement of enzyme cofactors, substrates, and products (including toxic or volatile intermediates) between the MCP interior and the cytoplasm of the cell using both passive electrostatic/steric and dynamic gated mechanisms. Evidence suggests that specialized shell proteins conduct electrons between the cytoplasm and the lumen of the MCP and/or help rebuild damaged iron-sulfur centers in the encapsulated enzymes. The MCP shell is elaborated through a family of small proteins whose structural core is known as a bacterial microcompartment (BMC) domain. BMC domain proteins oligomerize into flat, hexagonally shaped tiles, which assemble into extended protein sheets that form the facets of the shell. Shape complementarity along the edges allows different types of BMC domain proteins to form mixed sheets, while sequence variation provides functional diversification. Recent studies have also revealed targeting sequences that mediate protein encapsulation within MCPs, scaffolding proteins that organize lumen enzymes and the use of private cofactor pools (NAD/H and coenzyme A [HS-CoA]) to facilitate cofactor homeostasis. Although much remains to be learned, our growing understanding of MCPs is providing a basis for bioengineering of protein-based containers for the production of chemicals/pharmaceuticals and for use as molecular delivery vehicles. PMID:25184561

  10. Activated Macrophages as a Novel Determinant of Tumor Cell Radioresponse: The Role of Nitric Oxide-Mediated Inhibition of Cellular Respiration and Oxygen Sparing

    SciTech Connect

    Jiang Heng; De Ridder, Mark; Verovski, Valeri N.; Sonveaux, Pierre; Jordan, Benedicte F.; Law, Kalun; Monsaert, Christinne; Van den Berge, Dirk L.; Verellen, Dirk; Feron, Olivier; Gallez, Bernard; Storme, Guy A.

    2010-04-15

    Purpose: Nitric oxide (NO), synthesized by the inducible nitric oxide synthase (iNOS), is known to inhibit metabolic oxygen consumption because of interference with mitochondrial respiratory activity. This study examined whether activation of iNOS (a) directly in tumor cells or (b) in bystander macrophages may improve radioresponse through sparing of oxygen. Methods and Materials: EMT-6 tumor cells and RAW 264.7 macrophages were exposed to bacterial lipopolysaccharide plus interferon-gamma, and examined for iNOS expression by reverse transcription polymerase chain reaction, Western blotting and enzymatic activity. Tumor cells alone, or combined with macrophages were subjected to metabolic hypoxia and analyzed for radiosensitivity by clonogenic assay, and for oxygen consumption by electron paramagnetic resonance and a Clark-type electrode. Results: Both tumor cells and macrophages displayed a coherent picture of iNOS induction at transcriptional/translational levels and NO/nitrite production, whereas macrophages showed also co-induction of the inducible heme oxygenase-1, which is associated with carbon monoxide (CO) and bilirubin production. Activation of iNOS in tumor cells resulted in a profound oxygen sparing and a 2.3-fold radiosensitization. Bystander NO-producing, but not CO-producing, macrophages were able to block oxygen consumption by 1.9-fold and to radiosensitize tumor cells by 2.2-fold. Both effects could be neutralized by aminoguanidine, a metabolic iNOS inhibitor. An improved radioresponse was clearly observed at macrophages to tumor cells ratios ranging between 1:16 to 1:1. Conclusions: Our study is the first, as far as we are aware, to provide evidence that iNOS may induce radiosensitization through oxygen sparing, and illuminates NO-producing macrophages as a novel determinant of tumor cell radioresponse within the hypoxic tumor microenvironment.

  11. Bacterial vaginosis.

    PubMed Central

    Spiegel, C A

    1991-01-01

    Bacterial vaginosis (BV) is the most common of the vaginitides affecting women of reproductive age. It appears to be due to an alteration in the vaginal ecology by which Lactobacillus spp., the predominant organisms in the healthy vagina, are replaced by a mixed flora including Prevotella bivia, Prevotella disiens, Porphyromonas spp., Mobiluncus spp., and Peptostreptococcus spp. All of these organisms except Mobiluncus spp. are also members of the endogenous vaginal flora. While evidence from treatment trials does not support the notion that BV is sexually transmitted, recent studies have shown an increased risk associated with multiple sexual partners. It has also been suggested that the pathogenesis of BV may be similar to that of urinary tract infections, with the rectum serving as a reservoir for some BV-associated flora. The organisms associated with BV have also been recognized as agents of female upper genital tract infection, including pelvic inflammatory disease, and the syndrome BV has been associated with adverse outcome of pregnancy, including premature rupture of membranes, chorioamnionitis, and fetal loss; postpartum endometritis; cuff cellulitis; and urinary tract infections. The mechanisms by which the BV-associated flora causes the signs of BV are not well understood, but a role for H2O2-producing Lactobacillus spp. in protecting against colonization by catalase-negative anaerobic bacteria has been recognized. These and other aspects of BV are reviewed. PMID:1747864

  12. Investigating the Role of TNF-α and IFN-γ Activation on the Dynamics of iNOS Gene Expression in LPS Stimulated Macrophages

    PubMed Central

    Salim, Taha; Sershen, Cheryl L.; May, Elebeoba E.

    2016-01-01

    Macrophage produced inducible nitric oxide synthase (iNOS) is known to play a critical role in the proinflammatory response against intracellular pathogens by promoting the generation of bactericidal reactive nitrogen species. Robust and timely production of nitric oxide (NO) by iNOS and analogous production of reactive oxygen species are critical components of an effective immune response. In addition to pathogen associated lipopolysaccharides (LPS), iNOS gene expression is dependent on numerous proinflammatory cytokines in the cellular microenvironment of the macrophage, two of which include interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α). To understand the synergistic effect of IFN-γ and TNF-α activation, and LPS stimulation on iNOS expression dynamics and NO production, we developed a systems biology based mathematical model. Using our model, we investigated the impact of pre-infection cytokine exposure, or priming, on the system. We explored the essentiality of IFN-γ priming to the robustness of initial proinflammatory response with respect to the ability of macrophages to produce reactive species needed for pathogen clearance. Results from our theoretical studies indicated that IFN-γ and subsequent activation of IRF1 are essential in consequential production of iNOS upon LPS stimulation. We showed that IFN-γ priming at low concentrations greatly increases the effector response of macrophages against intracellular pathogens. Ultimately the model demonstrated that although TNF-α contributed towards a more rapid response time, measured as time to reach maximum iNOS production, IFN-γ stimulation was significantly more significant in terms of the maximum expression of iNOS and the concentration of NO produced. PMID:27276061

  13. Role of NADPH oxidase and iNOS in vasoconstrictor responses of vessels from hypertensive and normotensive rats

    PubMed Central

    Álvarez, Y; Briones, A M; Hernanz, R; Pérez-Girón, J V; Alonso, M J; Salaices, M

    2007-01-01

    Background and purpose: To analyse the influence of hypertension in the modulation induced by inducible NOS (iNOS)-derived NO and superoxide anion (O2 •−) of vasoconstrictor responses and the sources of O2 •− implicated. Experimental approach: Vascular reactivity experiments were performed in segments of aorta from normotensive, Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR); protein and mRNA expressions were respectively measured by western blot and quantitative reverse transcription-polymerase chain reaction and O2 •− production was evaluated by ethidium fluorescence. Key results: The contractile responses to phenylephrine (1 nM–30 μM) and 5-hydroxytryptamine (0.1–100 μM) were greater in aortic segments from SHR than WKY. The selective iNOS inhibitor, 1400W (10 μM), increased the phenylephrine contraction only in WKY segments; however, iNOS protein and mRNA expressions were greater in aorta from SHR than WKY. Superoxide dismutase (SOD, 150 U ml−1) reduced phenylephrine and 5-hydroxytryptamine responses only in aorta from SHR; the NAD(P)H oxidase inhibitor apocynin (0.3 mM) decreased phenylephrine and 5-hydroxytryptamine responses more in vessels from SHR than WKY. Co-incubation with SOD plus 1400W potentiated the phenylephrine and 5-hydroxytryptamine responses more in segments from SHR than WKY. O2 •− production was greater in aorta from SHR than WKY; apocynin abolished this difference. Conclusions and implications: Increased O2 •− formation from NADP(H) oxidase in vessels from hypertensive rats contributes to the vasoconstrictor responses and counteract the increase of NO from iNOS and the consequent modulation of these responses. PMID:17994107

  14. Development of a pyramidal wavefront sensor test-bench at INO

    NASA Astrophysics Data System (ADS)

    Turbide, Simon; Wang, Min; Gauvin, Jonny; Martin, Olivier; Savard, Maxime; Bourqui, Pascal; Veran, Jean-Pierre; Deschenes, William; Anctil, Genevieve; Chateauneuf, François

    2013-12-01

    The key technical element of the adaptive optics in astronomy is the wavefront sensing (WFS). One of the advantages of the pyramid wavefront sensor (P-WFS) over the widely used Shack-Hartmann wavefront sensor seems to be the increased sensitivity in closed-loop applications. A high-sensitivity and large dynamic-range WFS, such as P-WFS technology, still needs to be further investigated for proper justification in future Extremely Large Telescopes application. At INO, we have recently carried out the optical design, testing and performance evaluation of a P-WFS bench setup. The optical design of the bench setup mainly consists of the super-LED fiber source, source collimator, spatial light modulator (SLM), relay lenses, tip-tilt mirror, Fourier-transforming lens, and a four-faceted glass pyramid with a large vertex angle as well as pupil re-imaged optics. The phase-only SLM has been introduced in the bench setup to generate atmospheric turbulence with a maximum phase shift of more than 2π at each pixel (256 grey levels). Like a modified Foucault knife-edge test, the refractive pyramid element is used to produce four images of the entrance pupil on a CCD camera. The Fourier-transforming lens, which is used before the pyramid prism, is designed for telecentric output to allow dynamic modulation (rotation of the beam around the pyramid-prism center) from a tip-tilt mirror. Furthermore, a P-WFS diffraction-based model has been developed. This model includes most of the system limitations such as the SLM discrete voltage steps and the CCD pixel pitch. The pyramid effects (edges and tip) are considered as well. The modal wavefront reconstruction algorithm relies on the construction of an interaction matrix (one for each modulation's amplitude). Each column of the interaction matrix represents the combination of the four pupil images for a given wavefront aberration. The nice agreement between the data and the model suggest that the limitation of the system is not the P

  15. Bacterial concrete

    NASA Astrophysics Data System (ADS)

    Ramakrishnan, Venkataswamy; Ramesh, K. P.; Bang, S. S.

    2001-04-01

    Cracks in concrete are inevitable and are one of the inherent weaknesses of concrete. Water and other salts seep through these cracks, corrosion initiates, and thus reduces the life of concrete. So there was a need to develop an inherent biomaterial, a self-repairing material which can remediate the cracks and fissures in concrete. Bacterial concrete is a material, which can successfully remediate cracks in concrete. This technique is highly desirable because the mineral precipitation induced as a result of microbial activities is pollution free and natural. As the cell wall of bacteria is anionic, metal accumulation (calcite) on the surface of the wall is substantial, thus the entire cell becomes crystalline and they eventually plug the pores and cracks in concrete. This paper discusses the plugging of artificially cracked cement mortar using Bacillus Pasteurii and Sporosarcina bacteria combined with sand as a filling material in artificially made cuts in cement mortar which was cured in urea and CaCl2 medium. The effect on the compressive strength and stiffness of the cement mortar cubes due to the mixing of bacteria is also discussed in this paper. It was found that use of bacteria improves the stiffness and compressive strength of concrete. Scanning electron microscope (SEM) is used to document the role of bacteria in microbiologically induced mineral precipitation. Rod like impressions were found on the face of calcite crystals indicating the presence of bacteria in those places. Energy- dispersive X-ray (EDX) spectra of the microbial precipitation on the surface of the crack indicated the abundance of calcium and the precipitation was inferred to be calcite (CaCO3).

  16. Effect of electrical properties of glass electrodes on the performance of RPC detectors for the INO-ICAL experiment

    NASA Astrophysics Data System (ADS)

    Raveendrababu, K.; Behera, P. K.; Satyanarayana, B.

    2016-08-01

    The India-based Neutrino Observatory (INO) collaboration has chosen glass Resistive Plate Chambers (RPCs) as the active detector elements for the Iron Calorimeter (ICAL) experiment. In the present work, we study the electrical properties such as bulk resistivity and relative permittivity of the glasses from two different manufacturers and compared the performances of RPCs built using these glasses. We conclude that the glass electrodes with larger bulk resistivity and permittivity are better suited for manufacturing RPCs for the ICAL experiment, as these detectors could be operated at lower bias currents and voltages, and produce better time resolution compared to those built with glass electrodes of smaller bulk resistivity and permittivity.

  17. Mechanical properties and structure of Haliotis discus hannai Ino and Hemifusus tuba conch shells: a comparative study

    NASA Astrophysics Data System (ADS)

    Zhao, Jie; Chen, Chen; Liang, Yan; Wang, Jian

    2010-03-01

    Haliotis discus hannai Ino (abalone shell) and Hemifusus tuba conch shell have been studied for the purpose to comparatively investigate the mechanisms by which nature designs composites. It is shown that both shells are composed of aragonite and a small amount of proteins while the conch shell shows finer microstructure but lower strength than abalone shell. It is also shown that the fresh shells exhibits better property than those after heat-treatments. It is therefore supposed that the size of inorganic substance is not a dominant factor to improve strength, while both proteins in shells and the microstructure of inorganic matter also play important roles.

  18. The Heme Oxygenase-1 Inducer THI-56 Negatively Regulates iNOS Expression and HMGB1 Release in LPS-Activated RAW 264.7 Cells and CLP-Induced Septic Mice

    PubMed Central

    Kim, Young Min; Park, Sang Won; Kim, Hye Jung; Lee, Jae Heun; Chang, Ki Churl

    2013-01-01

    The nuclear DNA binding protein high mobility group box 1 (HMGB1) has recently been suggested to act as a late mediator of septic shock. The effect of ((S)-6,7-dihydroxy-1-(4-hydroxynaphthylmethyl)-1,2,3,4-tetrahydroisoquinoline alkaloid, also known as THI-56, in an experimental model of sepsis was investigated. THI-56 exhibited potent anti-inflammatory properties in response to LPS in RAW 264.7 cells. In particular, THI-56 significantly inhibited the expression of inducible nitric oxide synthase (iNOS) and the release of HMGB1 in activated macrophages. THI-56 activated NE-F2-regulated factor 2 (Nrf-2)/heme oxygenase 1 (HO-1). The specific knockdown of the HO-1 gene by HO-1 siRNA significantly reversed the inhibitory effects of THI-56 on iNOS expression and HMGB1 release in LPS-stimulated macrophages. Importantly, THI-56 administration protected animals from death induced by either a lethal dose of LPS or cecal ligation and puncture (CLP). Furthermore, the ALT, AST, BUN, creatinine, and HMGB1 levels in the blood were significantly increased in CLP-induced septic mice, and the administration of THI-56 reduced these levels in a concentration-dependent and zinc protoporphyrin IX (ZnPPIX)-sensitive manner. In addition, the administration of THI-56 significantly ameliorated not only lung damage but also macrophage infiltration in the livers of CLP-induced septic mice, and these effects were also abrogated in the presence of ZnPPIX. Thus, we conclude that THI-56 significantly attenuates the proinflammatory response induced by LPS and reduces organ damage in a CLP-induced sepsis model through the upregulation of Nrf-2/HO-1. PMID:24098466

  19. NPM-ALK up-regulates iNOS expression through a STAT3/microRNA-26a-dependent mechanism.

    PubMed

    Zhu, Haifeng; Vishwamitra, Deeksha; Curry, Choladda V; Manshouri, Roxsan; Diao, Lixia; Khan, Aarish; Amin, Hesham M

    2013-05-01

    NPM-ALK chimeric oncogene is aberrantly expressed in an aggressive subset of T-cell lymphomas that frequently occurs in children and young adults. The mechanisms underlying the oncogenic effects of NPM-ALK are not completely elucidated. Inducible nitric oxide synthase (iNOS) promotes the survival and maintains the malignant phenotype of cancer cells by generating NO, a highly active free radical. We tested the hypothesis that iNOS is deregulated in NPM-ALK(+) T-cell lymphoma and promotes the survival of this lymphoma. In line with this possibility, an iNOS inhibitor and NO scavenger decreased the viability, adhesion, and migration of NPM-ALK(+) T-cell lymphoma cells, and an NO donor reversed these effects. Moreover, the NO donor salvaged the viability of lymphoma cells treated with ALK inhibitors. In further support of an important role of iNOS, we found iNOS protein to be highly expressed in NPM-ALK(+) T-cell lymphoma cell lines and in 79% of primary tumours but not in human T lymphocytes. Although expression of iNOS mRNA was identified in NPM-ALK(+) T-cell lymphoma cell lines and tumours, iNOS mRNA was remarkably elevated in T lymphocytes, suggesting post-transcriptional regulation. Consistently, we found that miR-26a contains potential binding sites and interacts with the 3'-UTR of iNOS. In addition, miR-26a was significantly decreased in NPM-ALK(+) T-cell lymphoma cell lines and tumours compared with T lymphocytes and reactive lymph nodes. Restoration of miR-26a in lymphoma cells abrogated iNOS protein expression and decreased NO production and cell viability, adhesion, and migration. Importantly, the effects of miR-26a were substantially attenuated when the NO donor was simultaneously used to treat lymphoma cells. Our investigation of the mechanisms underlying the decrease in miR-26a in this lymphoma revealed novel evidence that STAT3, a major downstream substrate of NPM-ALK tyrosine kinase activity, suppresses MIR26A1 gene expression.

  20. Pravastatin inhibits fibrinogen- and FDP-induced inflammatory response via reducing the production of IL-6, TNF-α and iNOS in vascular smooth muscle cells.

    PubMed

    Lu, Peipei; Liu, Juntian; Pang, Xiaoming

    2015-10-01

    Atherosclerosis is a chronic inflammatory response of the arterial wall to pro‑atherosclerotic factors. As an inflammatory marker, fibrinogen directly participates in the pathogenesis of atherosclerosis. Our previous study demonstrated that fibrinogen and fibrin degradation products (FDP) produce a pro‑inflammatory effect on vascular smooth muscle cells (VSMCs) through inducing the production of interleukin‑6 (IL‑6), tumor necrosis factor‑α (TNF‑α) and inducible nitric oxide synthase (iNOS). In the present study, the effects of pravastatin on fibrinogen‑ and FDP‑induced expression of IL‑6, TNF‑α and iNOS were observed in VSMCs. The results showed that pravastatin dose‑dependently inhibited fibrinogen‑ and FDP‑stimulated expression of IL‑6, TNF‑α and iNOS in VSMCs at the mRNA and protein level. The maximal inhibition of protein expression of IL‑6, TNF‑α and iNOS was 46.9, 42.7 and 49.2% in fibrinogen‑stimulated VSMCs, and 50.2, 49.8 and 53.6% in FDP‑stimulated VSMCs, respectively. This suggests that pravastatin has the ability to relieve vascular inflammation via inhibiting the generation of IL‑6, TNF‑α and iNOS. The results of the present study may aid in further explaining the beneficial effects of pravastatin on atherosclerosis and related cardiovascular diseases. In addition, they suggest that application of pravastatin may be beneficial for prevention of atherosclerosis formation in hyperfibrinogenemia.

  1. Influence of intravascular low level He-Ne laser irradiation on iNOS, total-NOS, and ET-1 in acute spinal cord-injured rabbits

    NASA Astrophysics Data System (ADS)

    Yin, Zhenchun; Dong, Yinghai; Zhu, Jing

    2005-07-01

    Objective To research the influence of intravascular low level Laser irradiation (ILLLI) on total NOS, iNOS, and ET-1 in spinal cord following acute spinal cord injury (ASCI), and discuss the protective effects of ILLLI on neurons .Methods 72 rabbits were randomly divided into 3 groups: treatment group, injury group and control group. In treatment group and injury group, after laminectomy at the level of T-13, ASCI was performed by using Allen"s method with slight modification (6g×10cm) on rabbits. After injury, rabbits were treated immediately with He-Ne laser (power 5 mW, 1 hour per day for 10 days). At the day of 10th after treatment, total-NOS, iNOS, and ET-1 in spinal cord tissues were measured. Results The expression level of total-NOS, iNOS, and ET-1 in spinal cord in injury group were significantly higher than those in control group (P<0.05), while after ILLLI the level of these index in treatment group decreased statistically significantly compared with those in injury group (P<0.05). Conclusion ILLLI can significantly decrease the expression level of total-NOS, iNOS, and ET-1 in spinal cord. It indicates that ILLLI can relieve the overexpression of total-NOS, iNOS, and ET-1 ,and thus can perform protective effects on neurons in the course of secondary spinal cord injury (SSCI) following ASCI

  2. Modulation of adenovirus-mediated gene transfer by nitric oxide.

    PubMed

    Haddad, I Y; Sorscher, E J; Garver, R I; Hong, J; Tzeng, E; Matalon, S

    1997-05-01

    We assessed the role of .NO in recombinant adenovirus-mediated gene transfer both in vitro and in vivo. NIH3T3 fibroblasts, stably transfected with the human inducible nitric oxide synthase, but lacking tetrahydrobiopterin (NIH3T3/iNOS [inducibile nitric oxide synthase]), were infected with replication-deficient adenovirus (E1-deleted), containing either the luciferase or the Lac Z reporter genes (AdCMV-Luc and AdCMV-Lac Z; 1-10 plaque forming units [pfu]/cell). Incubation of infected cells with sepiapterin (50 microM), a precursor of tetrahydrobiopterin, progressively increased nitrate/nitrite levels in the medium and decreased both luciferase and beta-galactosidase protein expression to approximately 60% of their corresponding control values, 24 h later. NIH3T3/iNOS cells had normal ATP (adenosine 5'-triphosphate) levels and did not release LDH(lactic dehydrogenase) into the medium. Pretreatment of these cells with N(G)-monomethyl-L-arginine (L-NMMA; 1 mM), an inhibitor of iNOS, prevented the sepiapterin-mediated induction of .NO and restored gene transfer to baseline values. Incubation of NIH3T3/iNOS with 8-bromo-cGMP (400 microM) in the absence of sepiapterin, or exposure of AdCMV-Luc to large concentrations of .NO, did not alter the efficacy of gene transfer. .NO produced by NIH3T3/iNOS cells also suppressed beta-galactosidase expression in NIH3T3 cocultured cells stably transfected with beta-galactosidase gene, suggesting .NO inhibited gene expression at either the transriptional or posttranscriptional levels. To investigate the effects of inhaled .NO on gene transfer in vivo, CD1 mice received an intratracheal instillation of AdCMV-Luc (4 x 10(9) pfu in 80 microl of saline) and exposed to .NO (25 ppm in room air) for 72 h. At that time, no significant degree of lung inflammation was detected by histological examination. However, lung luciferase activity decreased by 53% as compared with air breathing controls (P < 0.05; n > or = 8). We concluded that

  3. Inhibition of nitric oxide is a good therapeutic target for bladder tumors that express iNOS.

    PubMed

    Belgorosky, Denise; Langle, Yanina; Prack Mc Cormick, Bárbara; Colombo, Lucas; Sandes, Eduardo; Eiján, Ana María

    2014-01-30

    Bladder cancer is the second cause of death for urological tumors in man. When the tumor is nonmuscle invasive, transurethral resection is curative. On the other hand, radical cystectomy is the treatment chosen for patients with invasive tumors, but still under treatment, these patients have high risk of dying, by the development of metastatic disease within 5 years. It is therefore important to identify a new therapeutic target to avoid tumor recurrences and tumor progression. Nitric oxide (NO) is an important biological messenger known to influence several types of cancers. In bladder cancer, production of NO and expression and activity of inducible NO synthase was associated to recurrence and progression. The objective of this work was to analyze if inhibition of nitric oxide production could be considered a therapeutic target for bladder tumors expressing iNOS. Using a bladder cancer murine model with different invasiveness grade we have demonstrated that NO inhibition was able to inhibit growth of bladder tumors expressing iNOS. Furthermore, invasive properties of MB49-I orthotopic growth was inhibited using NO inhibitors. This paper also shows that levels of NO in urine can be correlated with tumor size. In conclusion, inhibition of NO could be considered as a therapeutic target that prevents tumor growth and progression. Also, urine NO levels may be useful for measuring tumor growth.

  4. Bacterial differentiation.

    PubMed

    Shapiro, L; Agabian-Keshishian, N; Bendis, I

    1971-09-01

    technique can be used to select for mutants blocked in the various stages of morphogenesis. 3) Temperature-sensitive mutants of Caulobacter that are restricted in macromolecular synthesis and development at elevated temperatures have been isolated. 4) Genetic exchange in the Calflobacter genus has been demonstrated and is now being defined. Two questions related to control processes can now readily be approached experimentally. (i) Is the temporal progression of events occurring during bacterial differentiation controlled by regulator gene products? (ii) Is the differentiation cycle like a biosynthetic pathway where one event must follow another? The availability of temperature-sensitive mutants blocked at various stages of development permits access to both questions. An interesting feature of the differentiation cycle is that the polar organelle may represent a special segregated unit which is operative in the control of the differentiation process. Perhaps the sequential morphogenic changes exhibited by Caulobacter are dependent on the initial synthesis of this organelle. Because the ultimate expression of cell changes are dependent on selective protein synthesis, specific messenger RNA production-either from DNA present in an organelle or from the chromosome-may prove to be a controlling factor in cell differentiation. We have begun studies with RNA polymerase purified from Caulobacter crescentus to determine whether cell factors or alterations in the enzyme structure serve to change the specificity of transcription during the cell cycle. Control of sequential cell changes at the level of transcription has long been postulated and has recently been substantiated in the case of Bacillus sporulation (6). The Caulobacter bacteria now present another system in which direct analysis of these control mechanisms is feasible. PMID:5572165

  5. Controlled bile acid exposure to oesophageal mucosa causes up-regulation of nuclear γ-H2AX possibly via iNOS induction

    PubMed Central

    Jiang, Bo; Zhao, Shengqian; Tao, Zhen; Wen, Jin; Yang, Yancheng; Zheng, Yin; Yan, Hongling; Sheng, Ying; Gao, Aimin

    2016-01-01

    Using an in vitro model in which flatmounts of oesophagus was periodically exposed to bile acids, we demonstrate, using multiple methods, that the bile acid receptor TGR5, inducible nitric oxide synthase (iNOS) and γ-histone family 2A variant (γ-H2AX) are up-regulated. This indicates that bile acids cause up-regulation of iNOS, which further causes genotoxic stress as evidenced by increase of the highly sensitive marker, phosphorylated histone. In vitro nitric oxide (NO) assays showed increased production of nitric acid in the oesophageal epithelium exposed to the bile acids. This increase was inhibited in the presence of the nonspecific iNOS inhibitor aminoguanidine (AG). Cumulatively, the results of the present study provide suggestion that not only acid reflux, but also non-acid reflux of bile may cause genotoxic stress. These aspects merit to be tested in wide spectrum of Barrett epithelial tissues. PMID:27247425

  6. Lack of association of the iNOS gene polymorphism with risk of cancer: a systematic review and Meta-Analysis.

    PubMed

    Jiao, Jinghua; Wu, Jingyang; Huang, Desheng; Liu, Lei

    2015-01-01

    In order to investigate the association between the iNOS gene polymorphisms and susceptibility to cancer, a search of English papers was done using Pubmed, the Cochrane Library, Embase, ISI Web of Science, Google (scholar) database, and all Chinese reports were conducted using CBMDisc, Chongqing VIP database, and CNKI database. A total of eight studies were included in this meta-analysis including 1,920 cases and 2,373 controls. The results indicated that the polymorphisms in iNOS gene (C150T(Ser(608) Leu) polymorphism and polymorphic (CCTTT)n repeats) had no association with cancer risk for all genetic models. This meta-analysis suggested that the polymorphisms in the iNOS gene were not associated with cancer risk. PMID:26391304

  7. Controlled bile acid exposure to oesophageal mucosa causes up-regulation of nuclear γ-H2AX possibly via iNOS induction.

