Pesticidal and pest repellency activities of a plant derived triterpenoid 2α,3β,21β,23,28-penta hydroxyl 12-oleanene against Tribolium castaneum

Background

Tribolium castaneum (Herbst) is a major pest of stored grain-based products, and cause severe damage to cereal grains throughout the world. The present investigation was aimed to determine the pesticidal and pest repellent activities of 2α,3β,21β,23,28-penta hydroxyl 12-oleanene against T. castaneum. The compound 2α,3β,21β,23,28-penta hydroxyl 12-oleanene is a triterpenoid which was isolated from the roots of Laportea crenulata Gaud. Surface film technique was used for pesticidal screening, whereas, pest repellency property of the triterpenoid was determined by filter paper disc method.

Results

At 24 hours of exposure duration, significant mortality records (80% and 86%) were observed at doses 0.88 and 1.77 mg/cm². No significant change in mortality records was observed when duration of exposure was increased up to 48 hours. The triterpenoid showed significant repellency activity at doses 0.47 and 0.94 mg/cm².

Conclusion

These data suggest that the triterpenoid 2α,3β,21β,23,28-penta hydroxyl 12-oleanene possess both pesticidal and pest repellency activities against T. castaneum and can be used in controlling the pest of grain-based products.

Electronic supplementary material

The online version of this article (doi:10.1186/0717-6287-47-68) contains supplementary material, which is available to authorized users.     

Antitumor activity of IL-32β through the activation of lymphocytes,and the inactivation of NF-κB and STAT3 signals

Cytokine and activation of lymphocytes are critical for tumor growth. We investigated whether interleukin (IL)-32β overexpression changes other cytokine levels and activates cytotoxic lymphocyte, and thus modify tumor growth. Herein, IL-32β inhibited B16 melanoma growth in IL-32β-overexpressing transgenic mice (IL-32β mice), and downregulated the expressions of anti-apoptotic proteins (bcl-2, IAP, and XIAP) and cell growth regulatory proteins (Ki-67 antigen (Ki-67) and proliferating cell nuclear antigen (PCNA)), but upregulated the expressions of pro-apoptotic proteins (bax, cleaved caspase-3, and cleaved caspase-9). IL-32β also inhibited colon and prostate tumor growth in athymic nude mice inoculated with IL-32β-transfected SW620 colon or PC3 prostate cancer cells. The forced expression of IL-32β also inhibited cell growth in cultured colon and prostate cancer cells, and these inhibitory effects were abolished by IL-32 small interfering RNA (siRNA). IL-10 levels were elevated, but IL-1β, IL-6, and tumor necrosis factor-alpha (TNF-α) levels were reduced in the tumor tissues and spleens of IL-32β mice, and athymic nude mice. The number of cytotoxic T (CD8⁺) and natural killer (NK) cells in tumor tissues, spleen, and blood was significantly elevated in IL-32β mice and athymic nude mice inoculated with IL-32β-transfected cancer cells. Constituted activated NF-κB and STAT3 levels were reduced in the tumor tissues of IL-32β mice and athymic nude mice, as well as in IL-32β-transfected cultured cancer cells. These findings suggest that IL-32β inhibits tumor growth by increasing cytotoxic lymphocyte numbers, and by inactivating the NF-κB and STAT3 pathways through changing of cytokine levels in tumor tissues.     

Identification and characterization of PDGFRα+ mesenchymal progenitors in human skeletal muscle

Fatty and fibrous connective tissue formation is a hallmark of diseased skeletal muscle and deteriorates muscle function. We previously identified non-myogenic mesenchymal progenitors that contribute to adipogenesis and fibrogenesis in mouse skeletal muscle. In this study, we report the identification and characterization of a human counterpart to these progenitors. By using PDGFRα as a specific marker, mesenchymal progenitors can be identified in the interstitium and isolated from human skeletal muscle. PDGFRα⁺ cells represent a cell population distinct from CD56⁺ myogenic cells, and adipogenic and fibrogenic potentials were highly enriched in the PDGFRα⁺ population. Activation of PDGFRα stimulates proliferation of PDGFRα⁺ cells through PI3K-Akt and MEK2-MAPK signaling pathways, and aberrant accumulation of PDGFRα⁺ cells was conspicuous in muscles of patients with both genetic and non-genetic muscle diseases. Our results revealed the pathological relevance of PDGFRα⁺ mesenchymal progenitors to human muscle diseases and provide a basis for developing therapeutic strategy to treat muscle diseases.     

