Normal Growth Medium Research Articles

The Heme Oxygenase 1 Inducer (CoPP) Protects Human Cardiac Stem Cells against Apoptosis through Activation of the Extracellular Signal-regulated Kinase (ERK)/NRF2 Signaling Pathway and Cytokine Release

Intracoronary delivery of c-kit-positive human cardiac stem cells (hCSCs) is a promising approach to repair the infarcted heart, but it is severely limited by the poor survival of donor cells. Cobalt protoporphyrin (CoPP), a well known heme oxygenase 1 inducer, has been used to promote endogenous CO generation and protect against ischemia/reperfusion injury. Therefore, we determined whether preconditioning hCSCs with CoPP promotes CSC survival. c-kit-positive, lineage-negative hCSCs were isolated from human heart biopsies. Lactate dehydrogenase release assays demonstrated that preconditioning CSCs with CoPP markedly enhanced cell survival after oxidative stress induced by H(2)O(2), concomitant with up-regulation of heme oxygenase 1, COX-2, and anti-apoptotic proteins (BCL2, BCL2-A1, and MCL-1) and increased phosphorylation of NRF2. Apoptotic cytometric assays showed that pretreatment of CSCs with CoPP enhanced the cells' resistance to apoptosis induced by oxidative stress. Conversely, knocking down HO-1, COX-2, or NRF2 by shRNA gene silencing abrogated the cytoprotective effects of CoPP. Further, preconditioning CSCs with CoPP led to a global increase in release of cytokines, such as EGF, FGFs, colony-stimulating factors, and chemokine ligand. Conditioned medium from cells pretreated with CoPP conferred naive CSCs remarkable resistance to apoptosis, demonstrating that cytokines released by preconditioned cells play a key role in the anti-apoptotic effects of CoPP. Preconditioning CSCs with CoPP also induced an increase in the phosphorylation of Erk1/2, which are known to modulate multiple pro-survival genes. These results potentially provide a simple and effective strategy to enhance survival of CSCs after transplantation and, therefore, their efficacy in repairing infarcted myocardium.

Desiccation-induced non-radiative dissipation in isolated green lichen algae

Lichens are able to tolerate almost complete desiccation and can quickly resume metabolic activity after rehydration. In the desiccated state, photosynthesis is completely blocked and absorbed excitation energy cannot be used for electron transport, leading to a potential strong vulnerability for high light damage. Although desiccation and high insolation often occur simultaneously and many lichens colonize exposed habitats, these organisms show surprisingly little photodamage. In the desiccated state, variable chlorophyll fluorescence is lost, indicating a suspension of charge separation in photosystem II. At the same time, basal fluorescence (F (0)) is strongly quenched, which has been interpreted as an indication for high photoprotective non-radiative dissipation (NRD) of absorbed excitation energy. In an attempt to provide evidence for a photoprotective function of NRD in the desiccated state, isolated green lichen algae of the species Coccomyxa sp. and Trebouxia asymmetrica were used as experimental system. In contrast to experiments with intact lichens this system provided high reproducibility of the data without major optical artifacts on desiccation. The presence of 5mM trehalose during desiccation had no effect but culture of the algae in seawater enhanced F (0) quenching in T. asymmetrica together with a reduced depression of F (V)/F (M) after high light treatment. While this effect could not be induced using artificial seawater medium lacking trace elements, the addition of ZnCl(2) and NaI in small amounts to the normal growth medium led to qualitatively and quantitatively identical results as with pure seawater. It is concluded that NRD indicated by F (0) quenching is photoprotective. The formation of NRD in lichen algae is apparently partially dependent on the presence of specific micronutrients.

Regulatory role of β-arrestin-2 in cholesterol processing in cystic fibrosis epithelial cells

Cystic fibrosis (CF) cells exhibit an increase in the protein expression of β-arrestin-2 (βarr2) coincident with perinuclear accumulation of free cholesterol. Arrestins are proteins that both serve as broad signaling regulators and contribute to G-protein coupled receptor internalization after agonist stimulation. The hypothesis of this study is that βarr2 is an important component in the mechanisms leading to cholesterol accumulation characteristic of CF cells. To test this hypothesis, epithelial cells stably expressing GFP-tagged βarr2 (βarr2-GFP) and respective GFP-expressing control cells (cont-GFP) were analyzed by filipin staining. The βarr2-GFP cells show a late endosomal/lysosomal cholesterol accumulation that is identical to that seen in CF cells. This βarr2-mediated accumulation is sensitive to Rp-cAMPS treatment, and depleting βarr2 expression in CF-model cells by shRNA alleviates cholesterol accumulation compared with controls. Cftr/βarr2 double knockout mice also exhibit wild-type (WT) levels of cholesterol synthesis, and WT profiles of signaling protein expression have previously been shown to be altered in CF due to cholesterol-related pathways. These data indicate a significant regulatory role for βarr2 in the development of CF-like cholesterol accumulation and give further insight into cholesterol processing mechanisms. An impact of βarr2 expression on Niemann-Pick type C-1 (NPC1)-containing organelle movement is proposed as the mechanism of βarr2-mediated alterations on cholesterol processing. It is concluded that βarr2 expression contributes to altered cholesterol trafficking observed in CF cells.