    PubMed

    Jiang, Bo; Zhao, Shengqian; Tao, Zhen; Wen, Jin; Yang, Yancheng; Zheng, Yin; Yan, Hongling; Sheng, Ying; Gao, Aimin

    2016-08-01

    Using an in vitro model in which flatmounts of oesophagus was periodically exposed to bile acids, we demonstrate, using multiple methods, that the bile acid receptor TGR5, inducible nitric oxide synthase (iNOS) and γ-histone family 2A variant (γ-H2AX) are up-regulated. This indicates that bile acids cause up-regulation of iNOS, which further causes genotoxic stress as evidenced by increase of the highly sensitive marker, phosphorylated histone. In vitro nitric oxide (NO) assays showed increased production of nitric acid in the oesophageal epithelium exposed to the bile acids. This increase was inhibited in the presence of the nonspecific iNOS inhibitor aminoguanidine (AG). Cumulatively, the results of the present study provide suggestion that not only acid reflux, but also non-acid reflux of bile may cause genotoxic stress. These aspects merit to be tested in wide spectrum of Barrett epithelial tissues. PMID:27247425

  8. Specific Oligopeptides in Fermented Soybean Extract Inhibit NF-κB-Dependent iNOS and Cytokine Induction by Toll-Like Receptor Ligands

    PubMed Central

    Lee, Woo Hyung; Wu, Hong Min; Lee, Chan Gyu; Sung, Dae Il; Song, Hye Jung; Matsui, Toshiro

    2014-01-01

    Abstract The ethanol extract of fermented soybean from Glycine max (chungkookjang, CHU) has been claimed to have chemopreventive and cytoprotective effects. In the present study, we examined the inhibitory effect of CHU on inducible nitric oxide synthase (iNOS) and cytokine induction by toll-like receptor (TLR) ligands treatment and attempted to identify the responsible active components. Nitric oxide (NO) content and iNOS levels in the media or RAW264.7 cells were measured using the Griess reagent and real-time polymerase chain reaction assays. CHU treatment inhibited NO production and iNOS induction elicited by lipopolysaccharide (LPS, TLR4L) in a concentration-dependent manner. Tumor necrosis factor-α and interleukin-6 productions were also diminished. Peptidoglycans (TLR2/6L) and CpG-oligodeoxynucleotides (TLR9L) from CHU inhibited iNOS induction, but not poly I:C (TLR3L) or loxoribine (TLF7L). The anti-inflammatory effect resulted from the inhibition of nuclear factor-kappa B (NF-κB) through the inhibition of inhibitory-κB degradation. Of the representative components in CHU, specific oligopeptides (AFPG and GVAWWMY) had the ability to inhibit iNOS induction by LPS, whereas others failed to do so. Daidzein, an isoflavone used for comparative purposes, was active at a relatively higher concentration. In an animal model, oral administration of CHU to rats significantly diminished carrageenan-induced paw edema and iNOS induction. Our results demonstrate that CHU has anti-inflammatory effects against TLR ligands by inhibiting NF-κB activation, which may result from specific oligopeptide components in CHU. Since CHU is orally effective, dietary applications of CHU and/or the identified oligopeptides may be of use in the prevention of inflammatory diseases. PMID:25184943

  9. Achillea Millefolium L. Hydro- Alcoholic Extract Protects Pancreatic Cells by Down Regulating IL- 1β and iNOS Gene Expression in Diabetic Rats

    PubMed Central

    Zolghadri, Yalda; Fazeli, Mehdi; Kooshki, Marzieh; Shomali, Tahoora; Karimaghayee, Negar; Dehghani, Maryam

    2014-01-01

    Interleukin-1β (IL-1β) has a role in β- cell destruction in autoimmune diabetes by stimulating the expression of inducible nitric oxide synthase (iNOS) that generates the free radical nitric oxide. We aimed to investigate the effect of Achillea millefolium L, as a traditional hypoglycemic agent, on IL-1β and iNOS gene expression of pancreatic tissue in the STZ- induced diabetic rats. Forty adult male Wistar rats were randomly divided into four groups: 1. diabetic control; 2. diabetic rats treated with Achillea millefolium L. extract; 3. normal rats received only extract and 4. negative control (n= 10 each). Diabetes was induced by single i.p. injection of 45 mg/ kg streptozotocin (STZ). Rats in groups 2 and 3 were treated with i.p. injection of Achillea millefolium L. extract (100 mg/ kg/ day) for 14 days. Body weight, serum glucose and insulin levels were assayed at baseline and on days 3, 7, 10 and 14 of the experiment. Finally, the quantity of pancreatic IL-1β and iNOS mRNA was determined by real- time PCR. The mRNA expression level of IL-1β and iNOS genes, was significantly (p<0.001) increased in diabetic rats of group 1. Treatment with Achillea millefolium L. caused a significant (p<0.01) reduction in both IL-1β and iNOS genes expression. Moreover, rats in group 2 had higher insulin level associated with lower glucose level and higher body weight compared to control diabetic group. It seems that beneficial effect of Achillea millefolium L. on STZ- induced diabetes is at least partly due to amelioration of IL-1β and iNOS gene over expression which can have a β-cell protective effect. PMID:25635252

  10. Unexpected versatility in bacterial riboswitches.

    PubMed

    Mellin, J R; Cossart, Pascale

    2015-03-01

    Bacterial riboswitches are elements present in the 5'-untranslated regions (UTRs) of mRNA molecules that bind to ligands and regulate the expression of downstream genes. Riboswitches typically regulate the expression of protein-coding genes. However, mechanisms of riboswitch-mediated regulation have recently been shown to be more diverse than originally thought, with reports showing that riboswitches can regulate the expression of noncoding RNAs and control the access of proteins, such as transcription termination factor Rho and RNase E, to a nascent RNA. Riboswitches are also increasingly used in biotechnology, with advances in the engineering of synthetic riboswitches and the development of riboswitch-based sensors. In this review we address the emerging roles and mechanisms of riboswitch-mediated regulation in natura and recent progress in the development of riboswitch-based technology. PMID:25708284

  11. Set1/COMPASS and Mediator are repurposed to promote epigenetic transcriptional memory

    PubMed Central

    D'Urso, Agustina; Takahashi, Yoh-hei; Xiong, Bin; Marone, Jessica; Coukos, Robert; Randise-Hinchliff, Carlo; Wang, Ji-Ping; Shilatifard, Ali; Brickner, Jason H

    2016-01-01

    In yeast and humans, previous experiences can lead to epigenetic transcriptional memory: repressed genes that exhibit mitotically heritable changes in chromatin structure and promoter recruitment of poised RNA polymerase II preinitiation complex (RNAPII PIC), which enhances future reactivation. Here, we show that INO1 memory in yeast is initiated by binding of the Sfl1 transcription factor to the cis-acting Memory Recruitment Sequence, targeting INO1 to the nuclear periphery. Memory requires a remodeled form of the Set1/COMPASS methyltransferase lacking Spp1, which dimethylates histone H3 lysine 4 (H3K4me2). H3K4me2 recruits the SET3C complex, which plays an essential role in maintaining this mark. Finally, while active INO1 is associated with Cdk8- Mediator, during memory, Cdk8+ Mediator recruits poised RNAPII PIC lacking the Kin28 CTD kinase. Aspects of this mechanism are generalizable to yeast and conserved in human cells. Thus, COMPASS and Mediator are repurposed to promote epigenetic transcriptional poising by a highly conserved mechanism. DOI: http://dx.doi.org/10.7554/eLife.16691.001 PMID:27336723

  12. Set1/COMPASS and Mediator are repurposed to promote epigenetic transcriptional memory.

    PubMed

    D'Urso, Agustina; Takahashi, Yoh-Hei; Xiong, Bin; Marone, Jessica; Coukos, Robert; Randise-Hinchliff, Carlo; Wang, Ji-Ping; Shilatifard, Ali; Brickner, Jason H

    2016-01-01

    In yeast and humans, previous experiences can lead to epigenetic transcriptional memory: repressed genes that exhibit mitotically heritable changes in chromatin structure and promoter recruitment of poised RNA polymerase II preinitiation complex (RNAPII PIC), which enhances future reactivation. Here, we show that INO1 memory in yeast is initiated by binding of the Sfl1 transcription factor to the cis-acting Memory Recruitment Sequence, targeting INO1 to the nuclear periphery. Memory requires a remodeled form of the Set1/COMPASS methyltransferase lacking Spp1, which dimethylates histone H3 lysine 4 (H3K4me2). H3K4me2 recruits the SET3C complex, which plays an essential role in maintaining this mark. Finally, while active INO1 is associated with Cdk8(-) Mediator, during memory, Cdk8(+) Mediator recruits poised RNAPII PIC lacking the Kin28 CTD kinase. Aspects of this mechanism are generalizable to yeast and conserved in human cells. Thus, COMPASS and Mediator are repurposed to promote epigenetic transcriptional poising by a highly conserved mechanism. PMID:27336723

  13. Small bowel bacterial overgrowth

    MedlinePlus

    Overgrowth - intestinal bacteria; Bacterial overgrowth - intestine; Small intestinal bacterial overgrowth; SIBO ... Most of the time, the small intestine does not have a high number ... in the small intestine may use up the nutrients needed by the ...

  14. Measurement of integrated flux of cosmic ray muons at sea level using the INO-ICAL prototype detector

    SciTech Connect

    Pal, S.; Acharya, B.S.; Majumder, G.; Mondal, N.K.; Samuel, D.; Satyanarayana, B. E-mail: acharya@tifr.res.in E-mail: nkm@tifr.res.in E-mail: bsn@tifr.res.in

    2012-07-01

    The India-based Neutrino Observatory (INO) collaboration is planning to set-up a magnetized Iron-CALorimeter (ICAL) to study atmospheric neutrino oscillations with precise measurements of oscillations parameters. The ICAL uses 50 kton iron as target mass and about 28800 Resistive Plate Chambers (RPC) of 2 m × 2 m in area as active detector elements. As part of its R and D program, a prototype detector stack comprising 12 layers of RPCs of 1 m × 1 m in area has been set-up at Tata Institute of Fundamental Research (TIFR) to study the detector parameters using cosmic ray muons. We present here a study of muon flux measurement at sea level and lower latitude. (Site latitude: 18°54'N, longitude: 72°48'E.)

  15. 4-dimethylamino-3',4'-dimethoxychalcone downregulates iNOS expression and exerts anti-inflammatory effects.

    PubMed

    Herencia, F; Ferrándiz, M L; Ubeda, A; Guillén, I; Dominguez, J N; Charris, J E; Lobo, G M; Alcaraz, M J

    2001-01-01

    Reactive oxygen and nitrogen species contribute to the pathophysiology of inflammatory conditions. We have studied the effects of a novel superoxide scavenger, 4-dimethylamino-3', 4'-dimethoxychalcone (CH11) in macrophages and in vivo. CH11 has been shown to inhibit the chemiluminescence induced by zymosan in mouse peritoneal macrophages and the cytotoxic effects of superoxide. In the same cells, the modulation by superoxide of nitric oxide (NO) production in response to zymosan was investigated. CH11 was more effective than the membrane-permeable scavenger Tiron for inhibition of inducible nitric oxide synthase (iNOS) protein expression and nitrite production. We have shown that CH11 inhibited chemiluminescence in vivo, as well as cell migration, and eicosanoid and tumor necrosis factor-alpha (TNF-alpha) levels in the mouse air pouch injected with zymosan. This chalcone derivative also exerted anti-inflammatory effects in the carrageenan paw oedema.

  16. Activated protein C ameliorates LPS-induced acute kidney injury and downregulates renal INOS and angiotensin 2.

    PubMed

    Gupta, Akanksha; Rhodes, George J; Berg, David T; Gerlitz, Bruce; Molitoris, Bruce A; Grinnell, Brian W

    2007-07-01

    Endothelial dysfunction contributes significantly to acute renal failure (ARF) during inflammatory diseases including septic shock. Previous studies have shown that activated protein C (APC) exhibits anti-inflammatory properties and modulates endothelial function. Therefore, we investigated the effect of APC on ARF in a rat model of endotoxemia. Rats subjected to lipopolysaccharide (LPS) treatment exhibited ARF as illustrated by markedly reduced peritubular capillary flow and increased serum blood urea nitrogen (BUN) levels. Using quantitative two-photon intravital microscopy, we observed that at 3 h post-LPS treatment, rat APC (0.1 mg/kg iv bolus) significantly improved peritubular capillary flow [288 +/- 15 microm/s (LPS) vs. 734 +/- 59 microm/s (LPS+APC), P = 0.0009, n = 6], and reduced leukocyte adhesion (P = 0.003) and rolling (P = 0.01) compared with the LPS-treated group. Additional experiments demonstrated that APC treatment significantly improved renal blood flow and reduced serum BUN levels compared with 24-h post-LPS treatment. Biochemical analysis revealed that APC downregulated inducible nitric oxide synthase (iNOS) mRNA levels and NO by-products in the kidney. In addition, APC modulated the renin-angiotensin system by reducing mRNA expression levels of angiotensin-converting enzyme-1 (ACE1), angiotensinogen, and increasing ACE2 mRNA levels in the kidney. Furthermore, APC significantly reduced ANG II levels in the kidney compared with the LPS-treated group. Taken together, these data suggest that APC can suppress LPS-induced ARF by modulating factors involved in vascular inflammation, including downregulation of renal iNOS and ANG II systems. Furthermore, the data suggest a potential therapeutic role for APC in the treatment of ARF.

  17. Ginsenoside Rg3 regulates S-nitrosylation of the NLRP3 inflammasome via suppression of iNOS

    SciTech Connect

    Yoon, Sung-Jin; Park, Jun-Young; Choi, Song; Lee, Jin-Bong; Jung, Haiyoung; Kim, Tae-Don; Yoon, Suk Ran; Choi, Inpyo; Shim, Sungbo; Park, Young-Jun

    2015-08-07

    Ginsenoside Rg3, a specific biological effector, is well-known as a major bioactive ingredient of Panax ginseng. However, its role in the inflammasome activation process remains unclear. In this report, we demonstrate that ginsenosides 20(R)-Rg3 and 20(S)-Rg3 are capable of suppressing both lethal endotoxic shock and the S-nitrosylation of the NLRP3 inflammasome by inhibiting nitric oxide (NO) production through the regulation of inducible nitric oxide synthase (iNOS) expression. In response to lipopolysaccharide (LPS), the reducing effect of 20(S)-Rg3 and 20(R)-Rg3 on nitric oxide led to an increase in the survival time of mice after lethal endotoxin-induced shock, and excess levels of NO inhibited IL-1β production via the S-nitrosylation of the NLRP3 inflammasome. In addition, ginsenosides 20(R)-Rg3 and 20(S)-Rg3 had suppressive effects on the LPS- or UV-irradiation-induced reactive oxygen species (ROS) levels in macrophage and HaCaT cells and thereby prevented apoptosis of spleen cells in mice. Altogether, these results demonstrate that ginsenoside 20(R)-Rg3 and 20(S)-Rg3, a naturally occurring compound, might act as a dual therapeutic regulator for the treatment of inflammatory and oxidative stress-related diseases. - Highlights: • Ginsenosides Rg3 inhibits NO production through the regulation of iNOS expression. • Ginsenosides Rg3 inhibits the S-nitrosylation of the NLRP3 inflammasome. • Ginsenosides Rg3 suppress on the LPS- or UV-irradiation-induced ROS levels in cells.

  18. Effect of sildenafil citrate on interleukin-1β-induced nitric oxide synthesis and iNOS expression in SW982 cells

    PubMed Central

    Kim, Kyung-Ok; Park, Shin-Young; Han, Chang-Woo; Chung, Hyun Kee; Ryu, Dae-Hyun

    2008-01-01

    The purpose of this study was to identify the effect of sildenafil citrate on IL-1β-induced nitric oxide (NO) synthesis and iNOS expression in human synovial sarcoma SW982 cells. IL-1β stimulated the cells to generate NO in both dose- and time-dependent manners. The IL-1β-induced NO synthesis was inhibited by guanylate cyclase (GC) inhibitor, LY83583. When the cells were treated with 8-bromo-cGMP, a hydrolyzable analog of cGMP, NO synthesis was increased upto 5-fold without IL-1β treatment suggesting that cGMP is an essential component for increasing the NO synthesis. Synoviocytes and chondrocytes contain strong cGMP phosphodiesterase (PDE) activity, which has biochemical features of PDE5. When SW982 cells were pretreated with sildenafil citrate (Viagra), a PDE5 specific inhibitor, sildenafil citrate significantly inhibited IL-1β-induced NO synthesis and iNOS expressions. From this result, we noticed that PDE5 activity is required for IL-1β-induced NO synthesis and iNOS expressions in human synovial sarcoma cells, and sildenafil citrate may be able to suppress an inflammatory reaction of synovium through inhibition of NO synthesis and iNOS expression by cytokines. PMID:18587266

  19. Effect of ulinastatin on the expression of iNOS, MMP-2, and MMP-3 in degenerated nucleus pulposus cells of rabbits.

    PubMed

    Hua, Guo; Haiping, Zhang; Baorong, He; Dingjun, Hao

    2013-01-01

    We examined the effects of ulinastatin on the expression of inducible nitric oxide synthase (iNOS), matrix metalloproteinase-2 (MMP-2), and matrix metalloproteinase-3 (MMP-3) in degenerated nucleus pulposus (NP) cells of rabbits induced by interleukin (IL)-1β in vitro. An in vitro NP cell culture model was set up with enzyme digestion. NP cells from adult white rabbits were divided into six groups: the normal control group, the ulinastatin control group (320 U/mL ulinastatin), the induced group (10 ng/mL IL-1β), and three inhibition groups (IL-1β followed by 160, 320, or 640 U/mL ulinastatin). After a 2-day culture, the NP cells were collected for immunohistochemical staining for MMP-2 and MMP-3 and spectrophotometric analysis of the amount of iNOS. Immunohistochemical staining showed that the expression of MMP-2 and MMP-3 proteins in NP cells decreased in the inhibition groups compared with the induced group, which was in inverse proportion to the ulinastatin concentration. Spectrophotometric results showed that, compared with the induced group, the iNOS content in each inhibition group decreased, most significantly in the 320 U/mL group. Ulinastatin effectively inhibited the increased expression of MMP-2, MMP-3, and iNOS in degenerated NP cells induced by IL-1β in vitro. It suggests that ulinastatin may potentially be useful for clinical therapy of intervertebral disc degeneration.

  20. Constitutive intracellular production of iNOS and NO in human melanoma: possible role in regulation of growth and resistance to apoptosis.

    PubMed

    Grimm, Elizabeth A; Ellerhorst, Julie; Tang, Chi-Hui; Ekmekcioglu, Suhendan

    2008-09-01

    Human melanoma tumors cells are known to express the enzyme, inducible nitric oxide synthase (iNOS), which is responsible for cytokine induced nitric oxide (NO) production during immune responses. This constitutive expression of iNOS in many patients' tumor cells, as well as its strong association with poor patient survival, have led to the consideration of iNOS as a molecular marker of poor prognosis, as well as a possible target for therapy. The expression of iNOS in patient tumors was found to associate with nitrotyrosine, COX2, pSTAT3, and arginase. Using human melanoma patients' samples as well as cell lines, we have further evidence supporting intracellular NO production by detection of nitrotyrosine and also by use of DAF-2DA staining. Experiments were performed to scavenge the endogenous NO (with c-PTIO) resulting in melanoma cell growth inhibition; this was restored with SIN-1 (NO and O2-donor) providing data to support a functional role of this gas. Our goal is to understand the aberrant biology leading to this curious phenomenon, and to regulate it in favor of patient treatments.

  1. Looking for a heavy W-ino lightest supersymmetric particle in collider and dark matter experiments

    SciTech Connect

    Chattopadhyay, Utpal; Das, Debottam; Konar, Partha; Roy, D. P.

    2007-04-01

    We investigate the phenomenology of a wino lightest superparticle as obtained in anomaly mediated supersymmetry breaking and some string models. The Wilkinson Microwave Anisotropy Probe constraint on the dark matter relic density implies a wino lightest superparticle mass of 2.0-2.3 TeV. We find a viable signature for such a heavy wino at CLIC, operating at its highest center of mass energy of 5 TeV. One also expects a viable monochromatic {gamma}-ray signal from its pair-annihilation at the galactic center at least for cuspy dark matter halo profiles.

  2. Later phase cardioprotection of ischemic post-conditioning against ischemia/reperfusion injury depends on iNOS and PI3K-Akt pathway

    PubMed Central

    Wang, Gongming; Li, Xin; Wang, Hong; Wang, Yan; Zhang, Ligong; Zhang, Le; Liu, Bei; Zhang, Mengyuan

    2015-01-01

    Background: The cardioprotection of ischemic post-conditioning (IPO) has been well demonstrated after a short period of reperfusion. However, little is known about the long-term effects of IPO. This study aimed to investigate the long term cardioprotection of IPO in a rat myocardial ischemia/reperfusion model and to explore the potential mechanism. Methods and results: Rats were either sham-operated (Sham group) or underwent 30-min left anterior descending coronary artery ischemia followed by immediate reperfusion (I/R group) or post-conditioning with 5 cycles of 10-s ischemia and 10-s reperfusion (IPO group). At 24 h after reperfusion, infarct size reduced from 34.7±1.1% in I/R group to 24.9±1.3% in IPO group (P<0.05) and the iNOS expression in IPO group was 4.7-fold higher than in I/R group. iNOS inhibitor 1400 W (1 mg/kg, 5 min before postconditioning or reperfusion) prevented the increase in iNOS expression and abolished IPO-induced protection (34.4±1.0%, P>0.05 vs. I/R group). When rats were treated with PI3K inhibitor LY294002 5 min before reperfusion (0.3 mg/kg), p-Akt expression at R 3 h and iNOS expression at R 24 h were significantly inhibited. Moreover, the delayed infarct-sparing effect of IPO was absent in the presence of LY294002. Conclusion: IPO has prolonged cardioprotective effects and iNOS as an important downstream effector of PI3K-Akt pathway contributes to the delayed phase cardioprotection of IPO. PMID:26885260

  3. Impact of the Tumor Microenvironment on the Expression of Inflammatory Mediators in Cancer Cells.

    PubMed

    Riemann, A; Ihling, A; Reime, S; Gekle, M; Thews, O

    2016-01-01

    Hypoxia and extracellular acidosis are common features of solid malignant tumors. The aim of the study was to analyze whether these pathophysiological parameters affect the expression of inflammatory mediators in tumor cells. Therefore the mRNA expression of MCP-1 (monocyte chemotactic protein 1), iNOS and osteopontin was measured under hypoxic (pO2 1 mmHg) and acidotic (pH 6.6) conditions by qPCR in AT1 R-3327 prostate cancer cells. In addition, the underlying signaling cascades were analyzed by using inhibitors of the p38 and ERK1/2 MAP kinase pathways.Hypoxia led to a significant decrease of the expression of MCP-1 and osteopontin over the complete observation period of 24 h, whereas the iNOS expression after an initial reduction slightly increased. Acidotic conditions for up to 6 h increased the iNOS expression significantly which was functional as indicated by an elevated level of nitrate/nitrite formation by 30 %. Acidosis had almost no impact on the MCP-1 expression of tumor cells, whereas the osteopontin level tended to increase leading to a significantly elevated level after 24 h at pH 6.6. Inhibiting the p38 and ERK1/2 under control conditions revealed that the MAPKs play a significant role for the regulation of the expression of inflammatory mediators. MCP-1 expression could be lowered by inhibiting ERK1/2 whereas iNOS expression was dependent on both p38 and ERK1/2 MAPK. These results indicate that the adverse tumor microenvironment affects the expression of inflammatory mediators by tumors cells and may therefore modulate the immune response within the tumor tissue. PMID:27526131

  4. Small molecule control of bacterial biofilms

    PubMed Central

    Worthington, Roberta J.; Richards, Justin J.

    2012-01-01

    Bacterial biofilms are defined as a surface attached community of bacteria embedded in a matrix of extracellular polymeric substances that they have produced. When in the biofilm state, bacteria are more resistant to antibiotics and the host immune response than are their planktonic counterparts. Biofilms are increasingly recognized as being significant in human disease, accounting for 80% of bacterial infections in the body and diseases associated with bacterial biofilms include: lung infections of cystic fibrosis, colitis, urethritis, conjunctivitis, otitis, endocarditis and periodontitis. Additionally, biofilm infections of indwelling medical devices are of particular concern, as once the device is colonized infection is virtually impossible to eradicate. Given the prominence of biofilms in infectious diseases, there has been an increased effort toward the development of small molecules that will modulate bacterial biofilm development and maintenance. In this review, we highlight the development of small molecules that inhibit and/or disperse bacterial biofilms through non-microbicidal mechanisms. The review discuses the numerous approaches that have been applied to the discovery of lead small molecules that mediate biofilm development. These approaches are grouped into: 1) the identification and development of small molecules that target one of the bacterial signaling pathways involved in biofilm regulation, 2) chemical library screening for compounds with anti-biofilm activity, and 3) the identification of natural products that possess anti-biofilm activity, and the chemical manipulation of these natural products to obtain analogues with increased activity. PMID:22733439

  5. Small molecule control of bacterial biofilms.

    PubMed

    Worthington, Roberta J; Richards, Justin J; Melander, Christian

    2012-10-01

    Bacterial biofilms are defined as a surface attached community of bacteria embedded in a matrix of extracellular polymeric substances that they have produced. When in the biofilm state, bacteria are more resistant to antibiotics and the host immune response than are their planktonic counterparts. Biofilms are increasingly recognized as being significant in human disease, accounting for 80% of bacterial infections in the body and diseases associated with bacterial biofilms include: lung infections of cystic fibrosis patients, colitis, urethritis, conjunctivitis, otitis, endocarditis and periodontitis. Additionally, biofilm infections of indwelling medical devices are of particular concern, as once the device is colonized infection is virtually impossible to eradicate. Given the prominence of biofilms in infectious diseases, there has been an increased effort toward the development of small molecules that will modulate bacterial biofilm development and maintenance. In this review, we highlight the development of small molecules that inhibit and/or disperse bacterial biofilms through non-microbicidal mechanisms. The review discuses the numerous approaches that have been applied to the discovery of lead small molecules that mediate biofilm development. These approaches are grouped into: (1) the identification and development of small molecules that target one of the bacterial signaling pathways involved in biofilm regulation, (2) chemical library screening for compounds with anti-biofilm activity, and (3) the identification of natural products that possess anti-biofilm activity, and the chemical manipulation of these natural products to obtain analogues with increased activity. PMID:22733439

  6. Structural characterization and expression analysis of a novel cysteine protease inhibitor from Haliotis discus hannai Ino.

    PubMed

    Ding, Jianfeng; Li, Li; Zhang, Guofan

    2015-02-01

    The sequence of the cysteine protease inhibitor gene of Haliotis discus hannai (designated HdCpi) was determined using the RACE method. The full-length HdCpi cDNA is 1049 bp long, and contains an open reading frame of 813 bp, encoding a 271-amino-acid protein with a calculated molecular mass of 29.83 kDa and an isoelectric point of 8.57. The deduced amino acid sequence of HdCpi contains two cystatin-like domains, and each has the structural features of the cystatin family, including three evolutionarily conserved motifs known to interact with the active sites of cysteine peptidases: the Gly residue at the N-terminus (Gly(65) and Gly(160)), the Gln-X-Val-X-Gly motif (Q(106)IVSG(110) and Q(202)VVAG(206)), and the less conserved motif at the C-terminus (S(136)W(137) and A(254)W(255)). Many putative transcription-factor-binding sites involved in the immune system and cancer occur in the promoter region of HdCpi. Quantitative real-time RT-PCR detected HdCpi expression in all the tissues examined and in the gills of abalone challenged with the bacterium Vibrio anguillarum. HdCpi transcripts were expressed in the mantle, gill, digestive tract, hemocytes, and muscle, and increased HdCpi expression was observed after bacterial stimulation. These results suggest that HdCpi is a biologically active protease inhibitor that is likely to be involved in the antibacterial response of the abalone. PMID:25463299

  7. Structural characterization and expression analysis of a novel cysteine protease inhibitor from Haliotis discus hannai Ino.