Antioxidant,inhibition of α-glucosidase and suppression of nitric oxide production in LPS-induced murine macrophages by different fractions of Actinidia arguta stem

In traditional systems of medicine, fruits, leaves, and stems of Actinidia arguta (Sieb. et Zucc.) Planch. ex Miq. have been used to treat various inflammatory diseases. The present study determined the proximate composition, antioxidant, anti-inflammatory, and hypoglycemic potential of A. arguta stem. Phenolic composition of hot water extract and its sub-fractions was determined by Folin–Ciocalteu’s reagent method. In vitro antioxidant activities of the samples were evaluated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) radical scavenging assays. Anti-inflammatory activity of different fractions was investigated through the inhibition of nitric oxide (NO) production in lipopolysaccharide (1 μg/ml) stimulated RAW 264.7 cells. In addition, inhibition of α-glucosidase activity of hot water extract was determined using p-nitrophenyl-α-d-glucopyranoside (pNPG) as a substrate. Ethyl acetate (557.23 mg GAE/g) fraction contains higher level of total phenolic content. The antioxidant activity evaluated by DPPH radical scavenging assay showed a strong activity for ethyl acetate (IC₅₀ of 14.28 μg/ml) and n-butanol fractions (IC₅₀ of 48.27 μg/ml). Further, ethyl acetate fraction effectively inhibited NO production in RAW 264.7 cells induced by lipopolysaccharide (LPS) than other fractions (nitrite level to 32.14 μM at 200 μg/ml). In addition, hot water extract of A. arguta stem exhibited appreciable inhibitory activity against α-glucosidase enzyme with IC₅₀ of 1.71 mg/ml. The obtained results have important consequence of using A. arguta stem toward the development of effective anti-inflammatory drugs.     

TGF-β1 exposure induces epithelial to mesenchymal transition both in CSCs and non-CSCs of the A549 cell line,leading to an increase of migration ability in the CD133+ A549 cell fraction

Metastasis is the leading cause of death by cancer. Non-small-cell lung cancer (NSCLC) represents nearly 85% of primary malignant lung tumours. Recent researches have demonstrated that epithelial-to-mesenchymal transition (EMT) plays a key role in the early process of metastasis of cancer cells. Transforming growth factor-β1 (TGF-β1) is the major inductor of EMT. The aim of this study is to investigate TGF-β1''s effect on cancer stem cells (CSCs) identified as cells positive for CD133, side population (SP) and non-cancer stem cells (non-CSCs) identified as cells negative for CD133, and SP in the A549 cell line. We demonstrate that TGF-β1 induces EMT in both CSC and non-CSC A549 sublines, upregulating the expression of mesenchymal markers such as vimentin and Slug, and downregulating levels of epithelial markers such as e-cadherin and cytokeratins. CSC and non-CSC A549 sublines undergoing EMT show a strong migration and strong levels of MMP9 except for the CD133⁻ cell fraction. OCT4 levels are strongly upregulated in all cell fractions except CD133⁻ cells. On the contrary, wound size reveals that TGF-β1 enhances motility in wild-type A549 as well as CD133⁺ and SP⁺ cells. For CD133⁻ and SP⁻ cells, TGF-β1 exposure does not change the motility. Finally, assessment of growth kinetics reveals major colony-forming efficiency in CD133⁺ A549 cells. In particular, SP⁺ and SP⁻ A549 cells show more efficiency to form colonies than untreated corresponding cells, while for CD133⁻ cells no change in colony number was observable after TGF-β1 exposure. We conclude that it is possible to highlight different cell subpopulations with different grades of stemness. Each population seems to be involved in different biological mechanisms such as stemness maintenance, tumorigenicity, invasion and migration.     