Distinct expression and activity of GSK‐3α and GSK‐3β in prostate cancer

Glycogen synthase kinase (GSK-3) is upregulated in many types of tumor, including prostate cancer. GSK-3 inhibitors reduce prostate tumor cell growth; however, it is not clear if both isoforms, GSK-3α and GSK-3β, are involved. Here, we compared their expression in prostate tumors and used gene silencing to study their functions in 22Rv1 prostate cancer cells. Compared to normal prostate, GSK-3α and GSK-3β were upregulated in 25/79 and 24/79 cases of prostate cancer, respectively, with GSK-3α elevated in low Gleason sum score tumors and GSK-3β expressed in high Gleason tumors, and both isoforms correlating with high expression of the androgen receptor (AR). Gene silencing of GSK-3α and, to a lesser extent, GSK-3β reduced AR transcriptional activity. In addition, silencing of GSK-3β, but not GSK-3α, reduced Akt phosphorylation. Acute and chronic silencing of either isoform reduced 22Rv1 growth in colony formation assays; however, this did not correlate with effects on AR activity. The GSK-3 inhibitor CHIR99021 reduced 22Rv1 colony formation by 50% in normal growth medium and by 15% in hormone-depleted medium, suggesting that GSK-3 is required both for hormone-dependent and hormone-independent proliferation. In addition, CHIR99021 enhanced growth inhibition by the AR antagonists bicalutamide and MDV3100. Finally, expression of GSK3A and GSK3B mRNAs correlated with a gene expression signature for androgen-regulated genes. Our observations highlight the importance of the GSK-3/AR signaling axis in prostate cancer and support the case for development of isoform-specific GSK-3 inhibitors and their use, in combination with AR antagonists, to treat patients with prostate cancer.

Abstract 252: Altered caspase-8 localization and decreased caspase-8 sumoylation in TRAIL-resistant normal human fibroblasts

Abstract TNF-related apoptosis-inducing ligand (TRAIL)-mediated cell death is currently being utilized in newly developed cancer therapies. The molecular basis of normal cell resistance to TRAIL-mediated apoptosis, and thus therapeutic index, remains largely unexplored. We previously investigated TRAIL sensitivity in normal fibroblasts (HFF, WI-38, MRC-5) and found that normal fibroblasts have decreased protein expression of initiator caspase-8 that is required for complete TRAIL signaling. We examined caspase-8 stability in cycloheximide treated normal fibroblasts and SW480 human colon cancer cells. Normal fibroblast caspase-8 stability was found to be similar to SW480 cells. Additionally, we investigated caspase-8 modulation in normal fibroblasts to uncover any drug treatments or modified growth conditions that result in increased caspase-8 protein expression and enhanced susceptibility to TRAIL-mediated apoptosis. Normal fibroblasts treated for 24, 48, and 72 hours with non-lethal doses of histone deacetylase inhibitor Trichostatin A or DNA methylation inhibitor 5-azacytidine had no change in caspase-8 protein expression. Our results suggest epigenetic silencing of caspase-8 is not responsible for TRAIL resistance in normal fibroblasts. However, normal fibroblasts serum starved with media containing 0.5% fetal calf serum for 48 hours, resulted in increased caspase-8 protein expression but not effector caspase-3 expression. Serum starved normal fibroblasts treated with TRAIL had increased caspase-3 activity and an increase in the percentage of sub-G1 positive cells compared to TRAIL treated cells in normal growth media. No increase in TRAIL-mediated apoptosis was observed in serum starved SW480 cells. Since previous studies have detected caspase-8 targets in both the cytoplasm and nucleus, we examined caspase-8 localization in normal fibroblasts. Little caspase-8 was found in nuclear extracts isolated from normal fibroblasts. Alternatively, SW480 caspase-8 expression was observed in both cytoplasmic and nuclear fractions. Sumoylated caspase-8 (p75), previously found to be associated with caspase-8 nuclear localization, was noted in nuclear fractions in both normal fibroblasts and SW480 cells. Yet, like total caspase-8 expression, sumoylated caspase-8 expression was found to be decreased in normal fibroblasts compared to SW480 colon cancer cells. Thus, decreased nuclear caspase-8 may be a contributing factor in normal fibroblast resistance to TRAIL-mediated apoptosis. Our current studies are focused on further analysis of caspase-8 sumoylation/desumoylation as well as caspase-8 post-translational modifications that may suppress caspase activity and nuclear translocation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 252. doi:1538-7445.AM2012-252