    PubMed

    Ding, Jianfeng; Li, Li; Zhang, Guofan

    2015-02-01

    The sequence of the cysteine protease inhibitor gene of Haliotis discus hannai (designated HdCpi) was determined using the RACE method. The full-length HdCpi cDNA is 1049 bp long, and contains an open reading frame of 813 bp, encoding a 271-amino-acid protein with a calculated molecular mass of 29.83 kDa and an isoelectric point of 8.57. The deduced amino acid sequence of HdCpi contains two cystatin-like domains, and each has the structural features of the cystatin family, including three evolutionarily conserved motifs known to interact with the active sites of cysteine peptidases: the Gly residue at the N-terminus (Gly(65) and Gly(160)), the Gln-X-Val-X-Gly motif (Q(106)IVSG(110) and Q(202)VVAG(206)), and the less conserved motif at the C-terminus (S(136)W(137) and A(254)W(255)). Many putative transcription-factor-binding sites involved in the immune system and cancer occur in the promoter region of HdCpi. Quantitative real-time RT-PCR detected HdCpi expression in all the tissues examined and in the gills of abalone challenged with the bacterium Vibrio anguillarum. HdCpi transcripts were expressed in the mantle, gill, digestive tract, hemocytes, and muscle, and increased HdCpi expression was observed after bacterial stimulation. These results suggest that HdCpi is a biologically active protease inhibitor that is likely to be involved in the antibacterial response of the abalone.

  8. The role of macrophage mediators in respirable quartz-elicited inflammation

    NASA Astrophysics Data System (ADS)

    van Berlo, D.; Albrecht, C.; Knaapen, A. M.; van Schooten, F. J.; Schins, R. P. F.

    2009-02-01

    The instigation and persistence of an inflammatory response is widely considered to be critically important in quartz-induced lung cancer and fibrosis. Macrophages have been long recognised as a crucial player in pulmonary inflammation, but evidence for the role of type II epithelial cells is accumulating. Investigations were performed in the rat lung type II cell line RLE and the rat alveolar macrophage cell line NR8383 using Western blotting, NF-κB immunohistochemistry and qRT-PCR of the pro-inflammatory genes iNOS and COX-2, as well as the cellular stress gene HO-1. The direct effect of quartz on pro-inflammatory signalling cascades and gene expression in RLE cells was compared to the effect of conditioned media derived from quartz-treated NR8383 cells. Conditioned media activated the NF-κB signalling pathway and induced a far stronger upregulation of iNOS mRNA than quartz itself. Quartz elicited a stronger, progressive induction of COX-2 and HO-1 mRNA. Our results suggest a differentially mediated inflammatory response, in which reactive particles themselves induce oxidative stress and activation of COX-2, while mediators released from particle-activated macrophages trigger NF-κB activation and iNOS expression in type II cells.

  9. Tilmicosin and tylosin have anti-inflammatory properties via modulation of COX-2 and iNOS gene expression and production of cytokines in LPS-induced macrophages and monocytes.

    PubMed

    Cao, Xing-Yuan; Dong, Mei; Shen, Jian-Zhong; Wu, Bei-Bei; Wu, Cong-Ming; Du, Xiang-Dang; Wang, Zhuo; Qi, Yi-Tao; Li, Bing-Yu

    2006-05-01

    Macrolides have been reported to modify the host immune and inflammatory responses both in vivo and in vitro. We examined the in vitro effect of the macrolides tilmicosin and tylosin, which are only used in the veterinary clinic, on the production of nitric oxide (NO), prostaglandin E(2) (PGE(2)) and cytokines by lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages and mouse peripheral blood mononuclear cells (PBMCs). Compared with 5 microg/mL, tilmicosin and tylosin concentrations of 10 microg/mL and 20 microg/mL significantly decreased the production of 6-keto-prostaglandin F(1alpha) (6-keto-PGF(1alpha)), PGE(2), NO, tumour necrosis factor-alpha (TNF-alpha), interleukin (IL)-1beta and IL-6, and increased IL-10 production. Cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) gene expression were also significantly reduced. These results support the opinion that macrolides may exert an anti-inflammatory effect through modulating the synthesis of several mediators and cytokines involved in the inflammatory process.

  10. Demonstrating Bacterial Flagella.

    ERIC Educational Resources Information Center

    Porter, John R.; And Others

    1992-01-01

    Describes an effective laboratory method for demonstrating bacterial flagella that utilizes the Proteus mirabilis organism and a special harvesting technique. Includes safety considerations for the laboratory exercise. (MDH)

  11. Bacterial IgA protease-mediated degradation of agIgA1 and agIgA1 immune complexes as a potential therapy for IgA Nephropathy

    PubMed Central

    Wang, Li; Li, Xueying; Shen, Hongchun; Mao, Nan; Wang, Honglian; Cui, Luke; Cheng, Yuan; Fan, Junming

    2016-01-01

    Mesangial deposition of aberrantly glycosylated IgA1 (agIgA1) and its immune complexes is a key pathogenic mechanism of IgA nephropathy (IgAN). However, treatment of IgAN remains ineffective. We report here that bacteria-derived IgA proteases are capable of degrading these pathogenic agIgA1 and derived immune complexes in vitro and in vivo. By screening 14 different bacterial strains (6 species), we found that 4 bacterial IgA proteases from H. influenzae, N. gonorrhoeae and N. meningitidis exhibited high cleaving activities on serum agIgA1 and artificial galactose-depleted IgA1 in vitro and the deposited agIgA1-containing immune complexes in the mesangium of renal biopsy from IgAN patients and in a passive mouse model of IgAN in vitro. In the modified mouse model of passive IgAN with abundant in situ mesangial deposition of the agIgA-IgG immune complexes, a single intravenous delivery of IgA protease from H. influenzae was able to effectively degrade the deposited agIgA-IgG immune complexes within the glomerulus, demonstrating a therapeutic potential for IgAN. In conclusion, the bacteria-derived IgA proteases are biologically active enzymes capable of cleaving the circulating agIgA and the deposited agIgA-IgG immune complexes within the kidney of IgAN. Thus, the use of such IgA proteases may represent a novel therapy for IgAN. PMID:27485391

  12. Bacterial IgA protease-mediated degradation of agIgA1 and agIgA1 immune complexes as a potential therapy for IgA Nephropathy.

    PubMed

    Wang, Li; Li, Xueying; Shen, Hongchun; Mao, Nan; Wang, Honglian; Cui, Luke; Cheng, Yuan; Fan, Junming

    2016-01-01

    Mesangial deposition of aberrantly glycosylated IgA1 (agIgA1) and its immune complexes is a key pathogenic mechanism of IgA nephropathy (IgAN). However, treatment of IgAN remains ineffective. We report here that bacteria-derived IgA proteases are capable of degrading these pathogenic agIgA1 and derived immune complexes in vitro and in vivo. By screening 14 different bacterial strains (6 species), we found that 4 bacterial IgA proteases from H. influenzae, N. gonorrhoeae and N. meningitidis exhibited high cleaving activities on serum agIgA1 and artificial galactose-depleted IgA1 in vitro and the deposited agIgA1-containing immune complexes in the mesangium of renal biopsy from IgAN patients and in a passive mouse model of IgAN in vitro. In the modified mouse model of passive IgAN with abundant in situ mesangial deposition of the agIgA-IgG immune complexes, a single intravenous delivery of IgA protease from H. influenzae was able to effectively degrade the deposited agIgA-IgG immune complexes within the glomerulus, demonstrating a therapeutic potential for IgAN. In conclusion, the bacteria-derived IgA proteases are biologically active enzymes capable of cleaving the circulating agIgA and the deposited agIgA-IgG immune complexes within the kidney of IgAN. Thus, the use of such IgA proteases may represent a novel therapy for IgAN. PMID:27485391

  13. Lectin purified from Musca domestica pupa up-regulates NO and iNOS production via TLR4/NF-κB signaling pathway in macrophages.

    PubMed

    Cao, Xiaohong; Zhou, Minghui; Wang, Chunling; Hou, Lihua; Zeng, Bin

    2011-04-01

    The present study reported that nitric oxide (NO) was up-regulated by the induction of lectin purified from Musca domestica pupa (MPL) in macrophages without cytotoxicity. The mRNA expression and protein secretion of inducible nitric oxide synthase (iNOS) were strongly induced by MPL treatments. Subsequent investigation revealed that the nuclear factor-κB (NF-κB) inhibitory κB (IκB) in endochylema was inhibited and NF-κB translocated into the nucleus after MPL treatment. Meanwhile, the IKKβ was strongly induced and the production of the toll-like receptor 4 (TLR4) was significantly up-regulated. Moreover, MPL increased NO production via inducing the expression of iNOS through the activation of NF-κB, which suggested that MPL probably acted as an activating agent of the NF-κB activation.

  14. Interannual variation of stable isotopes in precipitation at Bangkok in response to El Ñino Southern Oscillation

    NASA Astrophysics Data System (ADS)

    Ichiyanagi, Kimpei; Yamanaka, Manabu D.

    2005-11-01

    Evidence for a close relationship between the interannual variation of stable isotopes in precipitation and the El Ñino Southern Oscillation (ENSO) is presented for Bangkok, Thailand. Anomalies of sea surface temperature in the Niño-3 region of the equatorial Pacific (ENSO index) and 18O in precipitation in Bangkok were positively correlated for May and October. The composite mean precipitation was much greater in the isotopic low phase than in the high phase for both May and October. This suggests that the amount of precipitation is the main factor determining the observed variation of stable isotopes in precipitation in Bangkok. Composite analyses of 850 hPa temperature, evaporation, outgoing longwave radiation (OLR), and precipitation showed that the variation in the amount of precipitation in Bangkok is a response to the ENSO-Asian summer monsoon coupling in May, and a direct response to ENSO in October. The composite mean d-excess values in both the low and high phases in October and in the low phase in May were about 10, and were less than 7 during the high phase in May. A large difference in the evaporation field between the low and high phases of May exists over the Indian Ocean, suggesting that evaporation was not in equilibrium during the high phase in May. Future studies will consider the precipitation amount effect based on daily or event-based sampling. Copyright

  15. Ginsenoside Rg3 regulates S-nitrosylation of the NLRP3 inflammasome via suppression of iNOS.

    PubMed

    Yoon, Sung-Jin; Park, Jun-Young; Choi, Song; Lee, Jin-Bong; Jung, Haiyoung; Kim, Tae-Don; Yoon, Suk Ran; Choi, Inpyo; Shim, Sungbo; Park, Young-Jun

    2015-08-01

    Ginsenoside Rg3, a specific biological effector, is well-known as a major bioactive ingredient of Panax ginseng. However, its role in the inflammasome activation process remains unclear. In this report, we demonstrate that ginsenosides 20(R)-Rg3 and 20(S)-Rg3 are capable of suppressing both lethal endotoxic shock and the S-nitrosylation of the NLRP3 inflammasome by inhibiting nitric oxide (NO) production through the regulation of inducible nitric oxide synthase (iNOS) expression. In response to lipopolysaccharide (LPS), the reducing effect of 20(S)-Rg3 and 20(R)-Rg3 on nitric oxide led to an increase in the survival time of mice after lethal endotoxin-induced shock, and excess levels of NO inhibited IL-1β production via the S-nitrosylation of the NLRP3 inflammasome. In addition, ginsenosides 20(R)-Rg3 and 20(S)-Rg3 had suppressive effects on the LPS- or UV-irradiation-induced reactive oxygen species (ROS) levels in macrophage and HaCaT cells and thereby prevented apoptosis of spleen cells in mice. Altogether, these results demonstrate that ginsenoside 20(R)-Rg3 and 20(S)-Rg3, a naturally occurring compound, might act as a dual therapeutic regulator for the treatment of inflammatory and oxidative stress-related diseases. PMID:26086107

  16. Heterosis and combining ability: a diallel cross of three geographically isolated populations of Pacific abalone Haliotis discus hannai Ino

    NASA Astrophysics Data System (ADS)

    Deng, Yuewen; Liu, Xiao; Zhang, Guofan; Wu, Fucun

    2010-11-01

    We conducted a complete diallel cross among three geographically isolated populations of Pacific abalone Haliotis discus hannai Ino to determine the heterosis and the combining ability of growth traits at the spat stage. The three populations were collected from Qingdao (Q) and Dalian (D) in China, and Miyagi (M) in Japan. We measured the shell length, shell width, and total weight. The magnitude of the general combining ability (GCA) variance was more pronounced than the specific combining ability (SCA) variance, which is evidenced by both the ratio of the genetic component in total variation and the GCA/SCA values. The component variances of GCA and SCA were significant for all three traits ( P<0.05), indicating the importance of additive and non-additive genetic effects in determining the expression of these traits. The reciprocal maternal effects (RE) were also significant for these traits ( P<0.05). Our results suggest that population D was the best general combiner in breeding programs to improve growth traits. The DM cross had the highest heterosis values for all three traits.

  17. Baicalin ameliorates isoproterenol-induced acute myocardial infarction through iNOS, inflammation, oxidative stress and P38MAPK pathway in rat

    PubMed Central

    Sun, Shen-Jie; Wu, Xiao-Peng; Song, Heng-Liang; Li, Gui-Qi

    2015-01-01

    Baicalin is one of the active ingredients in the skullcap, with a variety of pharmacological effects, such as blood pressure reduction, sedation, liver-protection, gallbladder-protection, anti-bacteria, anti-inflammation, etc. The aim of this study was to investigate the potential cardioprotective effects of baicalin ameliorates isoproterenol-induced acute myocardial infarction (AMI) through inducible nitric oxide synthase (iNOS), inflammation, oxidative stress and P38MAPK passageway in rat. Rat model of AMI was induced by isoproterenol (100 mg/kg) and then treated baicalin (various does of baicalin: 1 mg/kg, 10 mg/kg and 100 mg/kg, respectively) for 24 h. Infarct size, the heart weight to body weight ratio and creatine kinase (CK), the MB isoenzyme of creatine kinase (CK-MB), lactate dehydrogenase (LDH) and cardiac troponin T (cTnT) of rats with AMI induced by isoproterenol were used to evaluate curative effect of baicalin on AMI. Meanwhile, iNOS and phosphorylation-p38 MAPK (p-p38) protein expressions, inflammatory factor and oxidative stress were inspected using western blot and commercial kits, respectively. In the present study, pre-treatment with baicalin (10 or 100 mg/kg) significantly ameliorated infarct size, the heart weight to body weight ratio and CK, CK-MB, LDH and cTnT levels in rats with AMI induced by isoproterenol. iNOS protein expression, the serum TNF-α, IL-6, MDA and SOD levels and p-38 protein expressions were significantly suppressed by treatment with baicalin (10 or 100 mg/kg). These results suggest that acute treatment with baicalin ameliorates AMI, iNOS, inflammation, oxidative stress and P38MAPK pathway in rat with AMI induced by isoproterenol. PMID:26885181

  18. Trans-cinnamaldehyde improves memory impairment by blocking microglial activation through the destabilization of iNOS mRNA in mice challenged with lipopolysaccharide.

    PubMed

    Zhang, Liqing; Zhang, Zhangang; Fu, Yan; Yang, Pin; Qin, Zhenxia; Chen, Yongjun; Xu, Ying

    2016-11-01

    Microglia activation and neuroinflammation are critically involved in pathogenesis of neurodegenerative disorders. Patients with neurodegenerative disorders often suffer memory impairment and currently there is no effective treatment for inflammation-led memory impairment. Trans-cinnamaldehyde (TCA) isolated from medicinal herb Cinnamomum cassia has been shown to exhibit anti-inflammatory capability. However, the potential of TCA to be used to improve memory impairment under neuroinflammation has not been explored. Primary microglia stimulated by lipopolysaccharide (LPS) were used to evaluate the potential anti-neuroinflammatory effects of TCA by examining the production of nitric oxide (NO), expression of inducible nitric oxide synthase (iNOS), pro-inflammatory cytokines, and activation of MAPKs. A mouse model of LPS-induced memory impairment was established to assess the neuroprotective effects of TCA against memory deficit and synaptic plasticity inhibition by both behavioral tests and electrophysiological recordings. TCA pretreatment decreased LPS-induced morphological changes, NO production and IL-1β release in primary microglia. Decreased NO production was due to the accelerated degradation of iNOS mRNA in LPS-stimulated microglia through TCA's inhibitory effect on MEK1/2-ERK1/2 signaling pathway. TCA was able to reduce the levels of iNOS and phosphorylated ERK1/2 in hippocampus of mice challenged with LPS. Most importantly, TCA significantly lessened memory deficit and improved synaptic plasticity in LPS-challenged mice. This study demonstrates that TCA suppressed microglial activation by destabilizing iNOS mRNA, which leads to improved memory impairment in mice suffering neuroinflammation.

  19. Macrophages From Irradiated Tumors Express Higher Levels of iNOS, Arginase-I and COX-2, and Promote Tumor Growth

    SciTech Connect

    Tsai, C.-S.; Chen, F.-H.; Wang, C.-C.; Huang, H.-L.; Jung, Shih-Ming; Wu, C.-J.; Lee, C.-C.; McBride, William H.; Chiang, C.-S.; Hong, J.-H. . E-mail: jihong@adm.cgmh.org.tw

    2007-06-01

    Purpose: To investigate the effects of single and fractionated doses of radiation on tumors and tumor-associated macrophages (TAMs), and to elucidate the potential of TAMs to influence tumor growth. Methods and Materials: A murine prostate cell line, TRAMP-C1, was grown in C57Bl/6J mice to 4-mm tumor diameter and irradiated with either 25 Gy in a single dose, or 60 Gy in 15 fractions. The tumors were removed at the indicated times and assessed for a variety of markers related to TAM content, activation status, and function. Results: In tumors receiving a single radiation dose, arginase (Arg-I), and cycloxygenase-2 (COX-2) mRNA expression increased as a small transient wave within 24 h and a larger persistent wave starting after 3 days. Inducible nitric oxide synthase (iNOS) mRNA was elevated only after 3 days and continued to increase up to 3 weeks. After fractionated irradiation, Arg-1 and COX-2 mRNA levels increased within 5 days, whereas iNOS was increased only after 10 fractions of irradiation had been given. Increased levels of Arg-I, COX-2, and, to a lesser extent, iNOS protein were found to associate with TAMs 1-2 weeks after tumor irradiation. Function of TAMs were compared by mixing them with TRAMP-C1 cells and injecting them into mice; TRAMP-C1 cells mixed with TAMs from irradiated tumors appeared earlier and grew significantly faster than those mixed with TAMs from unirradiated tumors or TRAMP-C1 alone. Conclusions: Tumor-associated macrophages in the postirradiated tumor microenvironment express higher levels of Arg-1, COX-2, and iNOS, and promote early tumor growth in vivo.

  20. Vimentin in Bacterial Infections

    PubMed Central

    Mak, Tim N.; Brüggemann, Holger

    2016-01-01

    Despite well-studied bacterial strategies to target actin to subvert the host cell cytoskeleton, thus promoting bacterial survival, replication, and dissemination, relatively little is known about the bacterial interaction with other components of the host cell cytoskeleton, including intermediate filaments (IFs). IFs have not only roles in maintaining the structural integrity of the cell, but they are also involved in many cellular processes including cell adhesion, immune signaling, and autophagy, processes that are important in the context of bacterial infections. Here, we summarize the knowledge about the role of IFs in bacterial infections, focusing on the type III IF protein vimentin. Recent studies have revealed the involvement of vimentin in host cell defenses, acting as ligand for several pattern recognition receptors of the innate immune system. Two main aspects of bacteria-vimentin interactions are presented in this review: the role of vimentin in pathogen-binding on the cell surface and subsequent bacterial invasion and the interaction of cytosolic vimentin and intracellular pathogens with regards to innate immune signaling. Mechanistic insight is presented involving distinct bacterial virulence factors that target vimentin to subvert its function in order to change the host cell fate in the course of a bacterial infection. PMID:27096872

  1. Bacterial Influences on Animal Origins

    PubMed Central

    Alegado, Rosanna A.; King, Nicole

    2014-01-01

    Animals evolved in seas teeming with bacteria, yet the influences of bacteria on animal origins are poorly understood. Comparisons among modern animals and their closest living relatives, the choanoflagellates, suggest that the first animals used flagellated collar cells to capture bacterial prey. The cell biology of prey capture, such as cell adhesion between predator and prey, involves mechanisms that may have been co-opted to mediate intercellular interactions during the evolution of animal multicellularity. Moreover, a history of bacterivory may have influenced the evolution of animal genomes by driving the evolution of genetic pathways for immunity and facilitating lateral gene transfer. Understanding the interactions between bacteria and the progenitors of animals may help to explain the myriad ways in which bacteria shape the biology of modern animals, including ourselves. PMID:25280764

  2. The bacterial lipopeptide iturins induce Verticillium dahliae cell death by affecting fungal signalling pathways and mediate plant defence responses involved in pathogen-associated molecular pattern-triggered immunity.

    PubMed

    Han, Qin; Wu, Fengli; Wang, Xiaonan; Qi, Hong; Shi, Liang; Ren, Ang; Liu, Qinghai; Zhao, Mingwen; Tang, Canming

    2015-04-01

    Verticillium wilt in cotton caused by Verticillium dahliae is one of the most serious plant diseases worldwide. Because no known fungicides or cotton cultivars provide sufficient protection against this pathogen, V. dahliae causes major crop yield losses. Here, an isolated cotton endophytic bacterium, designated Bacillus amyloliquefaciens 41B-1, exhibited greater than 50% biocontrol efficacy against V. dahliae in cotton plants under greenhouse conditions. Through high-performance liquid chromatography and mass analysis of the filtrate, we found that the antifungal compounds present in the strain 41B-1 culture filtrate were a series of isoforms of iturins. The purified iturins suppressed V. dahliae microsclerotial germination in the absence or presence of cotton. Treatment with the iturins induced reactive oxygen species bursts, Hog1 mitogen-activated protein kinase (MAPK) activation and defects in cell wall integrity. The oxidative stress response and high-osmolarity glycerol pathway contribute to iturins resistance in V. dahliae. In contrast, the Slt2 MAPK pathway may be involved in iturins sensitivity in this fungus. In addition to antagonism, iturins could induce plant defence responses as activators and mediate pathogen-associated molecular pattern-triggered immunity. These findings suggest that iturins may affect fungal signalling pathways and mediate plant defence responses against V. dahliae.

  3. Macrophage-mediated inflammatory response decreases mycobacterial survival in mouse MSCs by augmenting NO production

    PubMed Central

    Yang, Kun; Wu, Yongjian; Xie, Heping; Li, Miao; Ming, Siqi; Li, Liyan; Li, Meiyu; Wu, Minhao; Gong, Sitang; Huang, Xi

    2016-01-01

    Mycobacterium tuberculosis (MTB) is a hard-to-eradicate intracellular microbe, which escapes host immune attack during latent infection. Recent studies reveal that mesenchymal stem cells (MSCs) provide a protective niche for MTB to maintain latency. However, the regulation of mycobacterial residency in MSCs in the infectious microenvironment remains largely unknown. Here, we found that macrophage-mediated inflammatory response during MTB infection facilitated the clearance of bacilli residing in mouse MSCs. Higher inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production were observed in mouse MSCs under macrophage-mediated inflammatory circumstance. Blocking NO production in MSCs increased the survival of intracellular mycobacteria, indicating NO-mediated antimycobacterial activity. Moreover, both nuclear factor κB (NF-κB) and Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathways were involved in iNOS expression and NO production in inflammatory microenvironment. Furthermore, pro-inflammatory cytokine interleukin-1β could trigger NO production in MSCs and exert anti-mycobacterial activity via NF-κB signaling pathway. Neutralization of interleukin-1β in macrophage-mediated inflammatory microenvironment dampened the ability of mouse MSCs to produce NO. Together, our findings demonstrated that macrophage-mediated inflammatory response during mycobacterial infection promotes the clearance of bacilli in mouse MSCs by increasing NO production, which may provide a better understanding of latent MTB infection. PMID:27251437

  4. Macrophage-mediated inflammatory response decreases mycobacterial survival in mouse MSCs by augmenting NO production.

    PubMed

    Yang, Kun; Wu, Yongjian; Xie, Heping; Li, Miao; Ming, Siqi; Li, Liyan; Li, Meiyu; Wu, Minhao; Gong, Sitang; Huang, Xi

    2016-01-01

    Mycobacterium tuberculosis (MTB) is a hard-to-eradicate intracellular microbe, which escapes host immune attack during latent infection. Recent studies reveal that mesenchymal stem cells (MSCs) provide a protective niche for MTB to maintain latency. However, the regulation of mycobacterial residency in MSCs in the infectious microenvironment remains largely unknown. Here, we found that macrophage-mediated inflammatory response during MTB infection facilitated the clearance of bacilli residing in mouse MSCs. Higher inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production were observed in mouse MSCs under macrophage-mediated inflammatory circumstance. Blocking NO production in MSCs increased the survival of intracellular mycobacteria, indicating NO-mediated antimycobacterial activity. Moreover, both nuclear factor κB (NF-κB) and Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathways were involved in iNOS expression and NO production in inflammatory microenvironment. Furthermore, pro-inflammatory cytokine interleukin-1β could trigger NO production in MSCs and exert anti-mycobacterial activity via NF-κB signaling pathway. Neutralization of interleukin-1β in macrophage-mediated inflammatory microenvironment dampened the ability of mouse MSCs to produce NO. Together, our findings demonstrated that macrophage-mediated inflammatory response during mycobacterial infection promotes the clearance of bacilli in mouse MSCs by increasing NO production, which may provide a better understanding of latent MTB infection.

  5. Bacterial hemagglutination by Neisseria gonorrhoeae.

    PubMed Central

    Koransky, J R; Scales, R W; Kraus, S J

    1975-01-01

    Direct bacterial hemagglutination was investigated with 20 clinical isolates of Neisseria gonorrhoeae. The hemagglutination tests were performed by both a macrotechnique with glass slides and a microtechnique with autotrays. Only organisms from form type 1 or 2 colonies caused hemagglutination. There was no statistical difference at a 10% or higher level in hemagglutination powers of type 1 and type 2 organisms, of male urethral and female cervical isolates, and of the eight major human blood types (ABO-Rh). Of seven erythrocyte species tested, only human cells were agglutinated. D-Mannose did not prevent the agglutination. Rabbit antigonococcal serum and high-titer antigonococcal human sera inhibited the hemagglutination. The results suggest the pili are the mediators of hemagglutination and that their specific agglutination of human erythrocytes may be a correlate of their adherence to human mucosal cells in natural infection. Also, although the procedure is presently insensitive, it is possible to detect human antigonococcal antibody by inhibition of direct bacterial hemagglutination. Images PMID:809353

  6. Oct-2 forms a complex with Oct-1 on the iNOS promoter and represses transcription by interfering with recruitment of RNA PolII by Oct-1.

    PubMed

    Bentrari, Fatima; Chantôme, Aurelie; Knights, Andrew; Jeannin, Jean-François; Pance, Alena

    2015-11-16

    Oct-1 (POU2f1) and Oct-2 (POU2f2) are members of the POU family of transcription factors. They recognize the same DNA sequence but fulfil distinct functions: Oct-1 is ubiquitous and regulates a variety of genes while Oct-2 is restricted to B-cells and neurones. Here we examine the interplay and regulatory mechanisms of these factors to control the inducible nitric oxide synthase (iNOS, NOS2). Using two breast cancer cell lines as a comparative model, we found that MCF-7 express iNOS upon cytokine stimulation while MDA-MB-231 do not. Oct-1 is present in both cell lines but MDA-MB-231 also express high levels of Oct-2. Manipulation of Oct-2 expression in these cell lines demonstrates that it is directly responsible for the repression of iNOS in MDA-MB-231. In MCF-7 cells Oct-1 binds the iNOS promoter, recruits RNA PolII and triggers initiation of transcription. In MDA-MB-231 cells, both Oct-1 and Oct-2 bind the iNOS promoter, forming a higher-order complex which fails to recruit RNA PolII, and as a consequence iNOS transcription does not proceed. Unravelling the mechanisms of transcription factor activity is paramount to the understanding of gene expression patterns that determine cell behaviour.