Mcl-1 downregulation by pro-inflammatory cytokines and palmitate is an early event contributing to β-cell apoptosis

Pancreatic β-cell apoptosis is a key feature of diabetes mellitus and the mitochondrial pathway of apoptosis is a major mediator of β-cell death. We presently evaluated the role of the myeloid cell leukemia sequence 1 (Mcl-1), an antiapoptotic protein of the Bcl-2 family, in β-cells following exposure to well-defined β-cell death effectors, for example, pro-inflammatory cytokines, palmitate and chemical endoplasmic reticulum (ER) stressors. All cytotoxic stresses rapidly and preferentially decreased Mcl-1 protein expression as compared with the late effect observed on the other antiapoptotic proteins, Bcl-2 and Bcl-xL. This was due to ER stress-mediated inhibition of translation through eIF2α phosphorylation for palmitate and ER stressors and through the combined action of translation inhibition and JNK activation for cytokines. Knocking down Mcl-1 using small interference RNAs increased apoptosis and caspase-3 cleavage induced by cytokines, palmitate or thapsigargin, whereas Mcl-1 overexpression partly prevented Bax translocation to the mitochondria, cytochrome c release, caspase-3 cleavage and apoptosis induced by the β-cell death effectors. Altogether, our data suggest that Mcl-1 downregulation is a crucial event leading to β-cell apoptosis and provide new insights into the mechanisms linking ER stress and the mitochondrial intrinsic pathway of apoptosis. Mcl-1 is therefore an attractive target for the design of new strategies in the treatment of diabetes.     

Key Residues in the Nicotinic Acetylcholine Receptor β2 Subunit Contribute to α-Conotoxin LvIA Binding

α-Conotoxin LvIA (α-CTx LvIA) is a small peptide from the venom of the carnivorous marine gastropod Conus lividus and is the most selective inhibitor of α3β2 nicotinic acetylcholine receptors (nAChRs) known to date. It can distinguish the α3β2 nAChR subtype from the α6β2* (* indicates the other subunit) and α3β4 nAChR subtypes. In this study, we performed mutational studies to assess the influence of residues of the β2 subunit versus those of the β4 subunit on the binding of α-CTx LvIA. Although two β2 mutations, α3β2[F119Q] and α3β2[T59K], strongly enhanced the affinity of LvIA, the β2 mutation α3β2[V111I] substantially reduced the binding of LvIA. Increased activity of LvIA was also observed when the β2-T59L mutant was combined with the α3 subunit. There were no significant difference in inhibition of α3β2[T59I], α3β2[Q34A], and α3β2[K79A] nAChRs when compared with wild-type α3β2 nAChR. α-CTx LvIA displayed slower off-rate kinetics at α3β2[F119Q] and α3β2[T59K] than at the wild-type receptor, with the latter mutant having the most pronounced effect. Taken together, these data provide evidence that the β2 subunit contributes to α-CTx LvIA binding and selectivity. The results demonstrate that Val¹¹¹ is critical and facilitates LvIA binding; this position has not previously been identified as important to binding of other 4/7 framework α-conotoxins. Thr⁵⁹ and Phe¹¹⁹ of the β2 subunit appear to interfere with LvIA binding, and their replacement by the corresponding residues of the β4 subunit leads to increased affinity.     

Biogeographic,climatic and spatial drivers differentially affect α-, β- and γ-diversities on oceanic archipelagos

Island biogeographic studies traditionally treat single islands as units of analysis. This ignores the fact that most islands are spatially nested within archipelagos. Here, we took a fundamentally different approach and focused on entire archipelagos using species richness of vascular plants on 23 archipelagos worldwide and their 174 constituent islands. We assessed differential effects of biogeographic factors (area, isolation, age, elevation), current and past climate (temperature, precipitation, seasonality, climate change velocity) and intra-archipelagic spatial structure (archipelago area, number of islands, area range, connectivity, environmental volume, inter-island distance) on plant diversity. Species diversity of each archipelago (γ) was additively partitioned into α, β, nestedness and replacement β-components to investigate the relative importance of environmental and spatial drivers. Multiple regressions revealed strong effects of biogeography and climate on α and γ, whereas spatial factors, particularly number of islands, inter-island distance and area range, were key to explain β. Structural equation models additionally suggested that γ is predominantly determined by indirect abiotic effects via its components, particularly β. This highlights that β and the spatial arrangement of islands are essential to understand insular ecology and evolution. Our methodological framework can be applied more widely to other taxa and archipelago-like systems, allowing new insights into biodiversity origin and maintenance.     