Development of in vitro Slow Growth Culture for Yarn (Dioscoreo alata L.)

Germplasm collections, the lifeblood of breeding programs, are traditionally maintained in the field. Field genebanks are expensive, subject to genetic erosion, and require several quarantine measures for safe movement of genetic materials. These problems are more serious in long-duration, non-flowering and vegetatively propagated crops like yarn. This study aimed to develop a tissue culture technique for in vitro conservation of yarn germplasm. ’VU-2’ and ‘Kinampay’ varieties were used in establishing the in vitro conservation technique which was then tested to other genotypes. With the tissue culture protocol for yarn propagation developed earlier, the plantlets became overgrown after 2-3 months, requiring frequent subculturing and increasing the cost of maintenance and the risk of microbial contamination. Slow growth culture was tested using MS medium added with 0-10 mg/L abscisic acid (ABA) or 0-7% mannitol or sorbitol. Expectedly, plantlet growth slowed down. However, ABA at higher levels increased mortality of cultures while sorbitol was less effective than mannitol in retarding growth. Mannitol at 4% was found to be the best slow growth medium to maintain the plantlets for 13 months, thereby saving at least 4 times the maintenance cost using the normal growth medium. Tissue viability, morphological stability and tuber yield were not affected. Other genotypes (VU-1, VU-3, VU-4, VU-5, PR5, PR7, PR10 and PR11) responded similarly to the slow growth culture condition.

Proteomic Identification of Betaig-h3 as a Lysophosphatidic Acid-Induced Secreted Protein of Human Mesenchymal Stem Cells: Paracrine Activation of A549 Lung Adenocarcinoma Cells by Betaig-h3

Lysophosphatidic acid (LPA) is enriched in the serum and malignant effusion of cancer patients and plays a key role in tumorigenesis and metastasis. LPA-activated mesenchymal stem cells promote tumorigenic potentials of cancer cells through a paracrine mechanism. LPA-conditioned medium (LPA CM) from human adipose tissue-derived mesenchymal stem cells (hASCs) elicited adhesion and proliferation of A549 human lung adenocarcinoma cells. To identify proteins involved in the LPA-stimulated paracrine functions of hASCs, we analyzed the LPA CM using liquid-chromatography tandem mass spectrometry-based shotgun proteomics. We identified βig-h3, an extracellular matrix protein that is implicated in tumorigenesis and metastasis, as an LPA-induced secreted protein in hASCs. LPA-induced βig-h3 expression was abrogated by pretreating hASCs with the LPA receptor(1/3) inhibitor Ki16425 or small interfering RNA-mediated silencing of endogenous LPA(1). LPA-induced βig-h3 expression was blocked by treating the cells with the Rho kinase inhibitor Y27632, implying that LPA-induced βig-h3 expression is mediated by the LPA(1)- Rho kinase pathway. Immunodepletion or siRNA-mediated silencing of βig-h3 abrogated LPA CM-stimulated adhesion and proliferation of A549 cells, whereas retroviral overexpression of βig-h3 in hASCs potentiated it. Furthermore, recombinant βig-h3 protein stimulated the proliferation and adhesion of A549 human lung adenocarcinoma cells. These results suggest that hASC-derived βig-h3 plays a key role in tumorigenesis by stimulating the adhesion and proliferation of cancer cells and it can be applicable as a biomarker and therapeutic target for lung cancer.