  7. Oct-2 forms a complex with Oct-1 on the iNOS promoter and represses transcription by interfering with recruitment of RNA PolII by Oct-1

    PubMed Central

    Bentrari, Fatima; Chantôme, Aurelie; Knights, Andrew; Jeannin, Jean-François; Pance, Alena

    2015-01-01

    Oct-1 (POU2f1) and Oct-2 (POU2f2) are members of the POU family of transcription factors. They recognize the same DNA sequence but fulfil distinct functions: Oct-1 is ubiquitous and regulates a variety of genes while Oct-2 is restricted to B-cells and neurones. Here we examine the interplay and regulatory mechanisms of these factors to control the inducible nitric oxide synthase (iNOS, NOS2). Using two breast cancer cell lines as a comparative model, we found that MCF-7 express iNOS upon cytokine stimulation while MDA-MB-231 do not. Oct-1 is present in both cell lines but MDA-MB-231also express high levels of Oct-2. Manipulation of Oct-2 expression in these cell lines demonstrates that it is directly responsible for the repression of iNOS in MDA-MB-231. In MCF-7 cells Oct-1 binds the iNOS promoter, recruits RNA PolII and triggers initiation of transcription. In MDA-MB-231 cells, both Oct-1 and Oct-2 bind the iNOS promoter, forming a higher-order complex which fails to recruit RNA PolII, and as a consequence iNOS transcription does not proceed. Unravelling the mechanisms of transcription factor activity is paramount to the understanding of gene expression patterns that determine cell behaviour. PMID:26271992

  8. Obesity, Inflammation, and Exercise Training: Relative Contribution of iNOS and eNOS in the Modulation of Vascular Function in the Mouse Aorta

    PubMed Central

    Silva, Josiane F.; Correa, Izabella C.; Diniz, Thiago F.; Lima, Paulo M.; Santos, Roger L.; Cortes, Steyner F.; Coimbra, Cândido C.; Lemos, Virginia S.

    2016-01-01

    Background: The understanding of obsesity-related vascular dysfunction remains controversial mainly because of the diseases associated with vascular injury. Exercise training is known to prevent vascular dysfunction. Using an obesity model without comorbidities, we aimed at investigating the underlying mechanism of vascular dysfunction and how exercise interferes with this process. Methods: High-sugar diet was used to induce obesity in mice. Exercise training was performed 5 days/week. Body weight, energy intake, and adipose tissues were assessed; blood metabolic and hormonal parameters were determined; and serum TNFα was measured. Blood pressure and heart rate were assessed by plethysmography. Changes in aortic isometric tension were recorded on myograph. Western blot was used to analyze protein expression. Nitric oxide (NO) was evaluated using fluorescence microscopy. Antisense oligodeoxynucleotides were used for inducible nitric oxide synthase isoform (iNOS) knockdown. Results: Body weight, fat mass, total cholesterol, low-density lipoprotein cholesterol fraction, insulin, and leptin were higher in the sedentary obese group (SD) than in the sedentary control animals (SS). Exercise training prevented these changes. No difference in glucose tolerance, insulin sensitivity, blood pressure, and heart rate was found. Decreased vascular relaxation and reduced endothelial nitric oxide synthase (eNOS) functioning in the SD group were prevented by exercise. Contractile response to phenylephrine was decreased in the aortas of the wild SD mice, compared with that of the SS group; however, no alteration was noted in the SD iNOS−/− animals. The decreased contractility was endothelium-dependent, and was reverted by iNOS inhibition or iNOS silencing. The aortas from the SD group showed increased basal NO production, serum TNFα, TNF receptor-1, and phospho-IκB. Exercise training attenuated iNOS-dependent reduction in contractile response in high-sugar diet–fed animals

  9. Obesity, Inflammation, and Exercise Training: Relative Contribution of iNOS and eNOS in the Modulation of Vascular Function in the Mouse Aorta

    PubMed Central

    Silva, Josiane F.; Correa, Izabella C.; Diniz, Thiago F.; Lima, Paulo M.; Santos, Roger L.; Cortes, Steyner F.; Coimbra, Cândido C.; Lemos, Virginia S.

    2016-01-01

    Background: The understanding of obsesity-related vascular dysfunction remains controversial mainly because of the diseases associated with vascular injury. Exercise training is known to prevent vascular dysfunction. Using an obesity model without comorbidities, we aimed at investigating the underlying mechanism of vascular dysfunction and how exercise interferes with this process. Methods: High-sugar diet was used to induce obesity in mice. Exercise training was performed 5 days/week. Body weight, energy intake, and adipose tissues were assessed; blood metabolic and hormonal parameters were determined; and serum TNFα was measured. Blood pressure and heart rate were assessed by plethysmography. Changes in aortic isometric tension were recorded on myograph. Western blot was used to analyze protein expression. Nitric oxide (NO) was evaluated using fluorescence microscopy. Antisense oligodeoxynucleotides were used for inducible nitric oxide synthase isoform (iNOS) knockdown. Results: Body weight, fat mass, total cholesterol, low-density lipoprotein cholesterol fraction, insulin, and leptin were higher in the sedentary obese group (SD) than in the sedentary control animals (SS). Exercise training prevented these changes. No difference in glucose tolerance, insulin sensitivity, blood pressure, and heart rate was found. Decreased vascular relaxation and reduced endothelial nitric oxide synthase (eNOS) functioning in the SD group were prevented by exercise. Contractile response to phenylephrine was decreased in the aortas of the wild SD mice, compared with that of the SS group; however, no alteration was noted in the SD iNOS−/− animals. The decreased contractility was endothelium-dependent, and was reverted by iNOS inhibition or iNOS silencing. The aortas from the SD group showed increased basal NO production, serum TNFα, TNF receptor-1, and phospho-IκB. Exercise training attenuated iNOS-dependent reduction in contractile response in high-sugar diet–fed animals

  10. ABC transporters: bacterial exporters.

    PubMed Central

    Fath, M J; Kolter, R

    1993-01-01

    The ABC transporters (also called traffic ATPases) make up a large superfamily of proteins which share a common function and a common ATP-binding domain. ABC transporters are classified into three major groups: bacterial importers (the periplasmic permeases), eukaryotic transporters, and bacterial exporters. We present a comprehensive review of the bacterial ABC exporter group, which currently includes over 40 systems. The bacterial ABC exporter systems are functionally subdivided on the basis of the type of substrate that each translocates. We describe three main groups: protein exporters, peptide exporters, and systems that transport nonprotein substrates. Prototype exporters from each group are described in detail to illustrate our current understanding of this protein family. The prototype systems include the alpha-hemolysin, colicin V, and capsular polysaccharide exporters from Escherichia coli, the protease exporter from Erwinia chrysanthemi, and the glucan exporters from Agrobacterium tumefaciens and Rhizobium meliloti. Phylogenetic analysis of the ATP-binding domains from 29 bacterial ABC exporters indicates that the bacterial ABC exporters can be divided into two primary branches. One branch contains the transport systems where the ATP-binding domain and the membrane-spanning domain are present on the same polypeptide, and the other branch contains the systems where these domains are found on separate polypeptides. Differences in substrate specificity do not correlate with evolutionary relatedness. A complete survey of the known and putative bacterial ABC exporters is included at the end of the review. PMID:8302219

  11. Bacterial vectors and delivery systems in cancer therapy.

    PubMed

    Gardlik, Roman; Fruehauf, Johannes H

    2010-10-01

    Live bacterial vectors may be useful tools for the development of novel cancer therapies that can be added to the repertoire of existing drugs. Several bacterial strains effectively colonize solid tumors and act as antitumor therapeutics. The naturally occurring tumor-colonizing characteristics of bacterial species such as Salmonella sp, Clostridium sp and Escherichia coli can be further modified by genetic manipulations, making these bacterial systems excellent vehicles for the production and targeted delivery of therapeutic molecules into cancer cells. This feature review summarizes recent research on cancer therapy using genetically modified bacteria. Different approaches - bactofection, DNA vaccination, and bacterially mediated protein and RNAi delivery - in which modified bacteria are used as anticancer therapeutics, are discussed.

  12. Plasmids spread very fast in heterogeneous bacterial communities.

    PubMed Central

    Dionisio, Francisco; Matic, Ivan; Radman, Miroslav; Rodrigues, Olivia R; Taddei, François

    2002-01-01

    Conjugative plasmids can mediate gene transfer between bacterial taxa in diverse environments. The ability to donate the F-type conjugative plasmid R1 greatly varies among enteric bacteria due to the interaction of the system that represses sex-pili formations (products of finOP) of plasmids already harbored by a bacterial strain with those of the R1 plasmid. The presence of efficient donors in heterogeneous bacterial populations can accelerate plasmid transfer and can spread by several orders of magnitude. Such donors allow millions of other bacteria to acquire the plasmid in a matter of days whereas, in the absence of such strains, plasmid dissemination would take years. This "amplification effect" could have an impact on the evolution of bacterial pathogens that exist in heterogeneous bacterial communities because conjugative plasmids can carry virulence or antibiotic-resistance genes. PMID:12524329

  13. L-arginine stimulates CAT-1-mediated arginine uptake and regulation of inducible nitric oxide synthase for the growth of chick intestinal epithelial cells.

    PubMed

    Yuan, Chao; Zhang, Xiaoyun; He, Qiang; Li, Junming; Lu, Jianjun; Zou, Xiaoting

    2015-01-01

    L-arginine (L-Arg) uptake is mediated by members of cationic amino acid transporter (CAT) family and may coincide with the induction of nitric oxide synthases (NOS). The present study was conducted to investigate the extracellular concentrations of L-Arg regulating the CAT-1, CAT-4 and inducible NOS (iNOS) in chick intestinal epithelial cells. The cells were cultured for 4 days in Arg-free Dulbecco's modified Eagle's medium containing 10, 100, 200, 400, or 600 μM L-Arg. Cell viability, nitric oxide (NO) concentrations, uptake and metabolism of L-[3H]-Arg as well as expression of CAT-1, CAT-4, and iNOS were determined. Our results showed that L-Arg enhances cell growth with a maximal response at 10-400 μM. Addition of 100, 200, or 400 μM L-Arg increased the L-[3H]-Arg uptake, which was associated with greater conversion of L-[3H]-citrulline and NO production in comparison with 10 μM L-Arg group. Increasing extracellular concentrations of L-Arg from 10 to 400 μM dose dependently increased the levels of CAT-1 mRNA and protein, while no effect on CAT-4 mRNA abundance was found. Furthermore, supplementation of 100, 200, or 400 μM L-Arg upregulated the expression of iNOS mRNA, and the relative protein levels for iNOS in 200 and 400 μM L-Arg groups were higher than those in 10 and 100 μM L-Arg groups. Collectively, we conclude that the CAT-1 isoform plays a role in L-Arg uptake, and L-Arg-mediated elevation of NO via iNOS promotes the growth of chick intestinal epithelial cells.

  14. Mechanisms of bacterial pathogenicity

    PubMed Central

    Wilson, J; Schurr, M; LeBlanc, C; Ramamurthy, R; Buchanan, K; Nickerson, C

    2002-01-01

    Pathogenic bacteria utilise a number of mechanisms to cause disease in human hosts. Bacterial pathogens express a wide range of molecules that bind host cell targets to facilitate a variety of different host responses. The molecular strategies used by bacteria to interact with the host can be unique to specific pathogens or conserved across several different species. A key to fighting bacterial disease is the identification and characterisation of all these different strategies. The availability of complete genome sequences for several bacterial pathogens coupled with bioinformatics will lead to significant advances toward this goal. PMID:11930024

  15. Bacterial challenges in food

    PubMed Central

    Collee, J. G.

    1974-01-01

    Qualitative and quantitative aspects of bacterial challenges that might be encountered in food are discussed with reference to recognized and relatively unrecognized hazards. Mechanisms of pathogenicity are reviewed and the populations at risk are noted. The bacterial content of food as it is served at table merits more study. The challenge of prevention by education is discussed. Indirect bacterial challenges in our food are considered. The real challenge of diagnosis depends upon an awareness of a complex range of conditions; the importance of effective communication with efficient laboratory and epidemiological services is stressed. There is an increasing need for care in the preparation and distribution of food. PMID:4467860

  16. The Fate of Bacterial Cocaine Esterase (CocE): An In Vivo Study of CocE-Mediated Cocaine Hydrolysis, CocE Pharmacokinetics, and CocE EliminationS⃞

    PubMed Central

    Brim, Remy L.; Noon, Kathleen R.; Collins, Gregory T.; Stein, Aron; Nichols, Joseph; Narasimhan, Diwa; Ko, Mei-Chuan; Woods, James H.

    2012-01-01

    Cocaine abuse and toxicity remain widespread problems in the United States. Currently cocaine toxicity is treated only symptomatically, because there is no Food and Drug Administration-approved pharmacotherapy for this indication. To address the unmet need, a stabilized mutant of bacterial cocaine esterase [T172R/G173Q-CocE (DM-CocE)], which hydrolyzes cocaine into inactive metabolites and has low immunogenic potential, has been developed and previously tested in animal models of cocaine toxicity. Here, we document the rapid cocaine hydrolysis by low doses of DM-CocE in vitro and in vivo, as well as the pharmacokinetics and distribution of the DM-CocE protein in rats. DM-CocE at 50.5 μg/kg effectively eliminated 4 mg/kg cocaine within 2 min in both male and female rats as measured by mass spectrometry. We expanded on these findings by using a pharmacologically relevant dose of DM-CocE (0.32 mg/kg) in rats and monkeys to hydrolyze convulsant doses of cocaine. DM-CocE reduced cocaine to below detection limits rapidly after injection; however, elimination of DM-CocE resulted in peripheral cocaine redistribution by 30 to 60 min. Elimination of DM-CocE was quantified by using [35S] labeling of the enzyme and was found to have a half-life of 2.1 h in rats. Minor urinary output of DM-CocE was also observed. Immunohistochemistry, Western blotting, and radiography all were used to elucidate the mechanism of DM-CocE elimination, rapid proteolysis, and recycling of amino acids into all tissues. This rapid elimination of DM-CocE is a desirable property of a therapeutic for cocaine toxicity and should reduce the likelihood of immunogenic or adverse reactions as DM-CocE moves toward clinical use. PMID:21990608

  17. The fate of bacterial cocaine esterase (CocE): an in vivo study of CocE-mediated cocaine hydrolysis, CocE pharmacokinetics, and CocE elimination.

    PubMed

    Brim, Remy L; Noon, Kathleen R; Collins, Gregory T; Stein, Aron; Nichols, Joseph; Narasimhan, Diwa; Ko, Mei-Chuan; Woods, James H; Sunahara, Roger K

    2012-01-01

    Cocaine abuse and toxicity remain widespread problems in the United States. Currently cocaine toxicity is treated only symptomatically, because there is no Food and Drug Administration-approved pharmacotherapy for this indication. To address the unmet need, a stabilized mutant of bacterial cocaine esterase [T172R/G173Q-CocE (DM-CocE)], which hydrolyzes cocaine into inactive metabolites and has low immunogenic potential, has been developed and previously tested in animal models of cocaine toxicity. Here, we document the rapid cocaine hydrolysis by low doses of DM-CocE in vitro and in vivo, as well as the pharmacokinetics and distribution of the DM-CocE protein in rats. DM-CocE at 50.5 μg/kg effectively eliminated 4 mg/kg cocaine within 2 min in both male and female rats as measured by mass spectrometry. We expanded on these findings by using a pharmacologically relevant dose of DM-CocE (0.32 mg/kg) in rats and monkeys to hydrolyze convulsant doses of cocaine. DM-CocE reduced cocaine to below detection limits rapidly after injection; however, elimination of DM-CocE resulted in peripheral cocaine redistribution by 30 to 60 min. Elimination of DM-CocE was quantified by using [³⁵S] labeling of the enzyme and was found to have a half-life of 2.1 h in rats. Minor urinary output of DM-CocE was also observed. Immunohistochemistry, Western blotting, and radiography all were used to elucidate the mechanism of DM-CocE elimination, rapid proteolysis, and recycling of amino acids into all tissues. This rapid elimination of DM-CocE is a desirable property of a therapeutic for cocaine toxicity and should reduce the likelihood of immunogenic or adverse reactions as DM-CocE moves toward clinical use. PMID:21990608

  18. Transcriptional up-regulation of a novel ferritin homolog in abalone Haliotis discus hannai Ino by dietary iron.

    PubMed

    Wu, Chenglong; Zhang, Wenbing; Mai, Kangsen; Xu, Wei; Wang, Xiaojie; Ma, Hongming; Liufu, Zhiguo

    2010-11-01

    A novel cDNA encoding ferritin (HdhNFT) was cloned from the hepatopancreas of abalone, Haliotis discus hannai Ino. The deduced protein contains 171 amino acid residues with a predicted molecular mass (MW) about 19.8 kDa and theoretical isoelectric point (pI) of 4.792. Amino acid alignment revealed that HdhNFT shared high similarity with other known ferritins. The HdhNFT contained a highly conserved motif for the ferroxidase center, which consists of seven residues of a typical vertebrate heavy-chain ferritin with a typical stem-loop structure. HdhNFT mRNA contains a 27 bp iron-responsive element (IRE) in the 5'-untranslated region. This IRE exhibited 82.14% similarity with abalone H. discus discus and 78.57% similarity with Pacific oyster Crassostrea gigas IREs. By real-time PCR assays, the mRNA transcripts of HdhNFT were found to be higher expressed in kidney, hepatopancreas, gill, mantle and muscle than in haemocytes and gonad. Moreover, mRNA expression levels of HdhNFT in the hepatopancreas and haemocytes were measured by real-time PCR in abalone fed with graded levels of dietary iron (29.2, 65.7, 1267.2 and 6264.7 mg/kg). Results showed that the expression of the HdhNFT mRNA increased with dietary iron contents. Furthermore, the maximum value of the HdhNFT mRNA was found in the treatment with 6264.7 mg/kg of dietary iron. These data indicated that dietary iron can up-regulate HdhNFT at transcriptional level in abalone.

  19. Bacterial Wound Culture

    MedlinePlus

    ... Home Visit Global Sites Search Help? Bacterial Wound Culture Share this page: Was this page helpful? Also known as: Aerobic Wound Culture; Anaerobic Wound Culture Formal name: Culture, wound Related ...

  20. Bacterial surface adaptation

    NASA Astrophysics Data System (ADS)

    Utada, Andrew

    2014-03-01

    Biofilms are structured multi-cellular communities that are fundamental to the biology and ecology of bacteria. Parasitic bacterial biofilms can cause lethal infections and biofouling, but commensal bacterial biofilms, such as those found in the gut, can break down otherwise indigestible plant polysaccharides and allow us to enjoy vegetables. The first step in biofilm formation, adaptation to life on a surface, requires a working knowledge of low Reynolds number fluid physics, and the coordination of biochemical signaling, polysaccharide production, and molecular motility motors. These crucial early stages of biofilm formation are at present poorly understood. By adapting methods from soft matter physics, we dissect bacterial social behavior at the single cell level for several prototypical bacterial species, including Pseudomonas aeruginosa and Vibrio cholerae.

  1. Bacterial Nail Infection (Paronychia)

    MedlinePlus

    ... in people who work in the health care industry. Chronic paronychia is most common in adult women and those who work in places where their hands are kept moist, such as food handlers. Signs and Symptoms Bacterial nail infection most ...

  2. Supplementation with Angelica keiskei inhibits expression of inflammatory mediators in the gastric mucosa of Helicobacter pylori-infected mice.

    PubMed

    Kim, Aryoung; Lim, Joo Weon; Kim, Hoguen; Kim, Hyeyoung

    2016-05-01

    Oxidative stress is involved in the pathogenesis of Helicobacter pylori-associated gastric ulceration and carcinogenesis. The oxidant-sensitive transcription factor, nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB), regulates expression of inflammatory mediators such as interferon γ (IFN-γ), cyclooxygenase 2 (COX-2), and inducible nitric oxide synthase (iNOS). These inflammatory mediators increased in gastric mucosal tissues from patients infected with H pylori. Angelica keiskei (AK), a green leafy vegetable, is rich in carotenoids and flavonoids and shows antioxidant and anti-inflammatory activities. Therefore, we hypothesized that AK may protect the gastric mucosa of H pylori-infected mice against inflammation. We determined lipid peroxide abundance, myeloperoxidase activity, expression levels of inflammatory mediators (IFN-γ, COX-2, and iNOS), NF-κB-DNA binding activity, and histologic changes in gastric mucosal tissues. The antioxidant N-acetylcysteine served as the positive control treatment. Supplementation with AK suppressed increases in lipid peroxide abundance, myeloperoxidase activity, induction of inflammatory mediators (IFN-γ, COX-2, and iNOS), activation of NF-κB, and degradation of nuclear factor of κ light polypeptide gene enhancer in B-cells inhibitor α in gastric mucosal tissue from H pylori-infected mice. Inhibition of H pylori-induced alterations by AK was similar to that by N-acetylcysteine. Taken together, these results suggest that supplementation with AK may prevent H pylori-induced gastric inflammation by inhibiting NF-κB-mediated induction of inflammatory mediators in the gastric mucosa of patients infected with H pylori. PMID:27101766

  3. Bistability and Bacterial Infections

    PubMed Central

    Malka, Roy; Shochat, Eliezer; Rom-Kedar, Vered

    2010-01-01

    Bacterial infections occur when the natural host defenses are overwhelmed by invading bacteria. The main component of the host defense is impaired when neutrophil count or function is too low, putting the host at great risk of developing an acute infection. In people with intact immune systems, neutrophil count increases during bacterial infection. However, there are two important clinical cases in which they remain constant: a) in patients with neutropenic-associated conditions, such as those undergoing chemotherapy at the nadir (the minimum clinically observable neutrophil level); b) in ex vivo examination of the patient's neutrophil bactericidal activity. Here we study bacterial population dynamics under fixed neutrophil levels by mathematical modelling. We show that under reasonable biological assumptions, there are only two possible scenarios: 1) Bacterial behavior is monostable: it always converges to a stable equilibrium of bacterial concentration which only depends, in a gradual manner, on the neutrophil level (and not on the initial bacterial level). We call such a behavior type I dynamics. 2) The bacterial dynamics is bistable for some range of neutrophil levels. We call such a behavior type II dynamics. In the bistable case (type II), one equilibrium corresponds to a healthy state whereas the other corresponds to a fulminant bacterial infection. We demonstrate that published data of in vitro Staphylococcus epidermidis bactericidal experiments are inconsistent with both the type I dynamics and the commonly used linear model and are consistent with type II dynamics. We argue that type II dynamics is a plausible mechanism for the development of a fulminant infection. PMID:20463954

  4. Type I Interferons Exert Anti-tumor Effect via Reversing Immunosuppression Mediated by Mesenchymal Stromal Cells

    PubMed Central

    Shou, Peishun; Chen, Qing; Jiang, Jingting; Xu, Chunliang; Zhang, Jimin; Zheng, Chunxing; Jiang, Menghui; Velletri, Tania; Cao, Wei; Huang, Yin; Yang, Qian; Han, Xiaoyan; Zhang, Liying; Wei, Lixin; Rabson, Arnold B.; Chin, Y. Eugene; Wang, Ying; Shi, Yufang

    2016-01-01

    Mesenchymal stromal cells (MSCs) are strongly immunosuppressive via producing nitric oxide (NO) and known to migrate into tumor sites to promote tumor growth, but the underlying mechanisms remain largely elusive. Here, we found that IFNα-secreting MSCs showed more dramatic inhibition effect on tumor progression than that of IFNα alone. Interestingly, IFNα-primed MSCs could also effectively suppress tumor growth. Mechanistically, we demonstrated that both IFNα and IFNβ (type I IFNs) reversed the immunosuppressive effect of MSCs on splenocyte proliferation. This effect of type I IFNs was exerted through inhibiting iNOS (inducible nitric oxide synthase) expression in IFNγ and TNFα-stimulated MSCs. Notably, only NO production was inhibited by IFNα; production of other cytokines or chemokines tested was not suppressed. Furthermore, IFNα promoted the switch from Stat1 homodimers to Stat1-Stat2 heterodimers. Studies using the luciferase reporter system and chromatin immunoprecipitation assay revealed that IFNα suppressed iNOS transcription through inhibiting the binding of Stat1 to iNOS promoter. Therefore, the synergistic anti-tumor effects of type I IFNs and MSCs were achieved by inhibiting NO production. This study provides essential information for understanding the mechanisms of MSC-mediated immunosuppression and for the development of better clinical strategies using IFNs and MSCs for cancer immunotherapy. PMID:27109100

  5. Integrated Analysis of COX-2 and iNOS Derived Inflammatory Mediators in LPS-Stimulated RAW Macrophages Pre-Exposed to Echium plantagineum L. Bee Pollen Extract

    PubMed Central

    Moita, Eduarda; Gil-Izquierdo, Angel; Sousa, Carla; Ferreres, Federico; Silva, Luís R.; Valentão, Patrícia; Domínguez-Perles, Raúl; Baenas, Nieves; Andrade, Paula B.

    2013-01-01

    Oxidative stress and inflammation play important roles in disease development. This study intended to evaluate the anti-inflammatory and antioxidant potential of Echium plantagineum L. bee pollen to support its claimed health beneficial effects. The hydromethanol extract efficiently scavenged nitric oxide (•NO) although against superoxide (O2•−) it behaved as antioxidant at lower concentrations and as pro-oxidant at higher concentrations. The anti-inflammatory potential was evaluated in LPS-stimulated macrophages. The levels of •NO and L-citrulline decreased for all extract concentrations tested, while the levels of prostaglandins, their metabolites and isoprostanes, evaluated by UPLC-MS, decreased with low extract concentrations. So, E. plantagineum bee pollen extract can exert anti-inflammatory activity by reducing •NO and prostaglandins. The extract is able to scavenge the reactive species •NO and O2•− and reduce markers of oxidative stress in cells at low concentrations. PMID:23520554

  6. Engineering nanoparticles to silence bacterial communication

    PubMed Central

    Miller, Kristen P.; Wang, Lei; Chen, Yung-Pin; Pellechia, Perry J.; Benicewicz, Brian C.; Decho, Alan W.

    2015-01-01

    The alarming spread of bacterial resistance to traditional antibiotics has warranted the study of alternative antimicrobial agents. Quorum sensing (QS) is a chemical cell-to-cell communication mechanism utilized by bacteria to coordinate group behaviors and establish infections. QS is integral to bacterial survival, and therefore provides a unique target for antimicrobial therapy. In this study, silicon dioxide nanoparticles (Si-NP) were engineered to target the signaling molecules [i.e., acylhomoserine lactones (HSLs)] used for QS in order to halt bacterial communication. Specifically, when Si-NP were surface functionalized with β-cyclodextrin (β-CD), then added to cultures of bacteria (Vibrio fischeri), whose luminous output depends upon HSL-mediated QS, the cell-to-cell communication was dramatically reduced. Reductions in luminescence were further verified by quantitative polymerase chain reaction (qPCR) analyses of luminescence genes. Binding of HSLs to Si-NPs was examined using nuclear magnetic resonance (NMR) spectroscopy. The results indicated that by delivering high concentrations of engineered NPs with associated quenching compounds, the chemical signals were removed from the immediate bacterial environment. In actively-metabolizing cultures, this treatment blocked the ability of bacteria to communicate and regulate QS, effectively silencing and isolating the cells. Si-NPs provide a scaffold and critical stepping-stone for more pointed developments in antimicrobial therapy, especially with regard to QS—a target that will reduce resistance pressures imposed by traditional antibiotics. PMID:25806030

  7. Rhododendron album Blume inhibits iNOS and COX-2 expression in LPS-stimulated RAW264.7 cells through the downregulation of NF-κB signaling.

    PubMed

    Park, Ji-Won; Kwon, Ok-Kyoung; Kim, Jung-Hee; Oh, Sei-Ryang; Kim, Jae-Hong; Paik, Jin-Hyub; Marwoto, Bambang; Widjhati, Rifatul; Juniarti, Fifit; Irawan, Doddy; Ahn, Kyung-Seop

    2015-04-01

    Rhododendron album Blume (RA) has traditionally been used as an herbal medicine and is considered to have anti-inflammatory properties. In the present study, we screened RA extracts with anti-inflammatory properties. The biological effects of an RA methanol extract (RAME) on inflammation were investigated in lipopolysaccharide (LPS)-stimulated mouse RAW264.7 cells. We investigated the effects of RAME on the production of nitric oxide (NO) and prostaglandin E2 (PGE2) in LPS-stimulated RAW264.7 cells. To explore the anti-inflammatory mechanisms of RAME, we measured the mRNA and protein expression of pro-inflammatory mediators induced by RAME in the LPS-stimulated RAW264.7 cells by RT-PCR and western blot analysis, respectively. RAME significantly inhibited the production of NO, PGE2, interleukin (IL)-6, IL-1β and tumor necrosis factor (TNF)-α in the LPS-stimulated RAW264.7 cells. It also suppressed the mRNA and protein expression of inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2) and mitogen-activated protein kinases (MAPKs) with a concomitant decrease in the nuclear translocation of nuclear factor-κB (NF-κB) in the LPS-stimulated RAW264.7 cells. These results indicate that RAME inhibits LPS-induced inflammatory responses. These effects were considered to be strongly associated with the suppression of NF-κB activation. We therefore suggest that RAME may be prove to be an effective therapeutic agent for the treatment of inflammatory diseases. PMID:25784296

  8. Platelet activating factor (PAF) antagonists on cytokine induction of iNOS and sPLA2 in immortalized astrocytes (DITNC).