ROS-PIASγ cross talk channelizes ATM signaling from resistance to apoptosis during chemosensitization of resistant tumors

With the existing knowledge of ATM''s role in therapeutic resistance, the present study aimed at identifying the molecular mechanisms that influence ATM to oscillate between chemoresistance and chemosensitivity. We observed that the redox status of tumors functions as a major determinant of ATM-dependent ‘resistance-to-apoptosis'' molecular switch. At a low reactive oxygen species (ROS) condition during genotoxic insult, the ATM/sumoylated-IKKγ interaction induced NFκB activation that resisted JNK-mediated apoptosis, whereas increasing cellular ROS restored ATM/JNK apoptotic signaling. A search for the upstream missing link revealed that high ROS induces oxidation and ubiquitin-mediated degradation of PIASγ, thereby disrupting PIASγ-IKKγ cross talk, a pre-requisite for IKKγ sumoylation and subsequent NFκB activation. Interruption in the PIASγ-mediated resistance pathway channels ATM signaling toward ATM/JNK pro-death circuitry. These in vitro results also translated to sensitive and resistant tumor allograft mouse models in which low ROS-induced resistance was over-ruled in PIASγ knockout tumors, while its overexpression inhibited high ROS-dependent apoptotic cues. Cumulatively, our findings identified an unappreciated yet critical combinatorial function of cellular ROS and PIASγ in regulating ATM-mediated chemosensitization of resistant tumors. Thus, therapeutic strategies employing ROS upregulation to inhibit PIASγ during genotoxic therapy may, in future, help to eliminate the problems of NFκB-mediated tumor drug resistance.     

Genetic disruption of Abl nuclear import reduces renal apoptosis in a mouse model of cisplatin-induced nephrotoxicity

DNA damage activates nuclear Abl tyrosine kinase to stimulate intrinsic apoptosis in cancer cell lines and mouse embryonic stem cells. To examine the in vivo function of nuclear Abl in apoptosis, we generated Abl-μNLS (μ, mutated in nuclear localization signals) mice. We show here that cisplatin-induced apoptosis is defective in the renal proximal tubule cells (RPTC) from the Abl^μ/μ mice. When injected with cisplatin, we found similar levels of platinum in the Abl^+/+ and the Abl^μ/μ kidneys, as well as similar initial inductions of p53 and PUMAα expression. However, the accumulation of p53 and PUMAα could not be sustained in the Abl^μ/μ kidneys, leading to reductions in renal apoptosis and tubule damage. Co-treatment of cisplatin with the Abl kinase inhibitor, imatinib, reduced the accumulation of p53 and PUMAα in the Abl^+/+ but not in the Abl^μ/μ kidneys. The residual apoptosis in the Abl^μ/μ mice was not further reduced in the Abl^μ/μ; p53^−/− double-mutant mice, suggesting that nuclear Abl and p53 are epistatic to each other in this apoptosis response. Although apoptosis and tubule damage were reduced, cisplatin-induced increases in phospho-Stat-1 and blood urea nitrogen were similar between the Abl^+/+ and the Abl^μ/μ kidneys, indicating that RPTC apoptosis is not the only factor in cisplatin-induced nephrotoxicity. These results provide in vivo evidence for the pro-apoptotic function of Abl, and show that its nuclear localization and tyrosine kinase activity are both required for the sustained expression of p53 and PUMAα in cisplatin-induced renal apoptosis.     