Vaccinia virus leads to ATG12–ATG3 conjugation and deficiency in autophagosome formation

The interactions between viruses and cellular autophagy have been widely reported. On the one hand, autophagy is an important innate immune response against viral infection. On the other hand, some viruses exploit the autophagy pathway for their survival and proliferation in host cells. Vaccinia virus is a member of the family of Poxviridae which includes the smallpox virus. The biogenesis of vaccinia envelopes, including the core envelope of the immature virus (IV), is not fully understood. In this study we investigated the possible interaction between vaccinia virus and the autophagy membrane biogenesis machinery. Massive LC3 lipidation was observed in mouse fibroblast cells upon vaccinia virus infection. Surprisingly, the vaccinia virus induced LC3 lipidation was shown to be independent of ATG5 and ATG7, as the atg5 and atg7 null mouse embryonic fibroblasts (MEFs) exhibited the same high levels of LC3 lipidation as compared with the wild-type MEFs. Mass spectrometry and immunoblotting analyses revealed that the viral infection led to the direct conjugation of ATG3, which is the E2-like enzyme required for LC3-phosphoethanonamine conjugation, to ATG12, which is a component of the E3-like ATG12–ATG5-ATG16 complex for LC3 lipidation. Consistently, ATG3 was shown to be required for the vaccinia virus induced LC3 lipidation. Strikingly, despite the high levels of LC3 lipidation, subsequent electron microscopy showed that vaccinia virus-infected cells were devoid of autophagosomes, either in normal growth medium or upon serum and amino acid deprivation. In addition, no autophagy flux was observed in virus-infected cells. We further demonstrated that neither ATG3 nor LC3 lipidation is crucial for viral membrane biogenesis or viral proliferation and infection. Together, these results indicated that vaccinia virus does not exploit the cellular autophagic membrane biogenesis machinery for their viral membrane production. Moreover, this study demonstrated that vaccinia virus instead actively disrupts the cellular autophagy through a novel molecular mechanism that is associated with aberrant LC3 lipidation and a direct conjugation between ATG12 and ATG3.

Cellular Aspects of the Regulation of the Transcriptional Activity of Nuclear Factor Kappa B (NF-κB) in Sensory Neurons in Vitro

Nuclear factor kappa B regulates the expression of a multitude of anti/proapoptotic genes, in nerve cells among others. Intranuclear acetylation processes play an important role in the mechanism regulating the activity of NF-κB. The aim of the present work was to identify the transcriptional activity of NF-κB in sensory neurons in vitro and to explain the possible regulation of NF-κB activity by modification of acetylation processes using a transgenic strain of reporter mice in which NF-κB activation leads to expression of the LacZ reporter gene. Expression of the reporter gene was found not to occur in transgenic neurons cultured either in normal growth medium or after stimulation with TNF-α, though it was induced in most neurons by in vitro exposure to the histone deacetylase inhibitor trichostatin (TSA). Thus, the transcriptional activity of NF-κB is restricted to sensory neurons be a repression mechanism mediated by histone deacetylases.

Receptor-like Protein-tyrosine Phosphatase α Enhances Cell Surface Expression of Neural Adhesion Molecule NB-3

Neural adhesion molecule NB-3 plays an important role in the apical dendrite development of layer V pyramidal neurons in the visual cortex, and receptor-like protein-tyrosine phosphatase α (PTPα) mediates NB-3 signaling in this process. Here we investigated the role of PTPα in regulating cell surface expression of NB-3. We found that cortical neurons from PTPα knock-out mice exhibited a lower level of NB-3 at the cell surface. When expressed in COS1 cells, NB-3 was enriched in the Golgi apparatus with a low level of cell surface expression. However, co-expression of PTPα increased the cell surface distribution of NB-3. Further analysis showed that PTPα facilitated Golgi exit of NB-3 and stabilized NB-3 protein at the cell surface by preventing its release from the plasma membrane. The extracellular region of PTPα but not its catalytic activity is necessary for its effect on NB-3 expression. Thus, the PTPα-mediated increase of NB-3 level at the cell surface represents a novel function of PTPα in NB-3 signaling in neural development.

Knockdown of DAPIT (Diabetes-associated Protein in Insulin-sensitive Tissue) Results in Loss of ATP Synthase in Mitochondria

It was found recently that a diabetes-associated protein in insulin-sensitive tissue (DAPIT) is associated with mitochondrial ATP synthase. Here, we report that the suppressed expression of DAPIT in DAPIT-knockdown HeLa cells causes loss of the population of ATP synthase in mitochondria. Consequently, DAPIT-knockdown cells show smaller mitochondrial ATP synthesis activity, slower growth in normal medium, and poorer viability in glucose-free medium than the control cells. The mRNA levels of α- and β-subunits of ATP synthase remain unchanged by DAPIT knockdown. These results indicate a critical role of DAPIT in maintaining the ATP synthase population in mitochondria and raise an intriguing possibility of active role of DAPIT in cellular energy metabolism.