    PubMed

    Wang, J H; Sun, G Y

    2000-05-01

    Platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) and its receptor are known to play important roles in modulating neuronal plasticity and inflammatory responses, particularly during neuronal injury. PAF receptors are widespread in different brain regions and are present on the cell surface as well as in intracellular membrane compartments. Astrocytes are immune active cells and are responsive to cytokines, which stimulate signaling cascades leading to transcriptional activation of genes and protein synthesis. Our recent studies indicate the ability of cytokines, e.g., tumor necrosis factor-alpha (TNFalpha), interleukin-1beta (IL-1beta) and interferon-gamma (IFNgamma), to induce the inducible nitric oxide (iNOS) and secretory phospholipase A2 (sPLA2) genes in immortalized astrocytes (DITNC) (Li et al., J. Interferon and Cytokine Res. 19: 121-127. 1999). The main objective for this study is to examine the effects of PAF antagonists on cytokine induction of iNOS and sPLA2 in these cells. Results show that BN50730, a synthetic PAF antagonist, but not BN52021, a natural PAF antagonist (ginkolide B) can dose-dependently inhibit cytokine induction of NO production and sPLA2 release. Inhibition of NO production by BN50730 corroborated well with the decrease in iNOS protein and mRNA levels as well as binding of NF-kappaB STAT- 1 to DNA, suggesting that BN50730 action is upstream of the transcriptional process. These results are in agreement with the role of intracellular PAF in regulating the cytokine signaling cascade in astrocytes and further suggest the possible use of BN50730 as a therapeutic agent for suppressing the inflammatory pathways elicited by cytokines. PMID:10905622

  9. D-limonene suppresses doxorubicin-induced oxidative stress and inflammation via repression of COX-2, iNOS, and NFκB in kidneys of Wistar rats.

    PubMed

    Rehman, Muneeb U; Tahir, Mir; Khan, Abdul Quaiyoom; Khan, Rehan; Oday-O-Hamiza; Lateef, Abdul; Hassan, Syed Kazim; Rashid, Sumaya; Ali, Nemat; Zeeshan, Mirza; Sultana, Sarwat

    2014-04-01

    D-limonene is a naturally occurring monoterpene and has been found to posses numerous therapeutic properties. In this study, we used D-limonene as a protective agent against the nephrotoxic effects of anticancer drug doxorubicin (Dox). Rats were given D-limonene at doses of 5% and 10% mixed with diet for 20 consecutive days. Dox was give at the dose of 20 mg/kg body weight intraperitoneally. The protective effects of D-limonene on Dox-induced oxidative stress and inflammation were investigated by assaying oxidative stress biomarkers, lipid peroxidation, serum toxicity markers, proinflammatory cytokines, and expression of nuclear factor kappa B (NFκB), cyclo-oxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) and Nitrite levels. Administration of Dox (20 mg/kg body weight) in rats enhanced renal lipid peroxidation; depleted glutathione content and anti-oxidant enzymes; elevated levels of kidney toxicity markers viz. kidney injury molecule-1 (KIM-1), blood urea nitrogen (BUN), and creatinine; enhanced expression of NFκB, COX-2, and iNOS and nitric oxide. Treatment with D-limonene prevented oxidative stress by restoring the levels of antioxidant enzymes, further both doses of 5% and 10% showed significant decrease in inflammatory response. Both the doses of D-limonene significantly decreased the levels of kidney toxicity markers KIM-1, BUN, and creatinine. D-limonene also effectively decreased the Dox induced overexpression of NF-κB, COX-2, and iNOS and nitric oxide. Data from the present study indicate the protective role of D-limonene against Dox-induced renal damage.

  10. The bacterial lipocalins.

    PubMed

    Bishop, R E

    2000-10-18

    The lipocalins were once regarded as a eukaryotic protein family, but new members have been recently discovered in bacteria. The first bacterial lipocalin (Blc) was identified in Escherichia coli as an outer membrane lipoprotein expressed under conditions of environmental stress. Blc is distinguished from most lipocalins by the absence of intramolecular disulfide bonds, but the presence of a membrane anchor is shared with two of its closest homologues, apolipoprotein D and lazarillo. Several common features of the membrane-anchored lipocalins suggest that each may play an important role in membrane biogenesis and repair. Additionally, Blc proteins are implicated in the dissemination of antibiotic resistance genes and in the activation of immunity. Recent genome sequencing efforts reveal the existence of at least 20 bacterial lipocalins. The lipocalins appear to have originated in Gram-negative bacteria and were probably transferred horizontally to eukaryotes from the endosymbiotic alpha-proteobacterial ancestor of the mitochondrion. The genome sequences also reveal that some bacterial lipocalins exhibit disulfide bonds and alternative modes of subcellular localization, which include targeting to the periplasmic space, the cytoplasmic membrane, and the cytosol. The relationships between bacterial lipocalin structure and function further illuminate the common biochemistry of bacterial and eukaryotic cells.

  11. Adhesive bacterial colonization of exposed traumatized tendon.

    PubMed

    Webb, L X; Hobgood, C D; Meredith, J W; Gristina, A G

    1987-05-01

    Recent studies of compromised or damaged tissues, as well as biomaterials, have shown that they provide a particularly fertile substratum for bacterial colonization. Colonization in these environments is mediated by a process of microbial adhesion to surfaces of the substrata. In this report, we present electron microscopic studies of a portion of damaged and infected tendon. These studies demonstrate colonies of bacteria surrounded by a ruthenium red-staining exopolysaccharide biofilm and adhesion to the surface of the tendon by means of an exopolysaccharide polymer. We suggest that this adhesive form of bacterial colonization may partially explain the resistance of exposed tendon to effective debridement by simple mechanical measures and to coverage with granulation tissue, partial-thickness skin grafts, and vascularized tissue grafts.

  12. Bacterial microbiome of lungs in COPD.

    PubMed

    Sze, Marc A; Hogg, James C; Sin, Don D

    2014-01-01

    Chronic obstructive pulmonary disease (COPD) is currently the third leading cause of death in the world. Although smoking is the main risk factor for this disease, only a minority of smokers develop COPD. Why this happens is largely unknown. Recent discoveries by the human microbiome project have shed new light on the importance and richness of the bacterial microbiota at different body sites in human beings. The microbiota plays a particularly important role in the development and functional integrity of the immune system. Shifts or perturbations in the microbiota can lead to disease. COPD is in part mediated by dysregulated immune responses to cigarette smoke and other environmental insults. Although traditionally the lung has been viewed as a sterile organ, by using highly sensitive genomic techniques, recent reports have identified diverse bacterial communities in the human lung that may change in COPD. This review summarizes the current knowledge concerning the lung microbiota in COPD and its potential implications for pathogenesis of the disease.

  13. A GPCR Handles Bacterial Sensing in Chemotaxis and Phagocytosis.

    PubMed

    Senoo, Hiroshi; Sesaki, Hiromi; Iijima, Miho

    2016-02-22

    In this issue of Developmental Cell, Pan et al. (2016) identified in cells of the social amoeba Dictyostelium a G protein-coupled receptor (GPCR) that recognizes a chemoattractant secreted by bacteria. This work uncovers a mechanism by which a single GPCR mediates pseudopod extension during cell migration and bacterial engulfment. PMID:26906729

  14. Mast cells: multitalented facilitators of protection against bacterial pathogens

    PubMed Central

    Trivedi, Nikita H; Guentzel, M Neal; Rodriguez, Annette R; Yu, Jieh-Juen; Forsthuber, Thomas G; Arulanandam, Bernard P

    2014-01-01

    Mast cells are crucial effector cells evoking immune responses against bacterial pathogens. The positioning of mast cells at the host–environment interface, and the multitude of pathogen-recognition receptors and preformed mediator granules make these cells potentially the earliest to respond to an invading pathogen. In this review, the authors summarize the receptors used by mast cells to recognize invading bacteria and discuss the function of immune mediators released by mast cells in control of bacterial infection. The interaction of mast cells with other immune cells, including macrophages, dendritic cells and T cells, to induce protective immunity is highlighted. The authors also discuss mast cell-based vaccine strategies and the potential application in control of bacterial disease. PMID:23390944

  15. Experimental Bacterial Endocarditis

    PubMed Central

    Durack, D. T.; Beeson, P. B.; Petersdorf, R. G.

    1973-01-01

    A simple and reliable model for endocarditis in rabbits has been studied and standardized. Non-bacterial thrombotic endocarditis was produced on either side of the heart by the presence of a polyethylene catheter. One day later, this was converted into bacterial endocarditis by single intravenous injections of streptococci, staphylococci, Proteus and Candida. No infection resulted from injection of L-forms or virus. Reduction of inoculum size or withdrawal of the catheter reduced the incidence of bacterial endocarditis, but the presence of a catheter in the heart for only a few minutes predisposed to infection. Left-sided Streptococcus viridans infection was uniformly fatal, with average survival of about two weeks. Right-sided infection was not always fatal; approximately 25% of infected vegetations healed spontaneously. The advantages of a standardized model for endocarditis which allows exact timing of infection are discussed. ImagesFigs. 1-2 PMID:4700697

  16. HIV-Enhancing Factors Are Secreted by Reproductive Epithelia upon Inoculation with Bacterial Vaginosis-Associated Bacteria.

    PubMed

    Eade, Colleen R; Diaz, Camila; Chen, Sixue; Cole, Amy L; Cole, Alexander M

    2015-01-01

    Bacterial vaginosis is a common reproductive infection in which commensal vaginal lactobacilli are displaced by a mixed population of pathogenic bacteria. Bacterial vaginosis increases susceptibility to HIV, and it has been suggested that host innate immune responses to pathogenic bacteria contribute to enhanced infection, yet the cellular mechanisms mediating the increased HIV susceptibility remain uncharacterized. We evaluated the HIV-enhancing effects of bacterial vaginosis by inoculating endocervical epithelia with Atopobium vaginae, a bacterial vaginosis-associated bacteria, and assaying secreted factors for HIV-enhancing activity. When epithelia and A. vaginae were cocultured, we observed increased HIV-enhancing activity mediated by secreted low molecular weight factors. From this complex mixture we identified several upregulated host proteins, which functioned in combination to enhance HIV infection. These studies suggest that the host immune response to bacterial vaginosis-associated bacteria results in the release of HIV-enhancing factors. The combined activity of bacterial vaginosis-induced proteins likely mediates HIV enhancement.

  17. [Diagnosis of bacterial vaginosis].

    PubMed

    Djukić, Slobodanka; Ćirković, Ivana; Arsić, Biljana; Garalejić, Eliana

    2013-01-01

    Bacterial vaginosis is a common, complex clinical syndrome characterized by alterations in the normal vaginal flora. When symptomatic, it is associated with a malodorous vaginal discharge and on occasion vaginal burning or itching. Under normal conditions, lactobacilli constitute 95% of the bacteria in the vagina. Bacterial vaginosis is associated with severe reduction or absence of the normal H2O2-producing lactobacilli and overgrowth of anaerobic bacteria and Gardnerella vaginalis, Atopobium vaginae, Mycoplasma hominis and Mobiluncus species. Most types of infectious disease are diagnosed by culture, by isolating an antigen or RNA/DNA from the microbe, or by serodiagnosis to determine the presence of antibodies to the microbe. Therefore, demonstration of the presence of an infectious agent is often a necessary criterion for the diagnosis of the disease. This is not the case for bacterial vaginosis, since the ultimate cause of the disease is not yet known. There are a variety of methods for the diagnosis of bacterial vaginosis but no method can at present be regarded as the best. Diagnosing bacterial vaginosis has long been based on the clinical criteria of Amsel, whereby three of four defined criteria must be satisfied. Nugent's scoring system has been further developed and includes validation of the categories of observable bacteria structures. Up-to-date molecular tests are introduced, and better understanding of vaginal microbiome, a clear definition for bacterial vaginosis, and short-term and long-term fluctuations in vaginal microflora will help to better define molecular tests within the broader clinical context. PMID:24073569

  18. Bacterial glycosyltransferase toxins.

    PubMed

    Jank, Thomas; Belyi, Yury; Aktories, Klaus

    2015-12-01

    Mono-glycosylation of host proteins is a common mechanism by which bacterial protein toxins manipulate cellular functions of eukaryotic target host cells. Prototypic for this group of glycosyltransferase toxins are Clostridium difficile toxins A and B, which modify guanine nucleotide-binding proteins of the Rho family. However, toxin-induced glycosylation is not restricted to the Clostridia. Various types of bacterial pathogens including Escherichia coli, Yersinia, Photorhabdus and Legionella species produce glycosyltransferase toxins. Recent studies discovered novel unexpected variations in host protein targets and amino acid acceptors of toxin-catalysed glycosylation. These findings open new perspectives in toxin as well as in carbohydrate research.

  19. Bacterial transfer RNAs

    PubMed Central

    Shepherd, Jennifer; Ibba, Michael

    2015-01-01

    Transfer RNA is an essential adapter molecule that is found across all three domains of life. The primary role of transfer RNA resides in its critical involvement in the accurate translation of messenger RNA codons during protein synthesis and, therefore, ultimately in the determination of cellular gene expression. This review aims to bring together the results of intensive investigations into the synthesis, maturation, modification, aminoacylation, editing and recycling of bacterial transfer RNAs. Codon recognition at the ribosome as well as the ever-increasing number of alternative roles for transfer RNA outside of translation will be discussed in the specific context of bacterial cells. PMID:25796611

  20. Bacterial ecologies in limonite.

    PubMed

    Vishniac, W

    1965-01-01

    Limonite (Fe2O3 . nH2O) may be a constituent of the Martian surface. We have prepared culture media with ferric hydroxide as an electron acceptor. One medium contained ethanol, another gaseous hydrogen and carbon dioxide. Bacterial growth without light and oxygen suggests that ferric iron serves as a terminal respiratory electron acceptor. The oxidation of ferrous hydroxide may be carried out by photosynthetic bacteria. A ferrous-ferric couple may thus support bacterial respiration and photosynthesis in the absence of oxygen. This cycle may account for the dark markings of Mars.

  1. Molecular mechanism of bacterial type 1 and P pili assembly.

    PubMed

    Busch, Andreas; Phan, Gilles; Waksman, Gabriel

    2015-03-01

    The formation of adhesive surface structures called pili or fimbriae ('bacterial hair') is an important contributor towards bacterial pathogenicity and persistence. To fight often chronic or recurrent bacterial infections such as urinary tract infections, it is necessary to understand the molecular mechanism of the nanomachines assembling such pili. Here, we focus on the so far best-known pilus assembly machinery: the chaperone-usher pathway producing the type 1 and P pili, and highlight the most recently acquired structural knowledge. First, we describe the subunits' structure and the molecular role of the periplasmic chaperone. Second, we focus on the outer-membrane usher structure and the catalytic mechanism of usher-mediated pilus biogenesis. Finally, we describe how the detailed understanding of the chaperone-usher pathway at a molecular level has paved the way for the design of a new generation of bacterial inhibitors called 'pilicides'. PMID:25624519

  2. Viral-bacterial interactions in acute otitis media.

    PubMed

    Marom, Tal; Nokso-Koivisto, Johanna; Chonmaitree, Tasnee

    2012-12-01

    Acute otitis media (AOM) is a polymicrobial disease, which usually occurs as a complication of viral upper respiratory tract infection (URI). While respiratory viruses alone may cause viral AOM, they increase the risk of bacterial middle ear infection and worsen clinical outcomes of bacterial AOM. URI viruses alter Eustachian tube (ET) function via decreased mucociliary action, altered mucus secretion and increased expression of inflammatory mediators among other mechanisms. Transient reduction in protective functions of the ET allows colonizing bacteria of the nasopharynx to ascend into the middle ear and cause AOM. Advances in research help us to better understand the host responses to viral URI, the mechanisms of viral-bacterial interactions in the nasopharynx and the development of AOM. In this review, we present current knowledge regarding viral-bacterial interactions in the pathogenesis and clinical course of AOM. We focus on the common respiratory viruses and their established role in AOM.

  3. Bacterial Catabolism of Dimethylsulfoniopropionate (DMSP)

    PubMed Central

    Reisch, Chris R.; Moran, Mary Ann; Whitman, William B.

    2011-01-01

    Dimethylsulfoniopropionate (DMSP) is a metabolite produced primarily by marine phytoplankton and is the main precursor to the climatically important gas dimethylsulfide (DMS). DMS is released upon bacterial catabolism of DMSP, but it is not the only possible fate of DMSP sulfur. An alternative demethylation/demethiolation pathway results in the eventual release of methanethiol, a highly reactive volatile sulfur compound that contributes little to the atmospheric sulfur flux. The activity of these pathways control the natural flux of sulfur released to the atmosphere. Although these biochemical pathways and the factors that regulate them are of great interest, they are poorly understood. Only recently have some of the genes and pathways responsible for DMSP catabolism been elucidated. Thus far, six different enzymes have been identified that catalyze the cleavage of DMSP, resulting in the release of DMS. In addition, five of these enzymes appear to produce acrylate, while one produces 3-hydroxypropionate. In contrast, only one enzyme, designated DmdA, has been identified that catalyzes the demethylation reaction producing methylmercaptopropionate (MMPA). The metabolism of MMPA is performed by a series of three coenzyme-A mediated reactions catalyzed by DmdB, DmdC, and DmdD. Interestingly, Candidatus Pelagibacter ubique, a member of the SAR11 clade of Alphaproteobacteria that is highly abundant in marine surface waters, possessed functional DmdA, DmdB, and DmdC enzymes. Microbially mediated transformations of both DMS and methanethiol are also possible, although many of the biochemical and molecular genetic details are still unknown. This review will focus on the recent discoveries in the biochemical pathways that mineralize and assimilate DMSP carbon and sulfur, as well as the areas for which a comprehensive understanding is still lacking. PMID:21886640

  4. Multiple genetic switches spontaneously modulating bacterial mutability

    PubMed Central

    2010-01-01

    Background All life forms need both high genetic stability to survive as species and a degree of mutability to evolve for adaptation, but little is known about how the organisms balance the two seemingly conflicting aspects of life: genetic stability and mutability. The DNA mismatch repair (MMR) system is essential for maintaining genetic stability and defects in MMR lead to high mutability. Evolution is driven by genetic novelty, such as point mutation and lateral gene transfer, both of which require genetic mutability. However, normally a functional MMR system would strongly inhibit such genomic changes. Our previous work indicated that MMR gene allele conversion between functional and non-functional states through copy number changes of small tandem repeats could occur spontaneously via slipped-strand mis-pairing during DNA replication and therefore may play a role of genetic switches to modulate the bacterial mutability at the population level. The open question was: when the conversion from functional to defective MMR is prohibited, will bacteria still be able to evolve by accepting laterally transferred DNA or accumulating mutations? Results To prohibit allele conversion, we "locked" the MMR genes through nucleotide replacements. We then scored changes in bacterial mutability and found that Salmonella strains with MMR locked at the functional state had significantly decreased mutability. To determine the generalizability of this kind of mutability 'switching' among a wider range of bacteria, we examined the distribution of tandem repeats within MMR genes in over 100 bacterial species and found that multiple genetic switches might exist in these bacteria and may spontaneously modulate bacterial mutability during evolution. Conclusions MMR allele conversion through repeats-mediated slipped-strand mis-pairing may function as a spontaneous mechanism to switch between high genetic stability and mutability during bacterial evolution. PMID:20836863

  5. Identification of a novel compound that inhibits iNOS and COX-2 expression in LPS-stimulated macrophages from Schisandra chinensis

    SciTech Connect

    Lee, You Jin; Park, Sun Young; Kim, Sun Gun; Park, Da Jung; Kang, Jum Soon; Lee, Sang Joon; Yoon, Sik; Kim, Young Hun; Bae, Yoe-Sik; Choi, Young-Whan

    2010-01-22

    A novel {alpha}-iso-cubebenol, which has anti-inflammatory effects in lipopolysaccharide (LPS)-treated RAW 264.7 macrophages, was isolated from the fruits of Schisandra chinensis. {alpha}-iso-cubebenol inhibited LPS-induced nitric oxide (NO) and prostaglandin E{sub 2} (PGE{sub 2}) production. Consistent with these findings, {alpha}-iso-cubebenol also reduced the LPS-induced expression of inducible nitric oxide synthase and cyclooxygenase-2 at the protein and mRNA levels in a concentration-dependent manner. {alpha}-iso-cubebenol also inhibited LPS-induced nuclear translocation of the NF-{kappa}B p65 subunit. Furthermore, {alpha}-iso-cubebenol suppressed the phosphorylation of ERK, JNK, and p38 kinase induced by LPS. Since the novel {alpha}-iso-cubebenol blocked the production of several pro-inflammatory mediators induced by LPS in macrophages, the molecule can be useful material for the development of anti-inflammatory agents against bacterial infections or endotoxin.

  6. BACTERIAL WATERBORNE PATHOGENS

    EPA Science Inventory

    Bacterial pathogens are examples of classical etiological agents of waterborne disease. While these agents no longer serve as major threats to U.S. water supplies, they are still important pathogens in areas with substandard sanitation and poor water treatment facilities. In th...

  7. Bacterial extracellular lignin peroxidase

    DOEpatents

    Crawford, Donald L.; Ramachandra, Muralidhara

    1993-01-01

    A newly discovered lignin peroxidase enzyme is provided. The enzyme is obtained from a bacterial source and is capable of degrading the lignin portion of lignocellulose in the presence of hydrogen peroxide. The enzyme is extracellular, oxidative, inducible by lignin, larch wood xylan, or related substrates and capable of attacking certain lignin substructure chemical bonds that are not degradable by fungal lignin peroxidases.

  8. The Bacterial Growth Curve.

    ERIC Educational Resources Information Center

    Paulton, Richard J. L.

    1991-01-01

    A procedure that allows students to view an entire bacterial growth curve during a two- to three-hour student laboratory period is described. Observations of the lag phase, logarithmic phase, maximum stationary phase, and phase of decline are possible. A nonpathogenic, marine bacterium is used in the investigation. (KR)

  9. Staining bacterial flagella easily.

    PubMed Central

    Heimbrook, M E; Wang, W L; Campbell, G

    1989-01-01

    A wet-mount technique for staining bacterial flagella is highly successful when a stable stain and regular slides and cover slips are used. Although not producing a permanent mount, the technique is simple for routine use when the number and arrangement of flagella are critical in identifying species of motile bacteria. Images PMID:2478573

  10. Modeling intraocular bacterial infections.

    PubMed

    Astley, Roger A; Coburn, Phillip S; Parkunan, Salai Madhumathi; Callegan, Michelle C

    2016-09-01

    Bacterial endophthalmitis is an infection and inflammation of the posterior segment of the eye which can result in significant loss of visual acuity. Even with prompt antibiotic, anti-inflammatory and surgical intervention, vision and even the eye itself may be lost. For the past century, experimental animal models have been used to examine various aspects of the pathogenesis and pathophysiology of bacterial endophthalmitis, to further the development of anti-inflammatory treatment strategies, and to evaluate the pharmacokinetics and efficacies of antibiotics. Experimental models allow independent control of many parameters of infection and facilitate systematic examination of infection outcomes. While no single animal model perfectly reproduces the human pathology of bacterial endophthalmitis, investigators have successfully used these models to understand the infectious process and the host response, and have provided new information regarding therapeutic options for the treatment of bacterial endophthalmitis. This review highlights experimental animal models of endophthalmitis and correlates this information with the clinical setting. The goal is to identify knowledge gaps that may be addressed in future experimental and clinical studies focused on improvements in the therapeutic preservation of vision during and after this disease. PMID:27154427

  11. Bacterial leaf spot

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bacterial leaf spot has been reported in Australia (Queensland), Egypt, El Salvador, India, Japan, Nicaragua, Sudan, and the United States (Florida, Iowa, Kansas, Maryland, and Wisconsin). It occasionally causes locally severe defoliation and post-emergence damping-off and stunting. The disease is...

  12. Dental pulp in mature replanted human teeth: morphological alterations and metalloproteineses-2 and -9, Annexin-5, BCL-2 and iNOS modulation.

    PubMed

    Leone, A; Angelova Volponi, A; Uzzo, M L; Spatola, G F; Jurjus, A; Vandevska-Radunovic, V

    2015-01-01

    Tooth replantation, as a treatment concept, has been subject to controversies regarding the mechanism as well as the various parameters underlying this process. This work aimed to study time-related changes in the pulp of replanted mature human premolars through the changes in the levels of certain factors involved in the underlying mechanisms of pulpal tissue healing after replantation. Eleven experimental mature teeth were extracted, immediately replanted in the original socket and left without any other intervention for 1, 2, 3 and 12 weeks before re-extraction. Three premolars served as control. All specimens were subject to histological analysis and the levels of MMP-2, MMP-9, Annexin V, iNOS and BCL-2 (anti-apoptotic family) were analyzed employing immunohistochemistry. The results showed degradation of the extracellular matrix (ECM), inflammatory cell infiltrate, loss in pulpo-dentine interface and loss of odontoblasts in the dental pulp tissue. This was accompanied by increase over time of MMP-9, Annexin V, iNOS and a decrease of BCL-2 and MMP-2, suggesting that apoptosis increased throughout the experimental period. PMID:26753662

  13. Transition from two to one integument in Prunus species: expression pattern of INNER NO OUTER (INO), ABERRANT TESTA SHAPE (ATS) and ETTIN (ETT).

    PubMed

    Lora, Jorge; Hormaza, José I; Herrero, Maria

    2015-10-01

    While gymnosperm ovules have one integument, in most angiosperms two integuments surround the ovules. Unitegmic ovules have arisen independently several times during the evolution of angiosperms, but the ultimate genetic cause of the presence of a single integument remains elusive. We compared species of the genus Prunus that have different numbers of integuments: bitegmic species, such as Prunus armeniaca (apricot) and Prunus persica (peach), and unitegmic species, such as Prunus incisa, analyzing the expression pattern of genes that are involved in integument development in Arabidopsis thaliana: INNER NO OUTER (INO), ABERRANT TESTA SHAPE (ATS) and ETTIN (ETT). Bitegmic and unitegmic species showed similar INO expression patterns, indicative of the conservation of an outer integument. However, expression of ETT, which occurs in the boundary of the outer and inner integuments, was altered in unitegmic ovules, which showed lack of ETT expression. These results strongly suggest that the presence of a single integument could be attributable to the amalgamation of two integuments and support the role of ETT in the fusion of the outer and inner integuments in unitegmic ovules, a situation that could be widespread in other unitegmic species of angiosperms.

  14. Effects of high tidal volume mechanical ventilation on production of cytokines, iNOS, and MIP-1β proteins in pigs.

    PubMed

    Vobruba, Václav; Klimenko, Oxana V; Kobr, Jiri; Cerna, Olga; Pokorna, Pavla; Mikula, Ivan; Hridel, Jan; Brantova, Olga; Martasek, Pavel

    2013-02-01

    The aim of this study was to investigate longitudinal changes of the pulmonary inflammatory process as a result of mechanical stress due to mechanical ventilation. The concentrations of IL-8, TNF-α, MIP-1β, nitrites/nitrates, and inducible nitric oxide synthases (iNOS) were investigated indicate in bronchoalveolar lavage (BAL). Twenty-three piglets were divided into three groups. Group I: animals breathing spontaneously; group II: mechanical ventilation (tidal volume (TV) = 7 mL/kg, PEEP = 5 cmH(2)O); group III: mechanical ventilation (TV = 15 mL/kg, PEEP = 0 cmH(2)0). Concentrations of BAL nitrites/nitrates from groups II and III increased during the first hour of mechanical ventilation (P = .03 and .02, respectively). The highest expression of iNOS was observed during the first hour in groups II and III. IL-8 concentration increased significantly in groups II and III. Production of TNF-α increased significantly in group III during the second and third hour (P = .01). Concentration of MIP-1β was significantly increased in groups II and III after the first hour (P = .012 and P = .008, respectively).