ATM-NFκB axis-driven TIGAR regulates sensitivity of glioma cells to radiomimetics in the presence of TNFα

Gliomas are resistant to radiation therapy, as well as to TNFα induced killing. Radiation-induced TNFα triggers Nuclear factor κB (NFκB)-mediated radioresistance. As inhibition of NFκB activation sensitizes glioma cells to TNFα-induced apoptosis, we investigated whether TNFα modulates the responsiveness of glioma cells to ionizing radiation-mimetic Neocarzinostatin (NCS). TNFα enhanced the ability of NCS to induce glioma cell apoptosis. NCS-mediated death involved caspase-9 activation, reduction of mitochondrial copy number and lactate production. Death was concurrent with NFκB, Akt and Erk activation. Abrogation of Akt and NFκB activation further potentiated the death inducing ability of NCS in TNFα cotreated cells. NCS-induced p53 expression was accompanied by increase in TP53-induced glycolysis and apoptosis regulator (TIGAR) levels and ATM phosphorylation. siRNA-mediated knockdown of TIGAR abrogated NCS-induced apoptosis. While DN-IκB abrogated NCS-induced TIGAR both in the presence and absence of TNFα, TIGAR had no effect on NFκB activation. Transfection with TIGAR mutant (i) decreased apoptosis and γH2AX foci formation (ii) decreased p53 (iii) elevated ROS and (iv) increased Akt/Erk activation in cells cotreated with NCS and TNFα. Heightened TIGAR expression was observed in GBM tumors. While NCS induced ATM phosphorylation in a NFκB independent manner, ATM inhibition abrogated TIGAR and NFκB activation. Metabolic gene profiling indicated that TNFα affects NCS-mediated regulation of several genes associated with glycolysis. The existence of ATM-NFκB axis that regulate metabolic modeler TIGAR to overcome prosurvival response in NCS and TNFα cotreated cells, suggests mechanisms through which inflammation could affect resistance and adaptation to radiomimetics despite concurrent induction of death.     

Combinatorial active-site variants confer sustained clavulanate resistance in BlaC β-lactamase from Mycobacterium tuberculosis

Bacterial resistance to β-lactam antibiotics is a global issue threatening the success of infectious disease treatments worldwide. Mycobacterium tuberculosis has been particularly resilient to β-lactam treatment, primarily due to the chromosomally encoded BlaC β-lactamase, a broad-spectrum hydrolase that renders ineffective the vast majority of relevant β-lactam compounds currently in use. Recent laboratory and clinical studies have nevertheless shown that specific β-lactam–BlaC inhibitor combinations can be used to inhibit the growth of extensively drug-resistant strains of M. tuberculosis, effectively offering new tools for combined treatment regimens against resistant strains. In the present work, we performed combinatorial active-site replacements in BlaC to demonstrate that specific inhibitor-resistant (IRT) substitutions at positions 69, 130, 220, and/or 234 can act synergistically to yield active-site variants with several thousand fold greater in vitro resistance to clavulanate, the most common clinical β-lactamase inhibitor. While most single and double variants remain sensitive to clavulanate, double mutants R220S-K234R and S130G-K234R are substantially less affected by time-dependent clavulanate inactivation, showing residual β-lactam hydrolytic activities of 46% and 83% after 24 h incubation with a clinically relevant inhibitor concentration (5 μg/ml, 25 µM). These results demonstrate that active-site alterations in BlaC yield resistant variants that remain active and stable over prolonged bacterial generation times compatible with mycobacterial proliferation. These results also emphasize the formidable adaptive potential of inhibitor-resistant substitutions in β-lactamases, potentially casting a shadow on specific β-lactam–BlaC inhibitor combination treatments against M. tuberculosis.     

Nuclear factor-κB modulates osteogenesis of periodontal ligament stem cells through competition with β-catenin signaling in inflammatory microenvironments