UVB를 조사한 HaCaT 세포의 세포사멸과 p53 및 GADD45 유전자 발현에 대한 파프리카 추출물 및 성분들의 효과

In the present study, the effects of paprika extract and its components including vitamin C, lycopene and beta-carotene on cell death in ultraviolet B (UVB)-exposed HaCaT cells were investigated. The cell viability upon treatment for 24 hr with either paprika extract or vitamin C alone was similar to or greater than that of the untreated control. However, the viability of the cells treated with lycopene or beta-carotene decreased to about 20% of that in the untreated control. When UVB-exposed cells were post-incubated for 24 hr in medium containing paprika extract or vitamin C, cell viability increased in a concentration dependent manner as compared to those post-incubated in a normal growth medium. In contrast, post-incubation of UVB-exposed cells with lycopene or beta-carotene decreased cell viability in a concentration dependent manner as compared to those post-incubated in a normal growth medium. The nuclear fragmentation analysis showed that paprika extract or vitamin C decreases UVB-induced apoptosis. The apoptotic nuclear fragmentation resulting from UVB exposure was also protected by the paprika extract or vitamin C post-incubation. However, the UVB-induced apoptotic nuclear fragmentation of the cells treated with lycopene or beta-carotene increased in a concentration dependent manner. Western blot analysis showed that either paprika extract or vitamin C treatment alone did not significantly change the level of p53 and GADD45 protein. Interestingly, post-incubation of UVB-exposed cells with paprika extract or vitamin C decreased the p53 and GADD45 protein level as compared to those post-incubated in a normal growth medium. In contrast, incubation of UVB-exposed or non-irradiated cells with lycopene or beta-carotene increased the p53 and GADD45 protein levels in a concentration dependent manner as compared to those incubated in a normal growth medium. All these results suggest that paprika extract and vitamin C help the survival of the UVB-exposed cells, while lycopene and beta-carotene potentiate the apoptotic death of UVB-exposed cells, in accordance with the respective changes in p53 and GADD45 protein levels.

Abstract 4006: Orphan nuclear receptor tailless TLX performs an oncogenic function in prostate cancer cells via its promotion of androgen-independent growth and induction of epithelial – mesenchymal transition phenotype

Abstract The Tailless TLX gene (NR2E1), which was initially identified as a vertebrate homolog to the Drosophila terminal-gap gene tailless (tll), is an orphan nuclear receptor and functions mainly as a constitutive transcriptional repressor. Functional studies show that TLX plays important roles in the maintenance and self-renewal of both embryonic and adult neural stem cells. Transgenic mice with TLX overexpression show that in vivo ectopic TLX expression can lead to uncontrolled expansion of a subgroup of neural stem cells and cause initiation of glioma. In a preliminary screening of expression patterns of nuclear receptors in prostatic cell lines and clinical prostate cancer tissues, we identified that TLX transcripts were significantly overexpressed in many prostate cancer cell lines. Furthermore, by immunohistochemistry, increased TLX nuclear immunosignals were detected in high Gleason-scored (≥4) and also hormone-refractory (castration- and flutamide- resistant) prostate cancer, paired neoplastic human prostatic tissues, suggesting that TLX up-regulation may be positively associated with the advanced progression of prostate cancer. Based on these observations, we hypothesize that TLX may play growth regulatory roles in prostate cancer development. In order to elucidate the functional significance of TLX in prostate cancer growth, we generated stable TLX-infectants in a hormone-sensitive prostate cancer line LNCaP, which expressed low endogenous TLX levels, for growth phenotypic characterization. Our results showed that ectopic TLX expression could enhance the cell proliferation of LNCaP-TLX cells in either normal growth medium or medium with charcoal-stripped fetal bovine serum, accompanied with a decreased p21 expression. The LNCaP-TLX transduced clones, which expressed decreased androgen receptor (AR) and prostatic specific antigen (PSA) expressions, were less sensitive to AR agonist R1881 and antagonist bicalutamide, suggesting that LNCaP-TLX clones were less dependent on androgen but resistant to anti-androgen. Lastly, we also found that LNCaP-TLX infectants become apparently fusiform-shaped and expressed evaluated levels of two epithelial-mesenchymal transition (EMT) markers, Twist and Vimentin, suggesting that an EMT phenotype was induced in LNCaP-TLX cells. Taken together, our results demonstrated for the first time orphan nuclear receptor TLX may play an oncogenic regulatory role in the advanced growth of prostate cancer. (This study was supported by a Direct Grant for Research CUHK, project code 2041534) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4006. doi:10.1158/1538-7445.AM2011-4006

Structural and Functional Analysis of Tomosyn Identifies Domains Important in Exocytotic Regulation