  15. Arctigenin Protects against Lipopolysaccharide-Induced Pulmonary Oxidative Stress and Inflammation in a Mouse Model via Suppression of MAPK, HO-1, and iNOS Signaling.

    PubMed

    Zhang, Wen-zhou; Jiang, Zheng-kui; He, Bao-xia; Liu, Xian-ben

    2015-08-01

    Arctigenin, a bioactive component of Arctium lappa (Nubang), has anti-inflammatory activity. Here, we investigated the effects of arctigenin on lipopolysaccharide (LPS)-induced acute lung injury. Mice were divided into four groups: control, LPS, LPS + DMSO, and LPS + Arctigenin. Mice in the LPS + Arctigenin group were injected intraperitoneally with 50 mg/kg of arctigenin 1 h before an intratracheal administration of LPS (5 mg/kg). Lung tissues and bronchoalveolar lavage fluids (BALFs) were collected. Histological changes of the lung were analyzed by hematoxylin and eosin staining. Arctigenin decreased LPS-induced acute lung inflammation, infiltration of inflammatory cells into BALF, and production of pro-inflammatory cytokines. Moreover, arctigenin pretreatment reduced the malondialdehyde level and increased superoxide dismutase and catalase activities and glutathione peroxidase/glutathione disulfide ratio in the lung. Mechanically, arctigenin significantly reduced the production of nitric oxygen and inducible nitric oxygen synthase (iNOS) expression, enhanced the expression of heme oxygenase-1, and decreased the phosphorylation of mitogen-activated protein kinases (MAPKs). Arctigenin has anti-inflammatory and antioxidative effects on LPS-induced acute lung injury, which are associated with modulation of MAPK, HO-1, and iNOS signaling.

  16. Arctigenin Protects against Lipopolysaccharide-Induced Pulmonary Oxidative Stress and Inflammation in a Mouse Model via Suppression of MAPK, HO-1, and iNOS Signaling.

    PubMed

    Zhang, Wen-zhou; Jiang, Zheng-kui; He, Bao-xia; Liu, Xian-ben

    2015-08-01

    Arctigenin, a bioactive component of Arctium lappa (Nubang), has anti-inflammatory activity. Here, we investigated the effects of arctigenin on lipopolysaccharide (LPS)-induced acute lung injury. Mice were divided into four groups: control, LPS, LPS + DMSO, and LPS + Arctigenin. Mice in the LPS + Arctigenin group were injected intraperitoneally with 50 mg/kg of arctigenin 1 h before an intratracheal administration of LPS (5 mg/kg). Lung tissues and bronchoalveolar lavage fluids (BALFs) were collected. Histological changes of the lung were analyzed by hematoxylin and eosin staining. Arctigenin decreased LPS-induced acute lung inflammation, infiltration of inflammatory cells into BALF, and production of pro-inflammatory cytokines. Moreover, arctigenin pretreatment reduced the malondialdehyde level and increased superoxide dismutase and catalase activities and glutathione peroxidase/glutathione disulfide ratio in the lung. Mechanically, arctigenin significantly reduced the production of nitric oxygen and inducible nitric oxygen synthase (iNOS) expression, enhanced the expression of heme oxygenase-1, and decreased the phosphorylation of mitogen-activated protein kinases (MAPKs). Arctigenin has anti-inflammatory and antioxidative effects on LPS-induced acute lung injury, which are associated with modulation of MAPK, HO-1, and iNOS signaling. PMID:25616905

  17. Fermented guava leaf extract inhibits LPS-induced COX-2 and iNOS expression in Mouse macrophage cells by inhibition of transcription factor NF-kappaB.

    PubMed

    Choi, Soo-Youn; Hwang, Joon-Ho; Park, Soo-Young; Jin, Yeong-Jun; Ko, Hee-Chul; Moon, Sang-Wook; Kim, Se-Jae

    2008-08-01

    The goal of this study was to elucidate the antiinflammatory activities of Psidium guajava L. (guava) leaf. To improve the functionality of guava leaf, it was fermented with Phellinus linteus mycelia, Lactobacillus plantarum and Saccharomyces cerevisiae. The ethanol extract from fermented guava leaf inhibited lipopolysaccharide (LPS)-induced nitric oxide (NO) and prostaglandin E(2) (PGE(2)) production. Western blot analysis showed that fermented guava leaf extract decreased LPS-induced inducible nitric oxide synthase (iNOS) and the cyclooxygenase-2 (COX-2) protein level in RAW 264.7 cells. To investigate the mechanism involved, the study examined the effect of fermented guava leaf extract on LPS-induced nuclear factor-kappaB (NF-kappaB) activation. Fermented guava leaf extract significantly inhibited LPS-induced NF-kappaB transcriptional activity. Immunochemical analysis revealed that fermented guava leaf extract suppressed LPS-induced degradation of I-kappaBalpha. Taken together, the data indicate that fermented guava leaf extract is involved in the inhibition of iNOS and COX-2 via the down-regulation of NF-kappaB pathway, revealing a partial molecular basis for the antiinflammatory properties of fermented guava leaf extract.

  18. Scabraside D Derived from Sea Cucumber Induces Apoptosis and Inhibits Metastasis via iNOS and STAT-3 Expression in Human Cholangiocarcinoma Xenografts.

    PubMed

    Assawasuparerk, Kanjana; Rawangchue, Thanakorn; Phonarknguen, Rassameepen

    2016-01-01

    Scabraside D, a sulfated triterpene glycoside, was extracted from the sea cucumber Holothuria scabra. It shows anti-proliferation in many of cancer cell lines, but the function and mechanisms of action of scabraside D in human cholangiocarcinoma (HuCCA) have not previously determined. In this study, we investigated the activity of scabraside D on HuCCA cell apoptosis, lymphangiogenesis and metastasis in a nude mouse model. Scabraside D induced signs of apoptosis, such as cell shrinkage, nuclear condensation, nuclear fragmentation and DNA fragmentation on TUNEL assays, while effectively decreasing expression of BCl-2 but increasing caspase-3 gene level expression. Immunohistochemistry revealed that scabraside D significantly reduced lymphatic vessel density (LVD). Moreover, scabraside D treatment significantly decreased VEGF-C, MMP-9 and uPA gene expression, which play important roles in the lymphangiogenesis and invasion of cancer cells in metastasis processes. Quantitative real-time PCR showed that scabraside D significantly decreased iNOS and STAT-3 gene expression. This study demonstrated that scabraside D plays a role in activation of HuCCA tumor apoptosis and inhibition of lymphangiogenesis, invasion and metastasis through decreasing BCl-2, MMP-9, uPA and VEGF-C and increasing caspase-3 expression by suppression of iNOS and STAT-3 expression. Therefore, scabraside D could be a promising candidate for cholangiocarcinoma treatment.

  19. Scabraside D Derived from Sea Cucumber Induces Apoptosis and Inhibits Metastasis via iNOS and STAT-3 Expression in Human Cholangiocarcinoma Xenografts.

    PubMed

    Assawasuparerk, Kanjana; Rawangchue, Thanakorn; Phonarknguen, Rassameepen

    2016-01-01

    Scabraside D, a sulfated triterpene glycoside, was extracted from the sea cucumber Holothuria scabra. It shows anti-proliferation in many of cancer cell lines, but the function and mechanisms of action of scabraside D in human cholangiocarcinoma (HuCCA) have not previously determined. In this study, we investigated the activity of scabraside D on HuCCA cell apoptosis, lymphangiogenesis and metastasis in a nude mouse model. Scabraside D induced signs of apoptosis, such as cell shrinkage, nuclear condensation, nuclear fragmentation and DNA fragmentation on TUNEL assays, while effectively decreasing expression of BCl-2 but increasing caspase-3 gene level expression. Immunohistochemistry revealed that scabraside D significantly reduced lymphatic vessel density (LVD). Moreover, scabraside D treatment significantly decreased VEGF-C, MMP-9 and uPA gene expression, which play important roles in the lymphangiogenesis and invasion of cancer cells in metastasis processes. Quantitative real-time PCR showed that scabraside D significantly decreased iNOS and STAT-3 gene expression. This study demonstrated that scabraside D plays a role in activation of HuCCA tumor apoptosis and inhibition of lymphangiogenesis, invasion and metastasis through decreasing BCl-2, MMP-9, uPA and VEGF-C and increasing caspase-3 expression by suppression of iNOS and STAT-3 expression. Therefore, scabraside D could be a promising candidate for cholangiocarcinoma treatment. PMID:27221911

  20. Transition from two to one integument in Prunus species: expression pattern of INNER NO OUTER (INO), ABERRANT TESTA SHAPE (ATS) and ETTIN (ETT).

    PubMed

    Lora, Jorge; Hormaza, José I; Herrero, Maria

    2015-10-01

    While gymnosperm ovules have one integument, in most angiosperms two integuments surround the ovules. Unitegmic ovules have arisen independently several times during the evolution of angiosperms, but the ultimate genetic cause of the presence of a single integument remains elusive. We compared species of the genus Prunus that have different numbers of integuments: bitegmic species, such as Prunus armeniaca (apricot) and Prunus persica (peach), and unitegmic species, such as Prunus incisa, analyzing the expression pattern of genes that are involved in integument development in Arabidopsis thaliana: INNER NO OUTER (INO), ABERRANT TESTA SHAPE (ATS) and ETTIN (ETT). Bitegmic and unitegmic species showed similar INO expression patterns, indicative of the conservation of an outer integument. However, expression of ETT, which occurs in the boundary of the outer and inner integuments, was altered in unitegmic ovules, which showed lack of ETT expression. These results strongly suggest that the presence of a single integument could be attributable to the amalgamation of two integuments and support the role of ETT in the fusion of the outer and inner integuments in unitegmic ovules, a situation that could be widespread in other unitegmic species of angiosperms. PMID:25991552

  1. Lipocortin 1 mediates the inhibition by dexamethasone of the induction by endotoxin of nitric oxide synthase in the rat.

    PubMed Central

    Wu, C C; Croxtall, J D; Perretti, M; Bryant, C E; Thiemermann, C; Flower, R J; Vane, J R

    1995-01-01

    Administration of Escherichia coli lipopolysaccharide (LPS; 10 mg/kg i.v.) to male Wistar rats caused within 240 min (i) a sustained fall (approximately 30 mmHg) in mean arterial blood pressure, (ii) a reduction (> 75%) in the pressor responses to norepinephrine (1 microgram/kg i.v.), and (iii) an induction of nitric oxide synthase (iNOS) as measured in the lung. Dexamethasone (1 mg/kg i.p. at 2 h prior to LPS) attenuated the hypotension and the vascular hyporeactivity to norepinephrine and reduced (by approximately 77%) the expression of iNOS in the lung. These effects of dexamethasone were prevented by pretreatment of LPS-treated rats with a neutralizing antiserum to lipocortin 1 (anti-LC1; 60 mg/kg s.c. at 24 h prior to LPS) but not by a control nonimmune sheep serum. Stimulation of J774.2 macrophages with LPS (1 microgram/ml for 24 h) caused the expression of iNOS and cyclooxygenase 2 (COX-2) protein and significantly increased nitrite generation; this was prevented by dexamethasone (0.1 microM at 1 h prior to LPS), which also increased cell surface lipocortin 1. Pretreatment of J774.2 cells with anti-LC1 (1:60 dilution at 4 h prior to LPS) also abolished the inhibitory effect of dexamethasone on iNOS expression and nitrite accumulation but not that on COX-2 expression. A lipocortin 1 fragment (residues 1-188 of human lipocortin 1; 20 micrograms/ml at 1 h prior to LPS) also blocked iNOS in J774.2 macrophages activated by LPS (approximately 78% inhibition), and this too was prevented by anti-LC1. We conclude that the extracellular release of endogenous lipocortin 1 (i) mediates the inhibition by dexamethasone of the expression of iNOS, but not of COX-2, and (ii) contributes substantially to the beneficial actions of dexamethasone in rats with endotoxic shock. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:7536934

  2. Phenotypic plasticity in bacterial plasmids.

    PubMed Central

    Turner, Paul E

    2004-01-01

    Plasmid pB15 was previously shown to evolve increased horizontal (infectious) transfer at the expense of reduced vertical (intergenerational) transfer and vice versa, a key trade-off assumed in theories of parasite virulence. Whereas the models predict that susceptible host abundance should determine which mode of transfer is selectively favored, host density failed to mediate the trade-off in pB15. One possibility is that the plasmid's transfer deviates from the assumption that horizontal spread (conjugation) occurs in direct proportion to cell density. I tested this hypothesis using Escherichia coli/pB15 associations in laboratory serial culture. Contrary to most models of plasmid transfer kinetics, my data show that pB15 invades static (nonshaking) bacterial cultures only at intermediate densities. The results can be explained by phenotypic plasticity in traits governing plasmid transfer. As cells become more numerous, the plasmid's conjugative transfer unexpectedly declines, while the trade-off between transmission routes causes vertical transfer to increase. Thus, at intermediate densities the plasmid's horizontal transfer can offset selection against plasmid-bearing cells, but at high densities pB15 conjugates so poorly that it cannot invade. I discuss adaptive vs. nonadaptive causes for the phenotypic plasticity, as well as potential mechanisms that may lead to complex transfer dynamics of plasmids in liquid environments. PMID:15166133

  3. Role of iNOS gene expression in the anti-inflammatory and tissue protective mechanisms of continuous wave at 630-905nm and 905nm superpulsed laser therapy

    NASA Astrophysics Data System (ADS)

    Mandel, Arkady; Moriyama, Yumi; Fong, Jamie; Dumoulin-White, Roger; Lilge, Lothar

    2012-03-01

    Up regulation of iNOS gene expression is playing a role in the initiation of the anti-inflammatory and tissue protective mechanisms related to nitric oxide (NO) for continuous wave red and infrared as well as 905nm superpulsed laser therapy (SPLT). The iNOS expression before and after laser therapy was evaluated in a zymosan-induced acute arthritis model, in knee joints of young (<15 weeks), middle aged (>15 weeks and < 35 weeks) and old (> 35 weeks) FVB/N-Tg (iNOS-luc) mice by bioluminescence imaging.

  4. Bacterial ratchet motors

    PubMed Central

    Di Leonardo, R.; Angelani, L.; Dell’Arciprete, D.; Ruocco, G.; Iebba, V.; Schippa, S.; Conte, M. P.; Mecarini, F.; De Angelis, F.; Di Fabrizio, E.

    2010-01-01

    Self-propelling bacteria are a nanotechnology dream. These unicellular organisms are not just capable of living and reproducing, but they can swim very efficiently, sense the environment, and look for food, all packaged in a body measuring a few microns. Before such perfect machines can be artificially assembled, researchers are beginning to explore new ways to harness bacteria as propelling units for microdevices. Proposed strategies require the careful task of aligning and binding bacterial cells on synthetic surfaces in order to have them work cooperatively. Here we show that asymmetric environments can produce a spontaneous and unidirectional rotation of nanofabricated objects immersed in an active bacterial bath. The propulsion mechanism is provided by the self-assembly of motile Escherichia coli cells along the rotor boundaries. Our results highlight the technological implications of active matter’s ability to overcome the restrictions imposed by the second law of thermodynamics on equilibrium passive fluids. PMID:20457936

  5. Flagella and bacterial pathogenicity.

    PubMed

    Duan, Qiangde; Zhou, Mingxu; Zhu, Liqian; Zhu, Guoqiang

    2013-01-01

    As locomotive organelles, flagella allow bacteria to move toward favorable environments. A flagellum consists of three parts: the basal structure (rotary motor), the hook (universal joint), and the filament (helical propeller). For ages, flagella have been generally regarded as important virulence factors, mainly because of their motility property. However, flagella are getting recognized to play multiple roles with more functions besides motility and chemotaxis. Recent evidence has pinpointed that the bacterial flagella participate in many additional processes including adhesion, biofilm formation, virulence factor secretion, and modulation of the immune system of eukaryotic cells. This mini-review summarizes data from recent studies that elucidated how flagella, as a virulence factor, contribute to bacterial pathogenicity.

  6. Flagella and bacterial pathogenicity.

    PubMed

    Duan, Qiangde; Zhou, Mingxu; Zhu, Liqian; Zhu, Guoqiang

    2013-01-01

    As locomotive organelles, flagella allow bacteria to move toward favorable environments. A flagellum consists of three parts: the basal structure (rotary motor), the hook (universal joint), and the filament (helical propeller). For ages, flagella have been generally regarded as important virulence factors, mainly because of their motility property. However, flagella are getting recognized to play multiple roles with more functions besides motility and chemotaxis. Recent evidence has pinpointed that the bacterial flagella participate in many additional processes including adhesion, biofilm formation, virulence factor secretion, and modulation of the immune system of eukaryotic cells. This mini-review summarizes data from recent studies that elucidated how flagella, as a virulence factor, contribute to bacterial pathogenicity. PMID:22359233

  7. Physics of Bacterial Morphogenesis

    PubMed Central

    Sun, Sean X.; Jiang, Hongyuan

    2011-01-01

    Summary: Bacterial cells utilize three-dimensional (3D) protein assemblies to perform important cellular functions such as growth, division, chemoreception, and motility. These assemblies are composed of mechanoproteins that can mechanically deform and exert force. Sometimes, small-nucleotide hydrolysis is coupled to mechanical deformations. In this review, we describe the general principle for an understanding of the coupling of mechanics with chemistry in mechanochemical systems. We apply this principle to understand bacterial cell shape and morphogenesis and how mechanical forces can influence peptidoglycan cell wall growth. We review a model that can potentially reconcile the growth dynamics of the cell wall with the role of cytoskeletal proteins such as MreB and crescentin. We also review the application of mechanochemical principles to understand the assembly and constriction of the FtsZ ring. A number of potential mechanisms are proposed, and important questions are discussed. PMID:22126993

  8. Formation of bacterial nanocells

    NASA Astrophysics Data System (ADS)

    Vainshtein, Mikhail; Kudryashova, Ekaterina; Suzina, Natalia; Ariskina, Elena; Voronkov, Vadim

    1998-07-01

    Existence of nanobacteria received increasing attention both in environmental microbiology/geomicro-biology and in medical microbiology. In order to study a production of nanoforms by typical bacterial cells. Effects of different physical factors were investigated. Treatment of bacterial cultures with microwave radiation, or culturing in field of electric current resulted in formation a few types of nanocells. The number and type of nanoforms were determined with type and dose of the treatment. The produced nanoforms were: i) globules, ii) clusters of the globules--probably produced by liaison, iii) nanocells coated with membrane. The viability of the globules is an object opened for doubts. The nanocells discovered multiplication and growth on solidified nutrient media. The authors suggest that formation of nanocells is a common response of bacteria to stress-actions produced by different agents.

  9. Reactive oxygen species mediate nitric oxide production through ERK/JNK MAPK signaling in HAPI microglia after PFOS exposure.

    PubMed

    Wang, Cheng; Nie, Xiaoke; Zhang, Yan; Li, Ting; Mao, Jiamin; Liu, Xinhang; Gu, Yiyang; Shi, Jiyun; Xiao, Jing; Wan, Chunhua; Wu, Qiyun

    2015-10-15

    Perfluorooctane sulfonate (PFOS), an emerging persistent contaminant that is commonly encountered during daily life, has been shown to exert toxic effects on the central nervous system (CNS). However, the molecular mechanisms underlying the neurotoxicity of PFOS remain largely unknown. It has been widely acknowledged that the inflammatory mediators released by hyper-activated microglia play vital roles in the pathogenesis of various neurological diseases. In the present study, we examined the impact of PFOS exposure on microglial activation and the release of proinflammatory mediators, including nitric oxide (NO) and reactive oxidative species (ROS). We found that PFOS exposure led to concentration-dependent NO and ROS production by rat HAPI microglia. We also discovered that there was rapid activation of the ERK/JNK MAPK signaling pathway in the HAPI microglia following PFOS treatment. Moreover, the PFOS-induced iNOS expression and NO production were attenuated after the inhibition of ERK or JNK MAPK by their corresponding inhibitors, PD98059 and SP600125. Interestingly, NAC, a ROS inhibitor, blocked iNOS expression, NO production, and activation of ERK and JNK MAPKs, which suggested that PFOS-mediated microglial NO production occurs via a ROS/ERK/JNK MAPK signaling pathway. Finally, by exposing SH-SY5Y cells to PFOS-treated microglia-conditioned medium, we demonstrated that NO was responsible for PFOS-mediated neuronal apoptosis.

  10. Bacterial, Fungal, Parasitic, and Viral Myositis

    PubMed Central

    Crum-Cianflone, Nancy F.

    2008-01-01

    Infectious myositis may be caused by a broad range of bacterial, fungal, parasitic, and viral agents. Infectious myositis is overall uncommon given the relative resistance of the musculature to infection. For example, inciting events, including trauma, surgery, or the presence of foreign bodies or devitalized tissue, are often present in cases of bacterial myositis. Bacterial causes are categorized by clinical presentation, anatomic location, and causative organisms into the categories of pyomyositis, psoas abscess, Staphylococcus aureus myositis, group A streptococcal necrotizing myositis, group B streptococcal myositis, clostridial gas gangrene, and nonclostridial myositis. Fungal myositis is rare and usually occurs among immunocompromised hosts. Parasitic myositis is most commonly a result of trichinosis or cystericercosis, but other protozoa or helminths may be involved. A parasitic cause of myositis is suggested by the travel history and presence of eosinophilia. Viruses may cause diffuse muscle involvement with clinical manifestations, such as benign acute myositis (most commonly due to influenza virus), pleurodynia (coxsackievirus B), acute rhabdomyolysis, or an immune-mediated polymyositis. The diagnosis of myositis is suggested by the clinical picture and radiologic imaging, and the etiologic agent is confirmed by microbiologic or serologic testing. Therapy is based on the clinical presentation and the underlying pathogen. PMID:18625683

  11. 3D printing of microscopic bacterial communities

    PubMed Central

    Connell, Jodi L.; Ritschdorff, Eric T.; Whiteley, Marvin; Shear, Jason B.

    2013-01-01

    Bacteria communicate via short-range physical and chemical signals, interactions known to mediate quorum sensing, sporulation, and other adaptive phenotypes. Although most in vitro studies examine bacterial properties averaged over large populations, the levels of key molecular determinants of bacterial fitness and pathogenicity (e.g., oxygen, quorum-sensing signals) may vary over micrometer scales within small, dense cellular aggregates believed to play key roles in disease transmission. A detailed understanding of how cell–cell interactions contribute to pathogenicity in natural, complex environments will require a new level of control in constructing more relevant cellular models for assessing bacterial phenotypes. Here, we describe a microscopic three-dimensional (3D) printing strategy that enables multiple populations of bacteria to be organized within essentially any 3D geometry, including adjacent, nested, and free-floating colonies. In this laser-based lithographic technique, microscopic containers are formed around selected bacteria suspended in gelatin via focal cross-linking of polypeptide molecules. After excess reagent is removed, trapped bacteria are localized within sealed cavities formed by the cross-linked gelatin, a highly porous material that supports rapid growth of fully enclosed cellular populations and readily transmits numerous biologically active species, including polypeptides, antibiotics, and quorum-sensing signals. Using this approach, we show that a picoliter-volume aggregate of Staphylococcus aureus can display substantial resistance to β-lactam antibiotics by enclosure within a shell composed of Pseudomonas aeruginosa. PMID:24101503

  12. Bacterial transformation of terpenoids

    NASA Astrophysics Data System (ADS)

    Grishko, V. V.; Nogovitsina, Y. M.; Ivshina, I. B.

    2014-04-01

    Data on the bacterial transformation of terpenoids published in the literature in the past decade are analyzed. Possible pathways for chemo-, regio- and stereoselective modifications of terpenoids are discussed. Considerable attention is given to new technological approaches to the synthesis of terpenoid derivatives suitable for the use in the perfume and food industry and promising as drugs and chiral intermediates for fine organic synthesis. The bibliography includes 246 references.

  13. Nitrative stress in cerebral endothelium is mediated by mGluR5 in hyperhomocysteinemia

    PubMed Central

    Mayo, Jamie N; Beard, Richard S; Price, Tulin O; Chen, Cheng-Hung; Erickson, Michelle A; Ercal, Nuran; Banks, William A; Bearden, Shawn E

    2012-01-01

    Hyperhomocysteinemia (HHcy) disrupts nitric oxide (NO) signaling and increases nitrative stress in cerebral microvascular endothelial cells (CMVECs). This is mediated, in part, by protein nitrotyrosinylation (3-nitrotyrosine; 3-NT) though the mechanisms by which extracellular homocysteine (Hcy) generates intracellular 3-NT are unknown. Using a murine model of mild HHcy (cbs+/− mouse), we show that 3-NT is significantly elevated in cerebral microvessels with concomitant reductions in serum NO bioavailability as compared with wild-type littermate controls (cbs+/+). Directed pharmacology identified a receptor-dependent mechanism for 3-NT formation in CMVECs. Homocysteine increased expression of inducible NO synthase (iNOS) and formation of 3-NT, both of which were blocked by inhibition of metabotropic glutamate receptor-5 (mGluR5) with the specific antagonist 2-methyl-6-(phenylethynyl) pyridine hydrochloride. Activation of mGluR5 is both sufficient and necessary to drive the nitrative stress because direct activation using the mGluR5-specific agonist (RS)-2-chloro-5-hydroxyphenylglycine also increased iNOS expression and 3-NT formation while knockdown of mGluR5 receptor expression by short hairpin RNA (shRNA) blocked their increase in response to Hcy. Nitric oxide derived from iNOS was required for Hcy-mediated formation of 3-NT because the effect was blocked by 1400W. These results provide the first evidence for a receptor-dependent process that explains how plasma Hcy levels control intracellular nitrative stress in cerebral microvascular endothelium. PMID:22186670

  14. Nitric oxide-mediated apoptosis of neutrophils through caspase-8 and caspase-3-dependent mechanism.

    PubMed

    Dubey, Megha; Nagarkoti, Sheela; Awasthi, Deepika; Singh, Abhishek K; Chandra, Tulika; Kumaravelu, J; Barthwal, Manoj K; Dikshit, Madhu

    2016-01-01

    Neutrophils play an indispensable role in killing of invading pathogens by enhancing reactive oxygen species (ROS) and NO generation, and subsequently undergoing apoptosis. Unlike ROS/NOX2, role of NO/NOS still remains undefined in the apoptosis of neutrophils (PMNs) and the present study attempts to decipher the importance of NO/NOS in the neutrophil apoptosis. Prolonged treatment of human PMNs or mice bone marrow derived neutrophils (BMDN) with NO led to enhanced ROS generation, caspase-8/caspase-3 cleavage, reduced mitochondrial membrane potential and finally cellular apoptosis. NO-induced ROS generation led to caspase-8 deglutathionylation and activation, which subsequently activated mitochondrial death pathway via BID (Bcl-2 family protein) cleavage. NO-mediated augmentation of caspase-8 and BID cleavage was significantly prevented in BMDN from neutrophil cytosolic factor-1 (NCF-1) knockout (KO) mice, implying the involvement of NOX2 in NO-induced apoptosis of PMNs. Furthermore, ROS, NO generation and inducible nitric oxide synthase (iNOS) expression were enhanced in a time-dependent manner in human PMNs and mice BMDN undergoing spontaneous apoptosis. Pharmacological and genetic ablation of iNOS in human PMNs and mice BMDN significantly reduced the levels of apoptosis. Impaired apoptosis of BMDN from iNOS KO mice was due to reduced caspase-8 activity which subsequently prevented caspase-3 and -9 activation. Altogether, our results suggest a crucial role of NO/iNOS in neutrophil apoptosis via enhanced ROS generation and caspase-8 mediated activation of mitochondrial death pathway. PMID:27584786

  15. Nitric oxide-mediated apoptosis of neutrophils through caspase-8 and caspase-3-dependent mechanism

    PubMed Central

    Dubey, Megha; Nagarkoti, Sheela; Awasthi, Deepika; Singh, Abhishek K; Chandra, Tulika; Kumaravelu, J; Barthwal, Manoj K; Dikshit, Madhu

    2016-01-01

    Neutrophils play an indispensable role in killing of invading pathogens by enhancing reactive oxygen species (ROS) and NO generation, and subsequently undergoing apoptosis. Unlike ROS/NOX2, role of NO/NOS still remains undefined in the apoptosis of neutrophils (PMNs) and the present study attempts to decipher the importance of NO/NOS in the neutrophil apoptosis. Prolonged treatment of human PMNs or mice bone marrow derived neutrophils (BMDN) with NO led to enhanced ROS generation, caspase-8/caspase-3 cleavage, reduced mitochondrial membrane potential and finally cellular apoptosis. NO-induced ROS generation led to caspase-8 deglutathionylation and activation, which subsequently activated mitochondrial death pathway via BID (Bcl-2 family protein) cleavage. NO-mediated augmentation of caspase-8 and BID cleavage was significantly prevented in BMDN from neutrophil cytosolic factor-1 (NCF-1) knockout (KO) mice, implying the involvement of NOX2 in NO-induced apoptosis of PMNs. Furthermore, ROS, NO generation and inducible nitric oxide synthase (iNOS) expression were enhanced in a time-dependent manner in human PMNs and mice BMDN undergoing spontaneous apoptosis. Pharmacological and genetic ablation of iNOS in human PMNs and mice BMDN significantly reduced the levels of apoptosis. Impaired apoptosis of BMDN from iNOS KO mice was due to reduced caspase-8 activity which subsequently prevented caspase-3 and -9 activation. Altogether, our results suggest a crucial role of NO/iNOS in neutrophil apoptosis via enhanced ROS generation and caspase-8 mediated activation of mitochondrial death pathway. PMID:27584786

  16. Piperine inhibits IL-β induced expression of inflammatory mediators in human osteoarthritis chondrocyte.

    PubMed

    Ying, Xiaozhou; Chen, Xiaowei; Cheng, Shaowen; Shen, Yue; Peng, Lei; Xu, Hua Zi

    2013-10-01

    Black pepper (Piper nigrum) is a common remedy in Traditional Chinese Medicine and possesses diverse biological activities including anti-inflammatory properties. Osteoarthritis (OA) is a degenerative joint disease with an inflammatory component that drives the degradation of cartilage extracellular matrix. The present study aimed to assess the effects of piperine, the active phenolic component in black pepper extract, on human OA chondrocytes. In this study, human OA chondrocytes were pretreated with piperine at 10, 50 or 100μg/ml and subsequently stimulated with IL-1β (5ng/ml) for 24h. Production of PGE2 and NO was evaluated by the Griess reaction and an ELISA. Gene expression of MMP-3, MMP-13, iNOS and COX-2 was measured by real-time PCR. MMP-3 and MMP-13 proteins in culture medium were determined using cytokine-specific ELISA. Western immunoblotting was used to analyze the iNOS and COX-2 protein production in the culture medium. The regulation of NF-kB activity and the degradation of IkB were explored using luciferase and Western immunoblotting, respectively. We found that piperine inhibited the production of PGE2 and NO induced by IL-1β. Piperine significantly decreased the IL-1β-stimulated gene expression and production of MMP-3, MMP-13, iNOS and COX-2 in human OA chondrocytes. Piperine inhibited the IL-1β-mediated activation of NF-κB by suppressing the degradation of its inhibitory protein IκBα in the cytoplasm. The present report is first to demonstrate the anti-inflammatory activity of piperine in human OA chondrocytes. Piperine can effectively abrogate the IL-1β-induced over-expression of inflammatory mediators; suggesting that piperine may be a potential agent in the treatment of OA.