Inflammation can influence multipotency and self-renewal of mesenchymal stem cells (MSCs), resulting in their awakened bone-regeneration ability. Human periodontal ligament tissue-derived MSCs (PDLSCs) have been isolated, and their differentiation potential was found to be defective due to β-catenin signaling indirectly regulated by inflammatory microenvironments. Nuclear factor-κB (NF-κB) is well studied in inflammation by many different groups. The role of NF-κB needs to be studied in PDLSCs, although genetic evidences have recently shown that NF-κB inhibits osteoblastic bone formation in mice. However, the mechanism as to how inflammation leads to the modulation of β-catenin and NF-κB signaling remains unclear. In this study, we investigated β-catenin and NF-κB signaling through regulation of glycogen synthase kinase 3β activity (GSK-3β, which modulates β-catenin and NF-κB signaling) using a specific inhibitor LiCl and a phosphatidylinositol 3-kinase (PI3K) inhibitor LY 294002. We identified that NF-κB signaling might be more important for the regulation of osteogenesis in PDLSCs from periodontitis compared with β-catenin. BAY 11-7082 (an inhibitor of NF-κB) could inhibit phosphorylation of p65 and partly rescue the differentiation potential of PDLSCs in inflammation. Our data indicate that NF-κB has a central role in regulating osteogenic differentiation of PDLSCs in inflammatory microenvironments. Given the molecular mechanisms of NF-κB in osteogenic differentiation governed by inflammation, it can be said that NF-κB helps in improving stem cell-mediated inflammatory bone disease therapy.     

Reduced IRE1α mediates apoptotic cell death by disrupting calcium homeostasis via the InsP3 receptor

The endoplasmic reticulum (ER) is not only a home for folding and posttranslational modifications of secretory proteins but also a reservoir for intracellular Ca²⁺. Perturbation of ER homeostasis contributes to the pathogenesis of various neurodegenerative diseases, such as Alzheimer''s and Parkinson diseases. One key regulator that underlies cell survival and Ca²⁺ homeostasis during ER stress responses is inositol-requiring enzyme 1α (IRE1α). Despite extensive studies on this ER membrane-associated protein, little is known about the molecular mechanisms by which excessive ER stress triggers cell death and Ca²⁺ dysregulation via the IRE1α-dependent signaling pathway. In this study, we show that inactivation of IRE1α by RNA interference increases cytosolic Ca²⁺ concentration in SH-SY5Y cells, leading to cell death. This dysregulation is caused by an accelerated ER-to-cytosolic efflux of Ca²⁺ through the InsP3 receptor (InsP3R). The Ca²⁺ efflux in IRE1α-deficient cells correlates with dissociation of the Ca²⁺-binding InsP3R inhibitor CIB1 and increased complex formation of CIB1 with the pro-apoptotic kinase ASK1, which otherwise remains inactivated in the IRE1α–TRAF2–ASK1 complex. The increased cytosolic concentration of Ca²⁺ induces mitochondrial production of reactive oxygen species (ROS), in particular superoxide, resulting in severe mitochondrial abnormalities, such as fragmentation and depolarization of membrane potential. These Ca²⁺ dysregulation-induced mitochondrial abnormalities and cell death in IRE1α-deficient cells can be blocked by depleting ROS or inhibiting Ca²⁺ influx into the mitochondria. These results demonstrate the importance of IRE1α in Ca²⁺ homeostasis and cell survival during ER stress and reveal a previously unknown Ca²⁺-mediated cell death signaling between the IRE1α–InsP3R pathway in the ER and the redox-dependent apoptotic pathway in the mitochondrion.     

The Nuclear Localization of Glycogen Synthase Kinase 3β Is Required Its Putative PY-Nuclear Localization Sequences

Glycogen synthase kinase-3β (GSK-3β), which is a member of the serine/threonine kinase family, has been shown to be crucial for cellular survival, differentiation, and metabolism. Here, we present evidence that GSK-3β is associated with the karyopherin β2 (Kap β2) (102-kDa), which functions as a substrate for transportation into the nucleus. A potential PY-NLS motif (¹⁰⁹IVRLRYFFY¹¹⁷) was observed, which is similar with the consensus PY NLS motif (R/K/H)X_2–5PY in the GSK-3β catalytic domain. Using a pull down approach, we observed that GSK-3β physically interacts with Kap β2 both in vivo and in vitro. Secondly, GSK-3β and Kap β2 were shown to be co-localized by confocal microscopy. The localization of GSK-3β to the nuclear region was disrupted by putative Kap β2 binding site mutation. Furthermore, in transient transfection assays, the Kap β2 binding site mutant induced a substantial reduction in the in vivo serine/threonine phosphorylation of GSK-3β, where- as the GSK-3β wild type did not. Thus, our observations indicated that Kap β2 imports GSK-3β through its putative PY NLS motif from the cytoplasm to the nucleus and increases its kinase activity.     