Tomosyn is a 130-kDa cytosolic R-SNARE protein that associates with Q-SNAREs and reduces exocytotic activity. Two paralogous genes, tomosyn-1 and -2, occur in mammals and produce seven different isoforms via alternative splicing. Here, we map the structural differences between the yeast homologue of m-tomosyn-1, Sro7, and tomosyn genes/isoforms to identify domains critical to the regulation of exocytotic activity to tomosyn that are outside the soluble N-ethylmaleimide-sensitive attachment receptor motif. Homology modeling of m-tomosyn-1 based on the known structure of yeast Sro7 revealed a highly conserved functional conformation but with tomosyn containing three additional loop domains that emanate from a β-propeller core. Notably, deletion of loops 1 and 3 eliminates tomosyn inhibitory activity on secretion without altering its soluble N-ethylmaleimide-sensitive attachment receptor pairing with syntaxin1A. By comparison, deletion of loop 2, which contains the hypervariable splice region, did not reduce the ability of tomosyn to inhibit regulated secretion. However, exon variation within the hypervariable splice region resulted in significant differences in protein accumulation of tomosyn-2 isoforms. Functional analysis of s-tomosyn-1, m-tomosyn-1, m-tomosyn-2, and xb-tomosyn-2 demonstrated that they exert similar inhibitory effects on elevated K(+)-induced secretion in PC12 cells, although m-tomosyn-2 was novel in strongly augmenting basal secretion. Finally, we report that m-tomosyn-1 is a target substrate for SUMO 2/3 conjugation and that mutation of this small ubiquitin-related modifier target site (Lys-730) enhances m-tomosyn-1 inhibition of secretion without altering interaction with syntaxin1A. Together these results suggest that multiple domains outside the R-SNARE of tomosyn are critical to the efficacy of inhibition by tomosyn on exocytotic secretion.

Partial XBP1 knockdown does not affect viability of oligodendrocyte precursor cells exposed to new models of hypoxia and ischemia in vitro

The endoplasmic reticulum (ER) stress signaling pathway has been implicated in tissue injury in several rodent models of brain ischemia. To understand better the effects of ischemia on white matter in particular, we developed several in vitro models of hypoxia and ischemia in oligodendrocyte precursors. For the first time, we present data showing that exposure of rat oligodendrocyte precursor cells (OPCs) to cobalt chloride (CoCl(2)), antimycin A (AA), or oxygen, glucose and nutrient deprivation (OGND) causes up-regulation of glucose-regulated protein 78/B-cell immunoglobulin-binding protein (Grp78/BiP), C/EBP homologous binding protein (CHOP), and spliced X-box-binding protein 1 (XBP1). To mimic the effects of ischemia, OPCs supplemented with 5% normal growth medium and 95% Hank's balanced salt solution were incubated in a hypoxia chamber set at 0.1% oxygen. Because the toxic effects of AA on OPCs more closely resembled those seen when OPCs were subjected to OGND, we found AA treatment preferable to CoCl(2) as an in vitro model. To investigate the role of XBP1 in survival following an ischemic insult, we generated a stable XBP1 knockdown OPC cell line and subjected it to simulated hypoxia or ischemia. Surprisingly, 65% XBP1 knockdown had no effect on viability following chemical treatment or OGND. These data strengthen the case for targeting the ER stress signalling pathway in an effort to develop new early treatments for ischemic stroke patients but at the same time demonstrate that partial knockdown of XBP1 is not sufficient to protect precursor oligodendrocytes from ischemic damage.

Microbial detoxification of pathotoxin produced by spot blotch pathogen Bipolaris sorokiniana infecting wheat

Bipolaris sorokiniana causes spot blotch in wheat and barley. The pathogen produces toxin (BS-toxin), which is a sesquiterpenoid belonging to eremophilane family. Isolates of Trichoderma spp., Chaetomium globosum and Pseudomonas fluorescens were tested for detoxification of BS-toxin amended in semi-synthetic medium at different concentrations. All the antagonists showed mycelial growth in toxin amended medium but their growth was less in comparison to growth in normal medium. The growth of biocontrol agents decreased with increasing concentration of toxin. Two isolates of C. globosum (Cg1 and Cg2), T.viride (TV5-2) and Pseudomonas fluorescens produced 4.9, 2.9, 3.6 g mycelium and 5.5 × 105 cfu /ml, respectively exhibiting 50% or less reduction in growth in BS-toxin amended medium at 1,000 ppm concentration. The biocontrol agents also reduced the severity of toxin-induced symptoms and electrolyte leakage from the wheat leaf tissues. Among the microbes tested, maximum reduction in electrolyte leakage was observed in C. globosum (Cg2) treated toxin samples. The spectral analysis also showed a remarkable decrease in optical density of Cg2 treated toxin at 294 nm. High Performance Liquid Chromatography (HPLC) analysis showed almost complete degradation of BS-toxin in C. globosum (Cg2) treated samples.