  17. Neglected bacterial zoonoses.

    PubMed

    Chikeka, I; Dumler, J S

    2015-05-01

    Bacterial zoonoses comprise a group of diseases in humans or animals acquired by direct contact with or by oral consumption of contaminated animal materials, or via arthropod vectors. Among neglected infections, bacterial zoonoses are among the most neglected given emerging data on incidence and prevalence as causes of acute febrile illness, even in areas where recognized neglected tropical diseases occur frequently. Although many other bacterial infections could also be considered in this neglected category, five distinct infections stand out because they are globally distributed, are acute febrile diseases, have high rates of morbidity and case fatality, and are reported as commonly as malaria, typhoid or dengue virus infections in carefully designed studies in which broad-spectrum diagnoses are actively sought. This review will focus attention on leptospirosis, relapsing fever borreliosis and rickettsioses, including scrub typhus, murine typhus and spotted fever group rickettsiosis. Of greatest interest is the lack of distinguishing clinical features among these infections when in humans, which confounds diagnosis where laboratory confirmation is lacking, and in regions where clinical diagnosis is often attributed to one of several perceived more common threats. As diseases such as malaria come under improved control, the real impact of these common and under-recognized infections will become evident, as will the requirement for the strategies and allocation of resources for their control. PMID:25964152

  18. Acute Bacterial Cholangitis

    PubMed Central

    Zimmer, Vincent; Lammert, Frank

    2015-01-01

    Background Acute bacterial cholangitis for the most part owing to common bile duct stones is common in gastroenterology practice and represents a potentially life-threatening condition often characterized by fever, abdominal pain, and jaundice (Charcot's triad) as well as confusion and septic shock (Reynolds' pentad). Methods This review is based on a systematic literature review in PubMed with the search items ‘cholangitis’, ‘choledocholithiasis’, ‘gallstone disease’, ‘biliary infection’, and ‘biliary sepsis’. Results Although most patients respond to empiric broad-spectrum antibiotic treatment, timely endoscopic biliary drainage depending on the severity of the disease is required to eliminate the underlying obstruction. Specific recommendations have been derived from the Tokyo guideline working group consensus 2006 and its update in 2013, albeit poorly evidence-based, providing a comprehensive overview of diagnosis, classification, risk stratification, and treatment algorithms in acute bacterial cholangitis. Conclusion Prompt clinical recognition and accurate diagnostic workup including adequate laboratory assessment and (aetiology-oriented) imaging are critical steps in the management of cholangitis. Treatment is directed at the two major interrelated pathophysiologic components, i.e. bacterial infection (immediate antimicrobial therapy) and bile duct obstruction (biliary drainage). As for the latter, transpapillary endoscopic drainage by stent or nasobiliary drain and/or same-session bile duct clearance, depending on individual disease severity, represent first-line treatment approaches. PMID:26468310

  19. Neglected bacterial zoonoses.

    PubMed

    Chikeka, I; Dumler, J S

    2015-05-01

    Bacterial zoonoses comprise a group of diseases in humans or animals acquired by direct contact with or by oral consumption of contaminated animal materials, or via arthropod vectors. Among neglected infections, bacterial zoonoses are among the most neglected given emerging data on incidence and prevalence as causes of acute febrile illness, even in areas where recognized neglected tropical diseases occur frequently. Although many other bacterial infections could also be considered in this neglected category, five distinct infections stand out because they are globally distributed, are acute febrile diseases, have high rates of morbidity and case fatality, and are reported as commonly as malaria, typhoid or dengue virus infections in carefully designed studies in which broad-spectrum diagnoses are actively sought. This review will focus attention on leptospirosis, relapsing fever borreliosis and rickettsioses, including scrub typhus, murine typhus and spotted fever group rickettsiosis. Of greatest interest is the lack of distinguishing clinical features among these infections when in humans, which confounds diagnosis where laboratory confirmation is lacking, and in regions where clinical diagnosis is often attributed to one of several perceived more common threats. As diseases such as malaria come under improved control, the real impact of these common and under-recognized infections will become evident, as will the requirement for the strategies and allocation of resources for their control.

  20. Neglected Bacterial Zoonoses

    PubMed Central

    Chikeka, Ijeuru; Dumler, J. Stephen

    2015-01-01

    Bacterial zoonoses comprise a group of diseases in humans or animals acquired by direct contact with or by oral consumption of contaminated animal materials, or via arthropod vectors. Among neglected infections, bacterial zoonoses are among the most neglected given emerging data on incidence and prevalence as causes of acute febrile illness, even in areas where recognized neglected tropical diseases occur frequently. While many other bacterial infections could also be considered in this neglected category, five distinct infections stand out because they are globally distributed, are acute febrile diseases, have high rates of morbidity and case fatality, and are reported as commonly as malaria, typhoid or dengue virus infections in carefully designed studies in which a broad spectrum diagnoses are actively sought. Thus, this review will focus attention on leptospirosis, relapsing fever borreliosis, and rickettsioses, including scrub typhus, murine typhus and spotted fever group rickettsiosis. Of greatest interest is the lack of distinguishing clinical features among these infections when in humans, which confounds diagnosis where laboratory confirmation is lacking, and in regions where clinical diagnosis is often attributed to one of several perceived more common threats. As diseases such as malaria come under improved control, the real impact of these common and under-recognized infections will become evident, as will the requirement for the strategies and allocation of resources for their control. PMID:25964152

  1. Zinc protoporphyrin inhibition of lipopolysaccharide-, lipoteichoic acid-, and peptidoglycan-induced nitric oxide production through stimulating iNOS protein ubiquitination

    SciTech Connect

    Chow, J.-M.; Lin, H.-Y.; Shen, S.-C.; Wu, M.-S.; Lin, C.-W.; Chiu, W.-T.; Lin, C.-H. Chen, Y.-C.

    2009-06-15

    In the present study, zinc protoporphyrin (ZnPP), but not ferric protoporphyrin (FePP), tin protoporphyrin (SnPP), or zinc chloride (ZnCl{sub 2}), at the doses of 0.5, 1, and 2 {mu}M, dose-dependently inhibited lipopolysaccharide- (LPS), lipoteichoic acid (LTA), and peptidoglycan (PGN)-induced inducible nitric oxide (iNOS) and nitric oxide (NO) production with an increase in heme oxygenase 1 (HO-1) protein in RAW264.7 macrophages in a serum-free condition. NO inhibition and HO-1 induction by ZnPP were blocked by the separate addition of fetal bovine serum (FBS) and bovine serum albumin (BSA). A decrease in the iNOS/NO ratio and an increase in HO-1 protein by ZnPP were identified in three different conditions including ZnPP pretreatment, ZnPP co-treatment, and ZnPP post-treatment with LPS and LTA. Activation of c-Jun N-terminal kinases (JNKs) and extracellular regulated kinases (ERKs) were detected in LPS-, LTA-, and PGN-treated RAW264.7 cells, and iNOS/NO production was blocked by adding the JNK inhibitor, SP600125, but not the ERK inhibitor, PD98059. However, ZnPP addition potentiated ERK and JNK protein phosphorylation stimulated by LPS, LTA, and PGN. Increases in total protein ubiquitination and ubiquitinated iNOS proteins were detected in ZnPP-treated macrophages elicited by LPS according to Western and immunoprecipitation/Western blotting assays, respectively. The decrease in LPS-induced iNOS protein by ZnPP was reversed by adding the proteasome inhibitors MG132 and lactacystin. The reduction in HO-1 protein induced by ZnPP via transfection of HO-1 small interfering RNA did not affect the inhibitory effect of ZnPP against LPS-induced iNOS/NO production and protein ubiquitination induced by ZnPP in macrophages. Data of the present study provide the first evidence to support ZnPP effectively inhibiting inflammatory iNOS/NO production through activation of protein ubiquitination in a HO-1-independent manner in macrophages.

  2. Momordin Ic induces HepG2 cell apoptosis through MAPK and PI3K/Akt-mediated mitochondrial pathways.

    PubMed

    Wang, Jing; Yuan, Li; Xiao, Haifang; Xiao, Chunxia; Wang, Yutang; Liu, Xuebo

    2013-06-01

    Momordin Ic is a natural triterpenoid saponin enriched in various Chinese and Japanese natural medicines such as the fruit of Kochia scoparia (L.) Schrad. So far, there is little scientific evidence for momordin Ic with regard to the anti-tumor activities. The aim of this work was to elucidate the anti-tumor effect of momordin Ic and the signal transduction pathways involved. We found that momordin Ic induced apoptosis in human hepatocellular carcinoma HepG2 cells, which were supported by DNA fragmentation, caspase-3 activation and PARP cleavage. Meanwhile, momordin Ic triggered reactive oxygen species (ROS) production together with collapse of mitochondrial membrane potential, cytochrome c release, down-regulation of Bcl-2 and up-regulation of Bax expression. The activation of p38 and JNK, inactivation of Erk1/2 and Akt were also demonstrated. Although ROS production rather than NO was stimulated, the expression of iNOS and HO-1 were altered after momordin Ic treatment for 4 h. Furthermore, the cytochrome c release, caspase-3 activation, Bax/Bcl-2 expression and PARP cleavage were promoted with LY294002 and U0126 intervention but were blocked by SB203580, SP600125, PI3K activator, NAC and 1,400 W pretreatment, demonstrating the mitochondrial disruption. Furthermore, momordin Ic combination with NAC influenced MAPK, PI3K/Akt and HO-1, iNOS pathways, MAPK and PI3K/Akt pathways also regulated the expression of HO-1 and iNOS. These results indicated that momordin Ic induced apoptosis through oxidative stress-regulated mitochondrial dysfunction involving the MAPK and PI3K-mediated iNOS and HO-1 pathways. Thus, momordin Ic might represent a potential source of anticancer candidate. PMID:23417763

  3. BACTERIALLY-MEDIATED DEGRADATION OF A CHIRAL DISINFECTION BYPRODUCT

    EPA Science Inventory

    Disinfection of drinking and waste waters, through chlorination, can result in the production of chlorinated organic compounds, many of which are regulated by the U.S. Environmental Protection Agency. Among these regulated compounds are the haloacetic acids, which exhibit toxic e...

  4. Perivascular macrophages mediate neutrophil recruitment during bacterial skin infection

    PubMed Central

    Abtin, Arby; Jain, Rohit; Mitchell, Andrew J.; Roediger, Ben; Brzoska, Anthony J.; Tikoo, Shweta; Cheng, Qiang; Ng, Lai Guan; Cavanagh, Lois L.; von Andrian, Ulrich H.; Hickey, Michael J.; Firth, Neville; Weninger, Wolfgang

    2014-01-01

    Transendothelial migration of neutrophils in post-capillary venules is a key event in the inflammatory response against pathogens and tissue damage. The precise regulation of this process is incompletely understood. We report that perivascular macrophages are critical for neutrophil migration into skin infected with the pathogen Staphylococcus aureus. Using multiphoton intravital microscopy we show that neutrophils extravasate from inflamed dermal venules in close proximity to perivascular macrophages, which are a major source of neutrophil chemoattractants. The virulence factor alpha-hemolysin lyses perivascular macrophages leading to decreased neutrophil transmigration. Our data illustrate a previously unrecognized role for perivascular macrophages in neutrophil recruitment to inflamed skin, and indicate that Staphylococcus aureus uses hemolysin-dependent killing of these cells as an immune evasion strategy. PMID:24270515

  5. Bacterial accumulation mediated by flow compression-expansion

    NASA Astrophysics Data System (ADS)

    Miño, Gastón L.; Altshuler, Ernesto; Lindner, Anke; Stocker, Roman; Condat, Carlos A.; Banchio, Adolfo J.; Marconi, Veronica I.; Clément, Eric

    2014-11-01

    Swimming bacteria can be concentrated using a suitable microfluidic device. The combination of flow rate and surface shape can have significant impact on the microorganisms' behavior. In those processes rheotaxis, accumulation caused by ratchets and even attachment to surfaces leading biofilm formation can be observed. Under these conditions, the transport of the active suspension is deeply modified, and differs significantly from passive suspensions. In this work, we present experimental evidence of Escherichia coli suspension flowing in a straight channel with a funnel-like constriction in the middle. This constriction is characterized by the aperture (wf) and its angle (Θf). We explore how the modification of wf and Θf affects the accumulation of bacteria in the channel. Concentrations of bacteria passing the constriction were observed for all the cases. However, the range of the flow rate that produced such accumulation varied with the geometry. In order to obtain a better understanding of this phenomenon, experiments are compared with a simple phenomenological model.

  6. Perivascular macrophages mediate neutrophil recruitment during bacterial skin infection.

    PubMed

    Abtin, Arby; Jain, Rohit; Mitchell, Andrew J; Roediger, Ben; Brzoska, Anthony J; Tikoo, Shweta; Cheng, Qiang; Ng, Lai Guan; Cavanagh, Lois L; von Andrian, Ulrich H; Hickey, Michael J; Firth, Neville; Weninger, Wolfgang

    2014-01-01

    Transendothelial migration of neutrophils in postcapillary venules is a key event in the inflammatory response against pathogens and tissue damage. The precise regulation of this process is incompletely understood. We report that perivascular macrophages are critical for neutrophil migration into skin infected with the pathogen Staphylococcus aureus. Using multiphoton intravital microscopy we showed that neutrophils extravasate from inflamed dermal venules in close proximity to perivascular macrophages, which are a major source of neutrophil chemoattractants. The virulence factor α-hemolysin produced by S. aureus lyses perivascular macrophages, which leads to decreased neutrophil transmigration. Our data illustrate a previously unrecognized role for perivascular macrophages in neutrophil recruitment to inflamed skin and indicate that S. aureus uses hemolysin-dependent killing of these cells as an immune evasion strategy. PMID:24270515

  7. Bacterial genetic methods to explore the biology of mariner transposons.

    PubMed

    Lampe, David J

    2010-05-01

    Mariners are small DNA mediated transposons of eukaryotes that fortuitously function in bacteria. Using bacterial genetics, it is possible to study a variety of properties of mariners, including transpositional ability, dominant-negative regulation, overexpresson inhibition, and the function of cis-acting sequences like the inverted terminal repeats. In conjunction with biochemical techniques, the structure of the transposase can be elucidated and the activity of the elements can be improved for genetic tool use. Finally, it is possible to uncover functional transposase genes directly from genomes given a suitable bacterial genetic screen.

  8. Bacterial nanomachines: the flagellum and type III injectisome.

    PubMed

    Erhardt, Marc; Namba, Keiichi; Hughes, Kelly T

    2010-11-01

    The bacterial flagellum and the virulence-associated injectisome are complex, structurally related nanomachines that bacteria use for locomotion or the translocation of virulence factors into eukaryotic host cells. The assembly of both structures and the transfer of extracellular proteins is mediated by a unique, multicomponent transport apparatus, the type III secretion system. Here, we discuss the significant progress that has been made in recent years in the visualization and functional characterization of many components of the type III secretion system, the structure of the bacterial flagellum, and the injectisome complex. PMID:20926516

  9. Bacterial nanomachines: the flagellum and type III injectisome.

    PubMed

    Erhardt, Marc; Namba, Keiichi; Hughes, Kelly T

    2010-11-01

    The bacterial flagellum and the virulence-associated injectisome are complex, structurally related nanomachines that bacteria use for locomotion or the translocation of virulence factors into eukaryotic host cells. The assembly of both structures and the transfer of extracellular proteins is mediated by a unique, multicomponent transport apparatus, the type III secretion system. Here, we discuss the significant progress that has been made in recent years in the visualization and functional characterization of many components of the type III secretion system, the structure of the bacterial flagellum, and the injectisome complex.

  10. Mediator Deathwork

    ERIC Educational Resources Information Center

    Walter, Tony

    2005-01-01

    The most discussed and analyzed form of deathwork is the dyadic "therapist" [double arrow] "client" relationship, but this far from exhausts the various types of professional work involving the dead. Mediator deathwork is where the professional gleans or constructs information about the dead, edits and polishes it, and publicly presents the edited…

  11. Metamorphosis of a Butterfly-Associated Bacterial Community

    PubMed Central

    Hammer, Tobin J.; McMillan, W. Owen; Fierer, Noah

    2014-01-01

    Butterflies are charismatic insects that have long been a focus of biological research. They are also habitats for microorganisms, yet these microbial symbionts are little-studied, despite their likely importance to butterfly ecology and evolution. In particular, the diversity and composition of the microbial communities inhabiting adult butterflies remain uncharacterized, and it is unknown how the larval (caterpillar) and adult microbiota compare. To address these knowledge gaps, we used Illumina sequencing of 16S rRNA genes from internal bacterial communities associated with multiple life stages of the neotropical butterfly Heliconius erato. We found that the leaf-chewing larvae and nectar- and pollen-feeding adults of H. erato contain markedly distinct bacterial communities, a pattern presumably rooted in their distinct diets. Larvae and adult butterflies host relatively small and similar numbers of bacterial phylotypes, but few are common to both stages. The larval microbiota clearly simplifies and reorganizes during metamorphosis; thus, structural changes in a butterfly's bacterial community parallel those in its own morphology. We furthermore identify specific bacterial taxa that may mediate larval and adult feeding biology in Heliconius and other butterflies. Although male and female Heliconius adults differ in reproductive physiology and degree of pollen feeding, bacterial communities associated with H. erato are not sexually dimorphic. Lastly, we show that captive and wild individuals host different microbiota, a finding that may have important implications for the relevance of experimental studies using captive butterflies. PMID:24466308

  12. Metamorphosis of a butterfly-associated bacterial community.

    PubMed

    Hammer, Tobin J; McMillan, W Owen; Fierer, Noah

    2014-01-01

    Butterflies are charismatic insects that have long been a focus of biological research. They are also habitats for microorganisms, yet these microbial symbionts are little-studied, despite their likely importance to butterfly ecology and evolution. In particular, the diversity and composition of the microbial communities inhabiting adult butterflies remain uncharacterized, and it is unknown how the larval (caterpillar) and adult microbiota compare. To address these knowledge gaps, we used Illumina sequencing of 16S rRNA genes from internal bacterial communities associated with multiple life stages of the neotropical butterfly Heliconius erato. We found that the leaf-chewing larvae and nectar- and pollen-feeding adults of H. erato contain markedly distinct bacterial communities, a pattern presumably rooted in their distinct diets. Larvae and adult butterflies host relatively small and similar numbers of bacterial phylotypes, but few are common to both stages. The larval microbiota clearly simplifies and reorganizes during metamorphosis; thus, structural changes in a butterfly's bacterial community parallel those in its own morphology. We furthermore identify specific bacterial taxa that may mediate larval and adult feeding biology in Heliconius and other butterflies. Although male and female Heliconius adults differ in reproductive physiology and degree of pollen feeding, bacterial communities associated with H. erato are not sexually dimorphic. Lastly, we show that captive and wild individuals host different microbiota, a finding that may have important implications for the relevance of experimental studies using captive butterflies.

  13. Exposure and post-exposure effects of endosulfan on Bufo bufo tadpoles: morpho-histological and ultrastructural study on epidermis and iNOS localization.

    PubMed

    Bernabò, Ilaria; Guardia, Antonello; La Russa, Daniele; Madeo, Giuseppe; Tripepi, Sandro; Brunelli, Elvira

    2013-10-15

    Endosulfan is a persistent organic pollutant (POP) that has lethal and sublethal effects on non-target organisms, including amphibians. In a laboratory study, we investigated direct and post-exposure effects of endosulfan on Bufo bufo tadpoles. For this purpose we exposed the tadpoles to a single short-term contamination event (96 h) at an environmentally-realistic concentration (200 μg endosulfan/L). This was followed by a recovery period of 10 days when the experimental animals were kept in pesticide-free water. The endpoints were assessed in terms of mortality, incidence of deformity, effects on behavior, and the morpho-functional features of the epidermis. We found that a short-term exposure to the tested concentration of endosulfan did not cause mortality but induced severe sublethal effects, such as hyperactivity, convulsions, and axis malformations. Following relocation to a pesticide-free environment, we noted two types of response within the experimental sample, in terms of morphological and behavioral traits. Moreover, by using both ultrastructural and a morpho-functional approach, we found that a short-term exposure to endosulfan negatively affected the amphibian epidermis. We also observed several histo-pathological alterations: increased mucous secretion, an increase in intercellular spaces and extensive cell degeneration, together with the induction of an inducible isoform of nitric oxide synthase (iNOS). Following the post-exposure period, we found large areas of epidermis in which degeneration phenomena were moderate or absent, as well as a further increase in iNOS immunoreactivity. Thus, after 10 days in a free-pesticide environment, the larval epidermis was able to partially replace elements that had been compromised due to a physiological and/or a pathological response to the pesticide. These results highlight the need for both exposure and post-exposure experiments, when attempting to assess pollutant effects.

  14. Dietary cardamom inhibits the formation of azoxymethane-induced aberrant crypt foci in mice and reduces COX-2 and iNOS expression in the colon.

    PubMed

    Sengupta, Archana; Ghosh, Samit; Bhattacharjee, Shamee

    2005-01-01

    Recently, considerable attention has been focused on identifying naturally occurring chemopreventive compounds capable of inhibiting, retarding, or reversing the multi-step carcinogenesis. The primary aim of the present study was to identify the effects of a commonly consumed spice, viz., cardamom against azoxymethane (AOM) induced colonic aberrant crypt foci (ACF) in Swiss Albino mice. The secondary aim, was to explore the ability of cardamom to modulate the status of proliferation and apoptosis, and to understand its role in altering cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) expression. Male Swiss albino mice were injected with AOM (dose: 5mg/Kg body weight) or saline (Group 1) weekly once for two weeks. The AOM-injected mice were randomly assigned to two groups (Groups 2 and 3). While all the groups were on standard lab chow, Group 3 received oral doses of 0.5% cardamom, in aqueous suspension, daily for 8 weeks. Following treatment, significant reduction in the incidences of aberrant crypt foci (p<0.05) was observed. This reduction in ACF was accompanied by suppression of cell proliferation (mean Brdu LI in carcinogen control =13.91+/-3.31, and 0.5% cardamom =2.723+/-0.830) and induction of apoptosis (mean AI in carcinogen control=1.547+/-0.42 and 0.5% cardamom = 6.61+/-0.55). Moreover, reduction of both COX-2 and iNOS expression was also observed. These results suggest that aqueous suspensions of cardamom have protective effects on experimentally induced colon carcinogenesis. Cardamom as a whole and its active components require further attention if the use of this spice is to be recommended for cancer prevention.