S4 Protein Sll1252 Is Necessary for Energy Balancing in Photosynthetic Electron Transport in Synechocystis sp. PCC 6803

Sll1252 was identified as a novel protein in photosystem II complexes from Synechocystis sp. PCC 6803. To investigate the function of Sll1252, the corresponding gene, sll1252, was deleted in Synechocystis 6803. Despite the homology of Sll1252 to YlmH, which functions in the cell division machinery in Streptococcus, the growth rate and cell morphology of the mutant were not affected in normal growth medium. Instead, it seems that cells lacking this polypeptide have increased sensitivity to Cl(-) depletion. The growth and oxygen evolving activity of the mutant cells was highly suppressed compared with those of wild-type cells when Cl(-) and/or Ca(2+) was depleted from the medium. Recovery of photosystem II from photoinhibition was suppressed in the mutant. Despite the defects in photosystem II, in the light, the acceptor side of photosystem II was more reduced and the donor side of photosystem I was more oxidized compared with wild-type cells, suggesting that functional impairments were also present in cytochrome b(6)/f complexes. The amounts of cytochrome c(550) and cytochrome f were smaller in the mutant in the Ca(2+)- and Cl(-)-depleted medium. Furthermore, the amount of IsiA protein was increased in the mutant, especially in the Cl(-)-depleted medium, indicating that the mutant cells perceive environmental stress to be greater than it is. The amount of accompanying cytochrome c(550) in purified photosystem II complexes was also smaller in the mutant. Overall, the Sll1252 protein appears to be closely related to redox sensing of the plastoquinone pool to balance the photosynthetic electron flow and the ability to cope with global environmental stresses.

Classical NF-κB Activation Negatively Regulates Noncanonical NF-κB-dependent CXCL12 Expression

Ligation of the lymphotoxin-β receptor (LTβR) by LIGHT (lymphotoxin-related inducible ligand that competes for glycoprotein D binding to herpes virus entry mediator on T cells (TNFSF14)) activates the noncanonical (NC) NF-κB (nuclear factor-κB) pathway and up-regulates CXCL12 gene expression by human umbilical vein endothelial cells (HUVEC). In contrast, TNF only activates classical NF-κB signaling and does not up-regulate CXCL12. To determine whether cross-talk between the classical and NC pathways affects CXCL12 expression, we investigated the effects of TNF on LIGHT signaling in HUVEC. We show here that TNF inhibits both basal and LIGHT-induced CXCL12 expression. Negative regulation by TNF requires the classical NF-κB pathway as inhibition of basal and induced CXCL12 was reversed in HUVEC-expressing dominant negative IκB (inhibitor of NF-κB) kinase (IKK)β (IKKβ(K44M)). TNF did not inhibit the NC NF-κB pathway activation as LIGHT-induced p100 processing to p52 was intact; however, TNF either alone or together with LIGHT up-regulated p100 and RelB expression and induced the nuclear localization of p100-RelB complexes. Enhanced p100 and RelB expression was inhibited by IKKβ(K44M), which led us to question whether the IκB function of elevated p100 mediates the inhibition of CXCL12 expression by TNF. We retrovirally transduced HUVEC to express p100 at a level similar to that up-regulated by TNF; however, basal and LIGHT-induced CXCL12 expression was normal in the transduced cells. In contrast, ectopic RelB expression recapitulated the effects of TNF on NC signaling and inhibited basal and LIGHT-induced CXCL12 expression by HUVEC. Our findings therefore demonstrate that TNF-induced classical NF-κB signaling up-regulates RelB expression that inhibits both basal and NC NF-κB-dependent CXCL12 expression.

N-Cadherin Expressing MM Cells Form Calcium-Dependent, Adherent Junctions That Likely Contribute to Focal Lesions and Nodular Growth of Multiple Myeloma Cells

AbstractAbstract 979E-cadherin-mediated adhesion regulates homeostasis in tissues of epithelial origin and homotypic N-cadherin interactions are central to the interaction of hematopoietic stem cells and the endosteal niche. Loss of E-cadherin in carcinomas is characteristic of the epithelial-to-mesenchymal transition and metastasis. The loss of E-cadherin, causes the release of b-catenin from the adherent complex, increased nuclear translocation, and increased transcriptional activity of b-catenin/TCF. We and others have demonstrated that alterations in the Wnt/b-catenin pathway exists in multiple myeloma, a malignancy of terminally differentiated antibody secreting plasma cells. The first evidence of this deregulation came from studies showing that MM cells secrete the potent Wnt/b-catenin signaling inhibitor DKK1. While it is now clear that DKK1 mediated suppression of Wnt/b-catenin in the bone marrow contributes to the decoupling of bone formation, the role of Wnt/b-catenin in normal plasma cell development and myelomagenesis is less clear and often controversial. Myeloma cells grow exclusively in the bone marrow. This growth is characterized as being interstitial or nodular, with most disease exhibiting a mixed pattern. Nodular growth, recognized as focal lesions (FL) on MRI, characterizes the conversion of MGUS to symptomatic MM. Consistently, DKK1 levels are highest in CD138 cells isolated from FL where nodular tumor growth and bone destruction may be linked by Wnt/b-catenin suppression in MM cells and local microenvironment. Given the central role of classical cadherins in promoting cell-cell adhesion and regulating b-catenin, we hypothesized that abnormal expression of cadherins might play a direct role in myelomagenesis.Cell lysates were prepared from 24 MM cell lines and from CD138+ cells from the BM of eight patients with MM. Immunoblotting was performed with antibodies specific to human N-cadherin protein. N-cadherin protein was observed in more than 82% of MM cell lines, with high protein levels in 55% of the cell lines. Similar levels of N-cadherin protein were seen in primary myeloma cells from the eight MM patients: two patients showed the highest levels of N-cadherin protein, two showed intermediate levels, and three weak levels; N-cadherin protein was absent in one patient. Similar results were obtained with three color-flow cytometry analysis of primary MM BM. To determine whether N-cadherin mediated MM cell interactions, a cell aggregation assay using GFP-expressing, N-cadherin+ MM cells observed under fluorescence microscopy was employed. When N-cadherin+ JJN3 cells were cultured in normal growth medium, they aggregated to form clusters. Similar results were observed in other MM cell lines, including OPM-2 and KMS-28-BM. Addition of a neutralizing N-cadherin antibody to these cultures significantly attenuated aggregation of JJN3 cells compared to control cells in normal medium or cells treated with control IgG. Homotypic N-cadherin interaction forms adherent junctions in a calcium dependent manner. To see if aggregation of MM cells results in the formation of adherent junctions, immunochemical staining was preformed to visualize the N-cadherin protein in OPM-2 cells. N-cadherin protein was clearly observed between myeloma cells in aggregation clusters that were significantly diminished in calcium-free medium. Taken together, these data suggest that N-cadherin induces homotypic adhesion of myeloma cells in a calcium-dependent manner and suggests that calcium release during bone resorption may enhance adherent junctions in MM that may in turn enhance plasma membrane localization of b-catenin. Studies are currently underway to determine whether DKK1 and N-cadherin adherent junctions cooperate to suppress b-catenin nuclear activity in MM cells and all converge to induce the nodular growth pattern and bone destruction often seen in N-cadherin-positive MM. Disclosures:No relevant conflicts of interest to declare.

Generation, validation and humanisation of a novel insulin resistant cell model

Insulin resistance is a characteristic of type 2 diabetes and is a major independent risk factor for progression to the disease. In particular, insulin resistance associates with increased body fat and almost certainly contributes to the dramatic increase in risk of type 2 diabetes associated with obesity. Therefore, in order to design truly effective insulin sensitising agents, targeted at the mechanism of disease development, we aimed to generate an obesity-related insulin resistant cell model. Rat hepatoma cells were grown in the presence of serum isolated from obese rodents or obese human volunteers, and the insulin sensitivity of the cells monitored over time by measuring a well-characterised insulin regulated gene promoter. Higher insulin concentrations were required to fully repress the gene in the cells grown in obese rodent serum compared with those grown in serum from lean rodents (almost a 10-fold shift in insulin sensitivity). This was reversed by restoration of normal growth medium, while the insulin resistance was prevented by pioglitazone or metformin. Meanwhile, growth of cells in serum collected from obese human volunteers with diabetes also reduced the insulin sensitivity of the rat cells. No clinical marker predicted the degree of insulin resistance that was generated by the human serum. We have developed a novel insulin resistant cell model for the study of the molecular development of obesity-linked insulin resistance, screen for compounds to overcome obesity-related insulin resistance and potentially search for novel serum biomarkers of insulin resistance.