  15. High-Resolution Global Analysis of the Influences of Bas1 and Ino4 Transcription Factors on Meiotic DNA Break Distributions in Saccharomyces cerevisiae

    PubMed Central

    Zhu, Xuan; Keeney, Scott

    2015-01-01

    Meiotic recombination initiates with DNA double-strand breaks (DSBs) made by Spo11. In Saccharomyces cerevisiae, many DSBs occur in “hotspots” coinciding with nucleosome-depleted gene promoters. Transcription factors (TFs) stimulate DSB formation in some hotspots, but TF roles are complex and variable between locations. Until now, available data for TF effects on global DSB patterns were of low spatial resolution and confined to a single TF. Here, we examine at high resolution the contributions of two TFs to genome-wide DSB distributions: Bas1, which was known to regulate DSB activity at some loci, and Ino4, for which some binding sites were known to be within strong DSB hotspots. We examined fine-scale DSB distributions in TF mutant strains by deep sequencing oligonucleotides that remain covalently bound to Spo11 as a byproduct of DSB formation, mapped Bas1 and Ino4 binding sites in meiotic cells, evaluated chromatin structure around DSB hotspots, and measured changes in global messenger RNA levels. Our findings show that binding of these TFs has essentially no predictive power for DSB hotspot activity and definitively support the hypothesis that TF control of DSB numbers is context dependent and frequently indirect. TFs often affected the fine-scale distributions of DSBs within hotspots, and when seen, these effects paralleled effects on local chromatin structure. In contrast, changes in DSB frequencies in hotspots did not correlate with quantitative measures of chromatin accessibility, histone H3 lysine 4 trimethylation, or transcript levels. We also ruled out hotspot competition as a major source of indirect TF effects on DSB distributions. Thus, counter to prevailing models, roles of these TFs on DSB hotspot strength cannot be simply explained via chromatin “openness,” histone modification, or compensatory interactions between adjacent hotspots. PMID:26245832

  16. Diverse mechanisms for inflammasome sensing of cytosolic bacteria and bacterial virulence.

    PubMed

    Zhao, Yue; Shao, Feng

    2016-02-01

    The inflammasomes are emerging cytosolic defenses against bacterial infections. The inflammasomes converge on inflammatory caspases activation that triggers pyroptosis, and interleukin-1β/18 maturation in the case of caspase-1 activation. The inflammasomes not only detect major bacterial molecules but also sense bacterial virulence activity. Among the canonical caspase-1-activating inflammasomes, the NAIP subfamily of NLR proteins serves as the receptors for bacterial flagellin and type III secretion apparatus; Pyrin indirectly senses Rho modification/inactivation by various bacterial agents; NLRP1 in mice/rats detects the protease activity of anthrax lethal toxin by serving as its substrate. Caspase-11 and caspase-4/5 directly recognize bacterial LPS and then become activated. Inflammasome sensing of cytosolic bacteria employs much more diversified biochemical mechanisms, compared with Toll-like receptors-mediated recognition on the membrane. PMID:26562791

  17. Bacterial infections in cirrhosis.

    PubMed

    Botwin, Gregory J; Morgan, Timothy R

    2014-09-01

    Bacterial infections occur in 25-35 % of cirrhotics admitted to hospital. Health-care associated and hospital acquired (nosocomial) infections are the most common epidemiology, with community acquired infections less common (15-30 %). Spontaneous bacterial peritonitis and urinary infections are the most common sites, with spontaneous bacteremia, pneumonia, cellulitis and other sites being less common. The risk of infection is increased among subjects with more severe liver disease and an infection in the past 6 months. Bacteria are isolated from approximately half of patients with a clinical diagnosis of infection. Gram-negative enterobacteriaceae are the most common organisms among community acquired infections; Gram-positive cocci are the most common organisms isolated among subjects with nosocomial infections. Up to 30 % of hospital associated infections are with multidrug resistant bacteria. Consequently, empiric antibiotic therapy that is recommended for community acquired infections is often inadequate for nosocomial infections. Infections worsen liver function. In-hospital and 1-year mortality of cirrhotics with infections is significantly higher than among cirrhotics without infection. In-hospital complications of infections, such as severe sepsis and septic shock, and mortality, are increased among subjects with multidrug-resistant infections as compared with cirrhotics with susceptible bacteria. Short-term antibiotic prophylaxis of cirrhotics with upper gastrointestinal bleeding and long-term antibiotic prophylaxis of selected cirrhotics with spontaneous bacterial peritonitis reduces infections and improves survival. Albumin administration to cirrhotics with SBP and evidence of advanced liver disease improves survival. The benefit of albumin administration to cirrhotics with infections other than SBP is under investigation. PMID:26201326

  18. Schisandra polysaccharide evokes immunomodulatory activity through TLR 4-mediated activation of macrophages.

    PubMed

    Zhao, Ting; Feng, Yun; Li, Jing; Mao, Riwen; Zou, Ye; Feng, Weiwei; Zheng, Daheng; Wang, Wei; Chen, Yao; Yang, Liuqing; Wu, Xiangyang

    2014-04-01

    Schisandra chinensis (Turcz.) Baill has been used in traditional Chinese medicine for centuries. Previous studies have shown that Schisandra polysaccharide (SCPP11) has robust antitumor activity in vivo. In this study, the immunomodulatory activity and mechanisms of action of SCPP11 were investigated further to reveal its mechanism of action against tumors. Results showed that SCPP11 increased the thymus and spleen indices, pinocytic activity of peritoneal macrophages, and hemolysin formation in CTX-induced immunosuppressed mice. Moreover, SCPP11 significantly increased immunoglobulin levels, cytokines levels in vivo and induced RAW264.7 cells to secrete cytokines in vitro. RAW264.7 cells pretreated with SCPP11 significantly inhibited the proliferation of HepG-2 cells. In addition, SCPP11 promoted both the expression of iNOS protein and of iNOS and TNF-α mRNA. TLR-4 is a possible receptor for SCPP11-mediated macrophage activation. Therefore, the data suggest that SCPP11 exerted its antitumor activity by improving immune system functions through TLR-4-mediated up-regulation of NO and TNF-α.

  19. Small intestinal bacterial overgrowth.

    PubMed

    Johnston, K L

    1999-03-01

    It is clear that the exact definition of small intestinal bacterial overgrowth (SIBO) needs to be reappraised in veterinary medicine. Antibiotic responsive enteropathies due to SIBO must be distinguished from those that are not associated with SIBO, such as those caused by a lack of immune tolerance. Once appropriate definitions and criteria for diagnosis are in place, the wide variety of diagnostic procedures that may facilitate the diagnosis can be evaluated with respect to their sensitivity and specificity, and statements about the prevalence and significance of this disorder can be made.

  20. Bacterial Skin Infections.

    PubMed

    Ibrahim, Fadi; Khan, Tariq; Pujalte, George G A

    2015-12-01

    Skin and soft tissue infections account for 0.5% of outpatient visits to primary care. Skin and soft tissue infections can usually be managed in an outpatient setting. However, there are certain circumstances as discussed in this article that require more urgent care or inpatient management. Primary care providers should be able to diagnose, manage, and provide appropriate follow-up care for these frequently seen skin infections. This article provides family physicians with a comprehensive review of the assessment and management of common bacterial skin infections. PMID:26612370

  1. Small Intestinal Bacterial Overgrowth

    PubMed Central

    Dukowicz, Andrew C.; Levine, Gary M.

    2007-01-01

    Small intestinal bacterial overgrowth (SIBO), defined as excessive bacteria in the small intestine, remains a poorly understood disease. Initially thought to occur in only a small number of patients, it is now apparent that this disorder is more prevalent than previously thought. Patients with SIBO vary in presentation, from being only mildly symptomatic to suffering from chronic diarrhea, weight loss, and malabsorption. A number of diagnostic tests are currently available, although the optimal treatment regimen remains elusive. Recently there has been renewed interest in SIBO and its putative association with irritable bowel syndrome. In this comprehensive review, we will discuss the epidemiology, pathogenesis, clinical manifestations, diagnosis, and treatment of SIBO. PMID:21960820

  2. Bacterial Skin Infections.

    PubMed

    Ibrahim, Fadi; Khan, Tariq; Pujalte, George G A

    2015-12-01

    Skin and soft tissue infections account for 0.5% of outpatient visits to primary care. Skin and soft tissue infections can usually be managed in an outpatient setting. However, there are certain circumstances as discussed in this article that require more urgent care or inpatient management. Primary care providers should be able to diagnose, manage, and provide appropriate follow-up care for these frequently seen skin infections. This article provides family physicians with a comprehensive review of the assessment and management of common bacterial skin infections.

  3. Bacterial terpene cyclases.

    PubMed

    Dickschat, Jeroen S

    2016-01-01

    Covering: up to 2015. This review summarises the accumulated knowledge about characterised bacterial terpene cyclases. The structures of identified products and of crystallised enzymes are included, and the obtained insights into enzyme mechanisms are discussed. After a summary of mono-, sesqui- and diterpene cyclases the special cases of the geosmin and 2-methylisoborneol synthases that are both particularly widespread in bacteria will be presented. A total number of 63 enzymes that have been characterised so far is presented, with 132 cited references. PMID:26563452

  4. The human parasite Leishmania amazonensis downregulates iNOS expression via NF-κB p50/p50 homodimer: role of the PI3K/Akt pathway

    PubMed Central

    Calegari-Silva, Teresa C.; Vivarini, Áislan C.; Miqueline, Marina; Dos Santos, Guilherme R. R. M.; Teixeira, Karina Luiza; Saliba, Alessandra Mattos; Nunes de Carvalho, Simone; de Carvalho, Laís; Lopes, Ulisses G.

    2015-01-01

    Leishmania amazonensis activates the NF-κB transcriptional repressor homodimer (p50/p50) and promotes nitric oxide synthase (iNOS) downregulation. We investigated the role of PI3K/Akt in p50/p50 NF-κB activation and the effect on iNOS expression in L. amazonensis infection. The increased occupancy of p50/p50 on the iNOS promoter of infected macrophages was observed and we demonstrated that both p50/p50 NF-κB induction and iNOS downregulation in infected macrophages depended on PI3K/Akt activation. Importantly, the intracellular growth of the parasite was also impaired during PI3K/Akt signalling inhibition and in macrophages knocked-down for Akt 1 expression. It was also observed that the increased nuclear levels of p50/p50 in L. amazonensis-infected macrophages were associated with reduced phosphorylation of 907 Ser p105, the precursor of p50. Corroborating these data, we demonstrated the increased levels of phospho-9 Ser GSK3β in infected macrophages, which is associated with GSK3β inhibition and, consequently, its inability to phosphorylate p105. Remarkably, we found that the levels of pPTEN 370 Ser, a negative regulator of PI3K, increased due to L. amazonensis infection. Our data support the notion that PI3K/Akt activity is sustained during the parasite infection, leading to NF-κB 105 phosphorylation and further processing to originate p50/p50 homodimers and the consequent downregulation of iNOS expression. PMID:26400473

  5. Neuroprotective and anti-apoptotic propensity of Bacopa monniera extract against sodium nitroprusside induced activation of iNOS, heat shock proteins and apoptotic markers in PC12 cells.

    PubMed

    Pandareesh, M D; Anand, T

    2014-05-01

    Sodium nitroprusside (SNP) is a widely used nitric oxide (NO) donor, known to exert nitrative stress by up-regulation of inducible nitric oxide synthase (iNOS). Nω-nitro-L-arginine-methyl esther (L-NAME) is a NO inhibitor, which inhibits iNOS expression, is used as positive control. The present study was designed to assess neuroprotective propensity of Bacopa monniera extract (BME) in SNP-induced neuronal damage and oxido-nitrative stress in PC12 cells via modulation of iNOS, heat shock proteins and apoptotic markers. Our results elucidate that pre-treatment of PC12 cells with BME ameliorates the mitochondrial and plasma membrane damage induced by SNP (200 μM) as evidenced by MTT and LDH assays. BME pre-treatment inhibited NO generation by down regulating iNOS expression. BME replenished the depleted antioxidant status induced by SNP treatment. SNP-induced damage to cellular, nuclear and mitochondrial integrity was also restored by BME, which was confirmed by ROS estimation, comet assay and mitochondrial membrane potential assays respectively. BME pre-treatment efficiently attenuated the SNP-induced apoptotic protein biomarkers such as Bax, Bcl-2, cytochrome-c and caspase-3, which orchestrate the proteolytic damage of the cell. Q-PCR results further elucidated up-regulation of neuronal cell stress markers like HO-1 and iNOS and down-regulation of BDNF upon SNP exposure was attenuated by BME pre-treatment. By considering all these findings, we report that BME protects PC12 cells against SNP-induced toxicity via its free radical scavenging and neuroprotective mechanism.

  6. The human parasite Leishmania amazonensis downregulates iNOS expression via NF-κB p50/p50 homodimer: role of the PI3K/Akt pathway.

    PubMed

    Calegari-Silva, Teresa C; Vivarini, Áislan C; Miqueline, Marina; Dos Santos, Guilherme R R M; Teixeira, Karina Luiza; Saliba, Alessandra Mattos; Nunes de Carvalho, Simone; de Carvalho, Laís; Lopes, Ulisses G

    2015-09-01

    Leishmania amazonensis activates the NF-κB transcriptional repressor homodimer (p50/p50) and promotes nitric oxide synthase (iNOS) downregulation. We investigated the role of PI3K/Akt in p50/p50 NF-κB activation and the effect on iNOS expression in L. amazonensis infection. The increased occupancy of p50/p50 on the iNOS promoter of infected macrophages was observed and we demonstrated that both p50/p50 NF-κB induction and iNOS downregulation in infected macrophages depended on PI3K/Akt activation. Importantly, the intracellular growth of the parasite was also impaired during PI3K/Akt signalling inhibition and in macrophages knocked-down for Akt 1 expression. It was also observed that the increased nuclear levels of p50/p50 in L. amazonensis-infected macrophages were associated with reduced phosphorylation of 907 Ser p105, the precursor of p50. Corroborating these data, we demonstrated the increased levels of phospho-9 Ser GSK3β in infected macrophages, which is associated with GSK3β inhibition and, consequently, its inability to phosphorylate p105. Remarkably, we found that the levels of pPTEN 370 Ser, a negative regulator of PI3K, increased due to L. amazonensis infection. Our data support the notion that PI3K/Akt activity is sustained during the parasite infection, leading to NF-κB 105 phosphorylation and further processing to originate p50/p50 homodimers and the consequent downregulation of iNOS expression. PMID:26400473

  7. Bacterial body plans

    PubMed Central

    Rieger, Tomáš; Neubauer, Zdeněk; Blahůšková, Anna; Cvrčková, Fatima

    2008-01-01

    The bacterium Serratia marcescens produces a plethora of multicellular shapes of different colorations on solid substrates, allowing immediate visual detection of varieties. Such a plasticity allows studies on multicellular community scale spanning two extremes, from well-elaborated individual colonies to undifferentiated cell mass. For a single strain and medium, we obtained a range of different multicellular bodies, depending on the layout of initial plating. Four principal factors affecting the morphogenetic pathways of such bodies can be distinguished: (1) amount, density and distribution pattern of founder cells; (2) the configuration of surrounding free medium; (3) the presence and character of other bacterial bodies sharing the same niche; and (4) self-perception, resulting in delimitation towards other bodies. The last feature results in an ability of well-formed multicellular individuals to maintain their identity upon a close mutual contact, as well as in spontaneous separation of cell masses in experimental chimeras. We propose an “embryo-like” colony model where multicellular bacterial bodies develop along genuine ontogenetic pathways inherent to the given species (clone), while external shaping forces (like nutrient gradients, pH, etc.,) exert not formative, but only regulative roles in the process. PMID:19513204

  8. The bacterial gliding machinery

    NASA Astrophysics Data System (ADS)

    Shrivastava, Abhishek

    Cells of Flavobacterium johnsoniae, a rod-shaped bacterium, glide over surfaces with speeds reaching up to 2 micrometer's. Gliding is powered by a protonmotive force. The adhesin SprB forms filaments about 160 nm long that move on the cell-surface along a looped track. Interaction of SprB filaments with a surface produces gliding. We tethered F. johnsoniae cells to glass by adding anti-SprB antibody. Tethered cells spun about fixed points, rotating at speeds of about 1 Hz. The torques required to sustain such speeds were large, comparable to those generated by the flagellar rotary motor. Using a flow cell apparatus, we changed load on the gliding motor by adding the viscous agent Ficoll to tethered cells. We found that a gliding motor runs at constant speed rather than constant torque. We attached gold nanoparticles to the SprB filament and tracked its motion. We fluorescently tagged a bacterial Type IX secretion system (T9SS) protein and imaged its dynamics. Fluorescently tagged T9SS protein localized near the point of tether, indicating that T9SS localizes with the gliding motor. Based on our results, we propose a model to explain bacterial gliding.

  9. Antimicrobials for bacterial bioterrorism agents.

    PubMed

    Sarkar-Tyson, Mitali; Atkins, Helen S

    2011-06-01

    The limitations of current antimicrobials for highly virulent pathogens considered as potential bioterrorism agents drives the requirement for new antimicrobials that are suitable for use in populations in the event of a deliberate release. Strategies targeting bacterial virulence offer the potential for new countermeasures to combat bacterial bioterrorism agents, including those active against a broad spectrum of pathogens. Although early in the development of antivirulence approaches, inhibitors of bacterial type III secretion systems and cell division mechanisms show promise for the future.

  10. Proteomic evidence of bacterial peptide translocation in afebrile patients with cirrhosis and ascites.

    PubMed

    Caño, Rocío; Llanos, Lucía; Zapater, Pedro; Pascual, Sonia; Bellot, Pablo; Barquero, Claudia; Pérez-Mateo, Miguel; Such, José; Francés, Rubén

    2010-05-01

    Bacterial translocation in patients with cirrhosis induces a marked proinflammatory activity that may be different against viable bacteria or bacterial products. The aim of this study is to identify new markers of bacterial translocation by investigating bacterial-driven peptides and correlate their presence with the inflammatory response. Patients with cirrhosis and ascites were included. An analysis by two-dimensional polyacrylamide gel electrophoresis of ascitic fluid total protein from patients (n = 47) and from frequently detected bacterial strains was performed. Two-dimensional maps were digitally compared. The identification of possible markers was performed by mass spectrometry. TNF-alpha, IFN-gamma, IL-12, nitric oxide, and proteins of the complement and lipopolysaccharide-binding protein levels were measured in ascitic fluid samples of patients by enzyme-linked immunosorbent assay. Patients were distributed according to the presence (group I, n = 16) and absence (group II, n = 31) of serum and ascitic fluid bacterial DNA. Among clinical and analytical differences between groups, only mean arterial pressure was significantly higher in patients from group II. Identified bacterial peptides were associated with bacterial protection against immune defenses and included glyceraldehyde-3-phosphate dehydrogenase A, Porin OmpC, and HSP60. Eight patients from group I also showed bacterial peptides, whereas none from group II did. All studied mediators of immune activation were significantly higher in patients with bacterial DNA than in patients without bacterial DNA. TNF-alpha, IFN-gamma, and proteins of the complement were significantly increased in patients with bacterial peptides versus those without bacterial peptides. Bacterial peptide translocation is present in the ascitic fluid of a subgroup of patients with advanced cirrhosis and is associated with an increased immune response.

  11. Nanoparticle approaches against bacterial infections.

    PubMed

    Gao, Weiwei; Thamphiwatana, Soracha; Angsantikul, Pavimol; Zhang, Liangfang

    2014-01-01

    Despite the wide success of antibiotics, the treatment of bacterial infections still faces significant challenges, particularly the emergence of antibiotic resistance. As a result, nanoparticle drug delivery platforms including liposomes, polymeric nanoparticles, dendrimers, and various inorganic nanoparticles have been increasingly exploited to enhance the therapeutic effectiveness of existing antibiotics. This review focuses on areas where nanoparticle approaches hold significant potential to advance the treatment of bacterial infections. These areas include targeted antibiotic delivery, environmentally responsive antibiotic delivery, combinatorial antibiotic delivery, nanoparticle-enabled antibacterial vaccination, and nanoparticle-based bacterial detection. In each area we highlight the innovative antimicrobial nanoparticle platforms and review their progress made against bacterial infections.

  12. Nanoparticle Approaches against Bacterial Infections

    PubMed Central

    Gao, Weiwei; Thamphiwatana, Soracha; Angsantikul, Pavimol; Zhang, Liangfang

    2014-01-01

    Despite the wide success of antibiotics, the treatment of bacterial infection still faces significant challenges, particularly the emergence of antibiotic resistance. As a result, nanoparticle drug delivery platforms including liposomes, polymeric nanoparticles, dendrimers, and various inorganic nanoparticles have been increasingly exploited to enhance the therapeutic effectiveness of existing antibiotics. This review focuses on areas where nanoparticle approaches hold significant potential to advance the treatment of bacterial infection. These areas include targeted antibiotic delivery, environmentally responsive antibiotic delivery, combinatorial antibiotic delivery, nanoparticle-enabled antibacterial vaccination, and nanoparticle-based bacterial detection. In each area we highlight the innovative antimicrobial nanoparticle platforms and review their progress made against bacterial infections. PMID:25044325

  13. Anti-Nociceptive Effect of Resveratrol During Inflammatory Hyperalgesia via Differential Regulation of pro-Inflammatory Mediators.

    PubMed

    Singh, Ajeet Kumar; Vinayak, Manjula

    2016-07-01

    Sensitization of nociceptive neurons by inflammatory mediators leads to hypersensitivity for normal painful stimuli which is termed hyperalgesia. Oxidative stress is an essential factor in pathological pain; therefore, antioxidants qualify as potential anti-hyperalgesic agents. The present study examines the efficacy of the natural antioxidant resveratrol in complete Freund's adjuvant (CFA) induced hyperalgesic rats. Thermal hyperalgesia was measured at different time points by paw withdrawal latency test and confirmed by c-Fos expression in spinal dorsal horn. The impact of resveratrol treatment on inflammatory mediators at peripheral (paw skin) and central (spinal cord) sites was determined during early (6 h) as well as late phase (48 h) of hyperalgesia. Intraplanter injection of CFA increased the level of cytokines IL-1β, TNF-α and IL-6 as well as inflammatory enzymes COX-2 and iNOS in paw skin in both phases. In case of spinal cord, the level of COX-2 was found to be elevated in both phases, whereas iNOS could not be detected. The cytokines were found to be elevated only in late phase in spinal cord. Administration of resveratrol (20 mg/kg) shifted the level of all inflammatory mediators towards normal, except cytokines in paw skin. The present study suggests that the anti-nociceptive effect of resveratrol is implicated at both peripheral and central sites in a tissue specific manner. Copyright © 2016 John Wiley & Sons, Ltd. PMID:27060370

  14. Effects of selective iNOS inhibition in sepsis: evaluation of lung tissue damage and blood gases.

    PubMed

    Ceran, Sami; Erikoglu, Mehmet; Sahin, Mustafa; Sunam, Güven Sadi; Gölcük, Murat; Pasaoğlu, Hatice; Avsar, Fatih; Hücümenoglu, Sema

    2008-01-01

    NO is an important mediator in the generalized inflammatory response of the body during sepsis and septic shock. We investigated the possible effects of L-arginine and aminoguanidine on plasma NO levels and the interaction between NO levels and lung tissue damage and blood gases in sepsis. Fifty Wistar male rats were used in this study and divided into five groups: group 1, sham group; group 2, CLP (sepsis); group 3, CLP + 10 mg/kg L-arginine administration; group 4, CLP +15 mg/kg aminoguanidine administration; group 5: CLP + L-arginine + aminoguanidine given in similar doses. Sepsis was induced by cecal ligation and puncture (CLP) method. Drugs were administered at postoperative hours 4 and 12. The levels in the aminoguanidine and aminoguanidine + L-arginine groups were similar to the sham group. Lung tissue damage in the sepsis and L-arginine groups was more severe than the other groups.

  15. Inhibition of bacterial and leukocyte adhesion under shear stress conditions by material surface chemistry.

    PubMed

    Patel, Jasmine D; Ebert, Michael; Stokes, Ken; Ward, Robert; Anderson, James M

    2003-01-01

    Biomaterial-centered infections, initiated by bacterial adhesion, persist due to a compromised host immune response. Altering implant materials with surface modifying endgroups (SMEs) may enhance their biocompatibility by reducing bacterial and inflammatory cell adhesion. A rotating disc model, which generates shear stress within physiological ranges, was used to characterize adhesion of leukocytes and Staphylococcus epidermidis on polycarbonate-urethanes and polyetherurethanes modified with SMEs (polyethylene oxide, fluorocarbon and dimethylsiloxane) under dynamic flow conditions. Bacterial adhesion in the absence of serum was found to be mediated by shear stress and surface chemistry, with reduced adhesion exhibited on materials modified with polydimethylsiloxane and polyethylene oxide SMEs. In contrast, bacterial adhesion was enhanced on materials modified with fluorocarbon SMEs. In the presence of serum, bacterial adhesion was primarily neither material nor shear dependent. However, bacterial adhesion in serum was significantly reduced to < or = 10% compared to adhesion in serum-free media. Leukocyte adhesion in serum exhibited a shear dependency with increased adhesion occurring in regions exposed to lower shear-stress levels of < or = 7 dyne/cm2. Additionally, polydimethylsiloxane and polyethylene oxide SMEs reduced leukocyte adhesion on polyether-urethanes. In conclusion, these results suggest that surface chemistry and shear stress can mediate bacterial and cellular adhesion. Furthermore, materials modified with polyethylene oxide SMEs are capable of inhibiting bacterial adhesion, consequently minimizing the probability of biomaterial-centered infections.

  16. Bacterial cellulose membranes used as artificial substitutes for dural defection in rabbits.

    PubMed

    Xu, Chen; Ma, Xia; Chen, Shiwen; Tao, Meifeng; Yuan, Lutao; Jing, Yao

    2014-06-16

    To improve the efficacy and safety of dural repair in neurosurgical procedures, a new dural material derived from bacterial cellulose (BC) was evaluated in a rabbit model with dural defects. We prepared artificial dura mater using bacterial cellulose which was incubated and fermented from Acetobacter xylinum. The dural defects of the rabbit model were repaired with BC membranes. All surgeries were performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering. All animals were humanely euthanized by intravenous injection of phenobarbitone, at each time point, after the operation. Then, the histocompatibility and inflammatory effects of BC were examined by histological examination, real-time fluorescent quantitative polymerase chain reaction (PCR) and Western Blot. BC membranes evenly covered the surface of brain without adhesion. There were seldom inflammatory cells surrounding the membrane during the early postoperative period. The expression of inflammatory cytokines IL-1β, IL-6 and TNF-α as well as iNOS and COX-2 were lower in the BC group compared to the control group at 7, 14 and 21 days after implantation. BC can repair dural defects in rabbit and has a decreased inflammatory response compared to traditional materials. However, the long-term effects need to be validated in larger animals.

  17. Bacterial Cellulose Membranes Used as Artificial Substitutes for Dural Defection in Rabbits

    PubMed Central

    Xu, Chen; Ma, Xia; Chen, Shiwen; Tao, Meifeng; Yuan, Lutao; Jing, Yao

    2014-01-01

    To improve the efficacy and safety of dural repair in neurosurgical procedures, a new dural material derived from bacterial cellulose (BC) was evaluated in a rabbit model with dural defects. We prepared artificial dura mater using bacterial cellulose which was incubated and fermented from Acetobacter xylinum. The dural defects of the rabbit model were repaired with BC membranes. All surgeries were performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering. All animals were humanely euthanized by intravenous injection of phenobarbitone, at each time point, after the operation. Then, the histocompatibility and inflammatory effects of BC were examined by histological examination, real-time fluorescent quantitative polymerase chain reaction (PCR) and Western Blot. BC membranes evenly covered the surface of brain without adhesion. There were seldom inflammatory cells surrounding the membrane during the early postoperative period. The expression of inflammatory cytokines IL-1β, IL-6 and TNF-α as well as iNOS and COX-2 were lower in the BC group compared to the control group at 7, 14 and 21 days after implantation. BC can repair dural defects in rabbit and has a decreased inflammatory response compared to traditional materials. However, the long-term effects need to be validated in larger animals. PMID:24937688

  18. Ribonucleotides in Bacterial DNA

    PubMed Central

    Schroeder, Jeremy W.; Randall, Justin R.; Matthews, Lindsay A.; Simmons, Lyle A.

    2014-01-01

    In all living cells, DNA is the storage medium for genetic information. Being quite stable, DNA is well-suited for its role in storage and propagation of information, but RNA is also covalently included in DNA through various mechanisms. Recent studies also demonstrate useful aspects of including ribonucleotides in the genome during repair. Therefore, our understanding of the consequences of RNA inclusion into bacterial genomic DNA is just beginning, but with its high frequency of occurrence the consequences and potential benefits are likely to be numerous and diverse. In this review, we discuss the processes that cause ribonucleotide inclusion in genomic DNA, the pathways important for ribonucleotide removal and the consequences that arise should ribonucleotides remain nested in genomic DNA. PMID:25387798

  19. Exploring bacterial lignin degradation.

    PubMed

    Brown, Margaret E; Chang, Michelle C Y

    2014-04-01

    Plant biomass represents a renewable carbon feedstock that could potentially be used to replace a significant level of petroleum-derived chemicals. One major challenge in its utilization is that the majority of this carbon is trapped in the recalcitrant structural polymers of the plant cell wall. Deconstruction of lignin is a key step in the processing of biomass to useful monomers but remains challenging. Microbial systems can provide molecular information on lignin depolymerization as they have evolved to break lignin down using metalloenzyme-dependent radical pathways. Both fungi and bacteria have been observed to metabolize lignin; however, their differential reactivity with this substrate indicates that they may utilize different chemical strategies for its breakdown. This review will discuss recent advances in studying bacterial lignin degradation as an approach to exploring greater diversity in the environment. PMID:24780273

  20. Bacterial polyhydroxyalkanoates: Still fabulous?

    PubMed

    Możejko-Ciesielska, Justyna; Kiewisz, Robert

    2016-11-01

    Bacterial polyhydroxyalkanoates (PHA) are polyesters accumulated as carbon and energy storage materials under limited growth conditions in the presence of excess carbon sources. They have been developed as biomaterials with unique properties for the past many years being considered as a potential substitute for conventional non-degradable plastics. Due to the increasing concern towards global climate change, depleting petroleum resource and problems with an utilization of a growing number of synthetic plastics, PHAs have gained much more attention from industry and research. These environmentally friendly microbial polymers have great potential in biomedical, agricultural, and industrial applications. However, their production on a large scale is still limited. This paper describes the backgrounds of PHAs and discussed the current state of knowledge on the polyhydroxyalkanoates. Ability of bacteria to convert different carbon sources to PHAs, the opportunities and challenges of their introduction to global market as valuable renewable products have been also discussed.

  1. Epigenetics and Bacterial Infections

    PubMed Central

    Bierne, Hélène; Hamon, Mélanie; Cossart, Pascale

    2012-01-01

    Epigenetic mechanisms regulate expressi