Differential activity of GSK-3 isoforms regulates NF-κB and TRAIL- or TNFα induced apoptosis in pancreatic cancer cells

While TRAIL is a promising anticancer agent due to its ability to selectively induce apoptosis in neoplastic cells, many tumors, including pancreatic ductal adenocarcinoma (PDA), display intrinsic resistance, highlighting the need for TRAIL-sensitizing agents. Here we report that TRAIL-induced apoptosis in PDA cell lines is enhanced by pharmacological inhibition of glycogen synthase kinase-3 (GSK-3) or by shRNA-mediated depletion of either GSK-3α or GSK-3β. In contrast, depletion of GSK-3β, but not GSK-3α, sensitized PDA cell lines to TNFα-induced cell death. Further experiments demonstrated that TNFα-stimulated IκBα phosphorylation and degradation as well as p65 nuclear translocation were normal in GSK-3β-deficient MEFs. Nonetheless, inhibition of GSK-3β function in MEFs or PDA cell lines impaired the expression of the NF-κB target genes Bcl-xL and cIAP2, but not IκBα. Significantly, the expression of Bcl-xL and cIAP2 could be reestablished by expression of GSK-3β targeted to the nucleus but not GSK-3β targeted to the cytoplasm, suggesting that GSK-3β regulates NF-κB function within the nucleus. Consistent with this notion, chromatin immunoprecipitation demonstrated that GSK-3 inhibition resulted in either decreased p65 binding to the promoter of BIR3, which encodes cIAP2, or increased p50 binding as well as recruitment of SIRT1 and HDAC3 to the promoter of BCL2L1, which encodes Bcl-xL. Importantly, depletion of Bcl-xL but not cIAP2, mimicked the sensitizing effect of GSK-3 inhibition on TRAIL-induced apoptosis, whereas Bcl-xL overexpression ameliorated the sensitization by GSK-3 inhibition. These results not only suggest that GSK-3β overexpression and nuclear localization contribute to TNFα and TRAIL resistance via anti-apoptotic NF-κB genes such as Bcl-xL, but also provide a rationale for further exploration of GSK-3 inhibitors combined with TRAIL for the treatment of PDA.     

Oligomeric amyloid β induces IL-1β processing via production of ROS: implication in Alzheimer's disease

Alzheimer''s disease (AD) is a chronic neurodegenerative disease characterized by progressive neuronal loss and cognitive decline. Oligomeric amyloid β (oAβ) is involved in the pathogenesis of AD by affecting synaptic plasticity and inhibiting long-term potentiation. Although several lines of evidence suggests that microglia, the resident immune cells in the central nervous system (CNS), are neurotoxic in the development of AD, the mechanism whether or how oAβ induces microglial neurotoxicity remains unknown. Here, we show that oAβ promotes the processing of pro-interleukin (IL)-1β into mature IL-1β in microglia, which then enhances microglial neurotoxicity. The processing is induced by an increase in activity of caspase-1 and NOD-like receptor family, pyrin domain containing 3 (NLRP3) via mitochondrial reactive oxygen species (ROS) and partially via NADPH oxidase-induced ROS. The caspase-1 inhibitor Z-YVAD-FMK inhibits the processing of IL-1β, and attenuates microglial neurotoxicity. Our results indicate that microglia can be activated by oAβ to induce neuroinflammation through processing of IL-1β, a pro-inflammatory cytokine, in AD.     

Reconstitution of the recombinant human γ-tubulin ring complex

Cryo-electron microscopy recently resolved the structure of the vertebrate γ-tubulin ring complex (γ-TuRC) purified from Xenopus laevis egg extract and human cells to near-atomic resolution. These studies clarified the arrangement and stoichiometry of γ-TuRC components and revealed that one molecule of actin and the small protein MZT1 are embedded into the complex. Based on this structural census of γ-TuRC core components, we developed a recombinant expression system for the reconstitution and purification of human γ-TuRC from insect cells. The recombinant γ-TuRC recapitulates the structure of purified native γ-TuRC and has similar functional properties in terms of microtubule nucleation and minus end capping. This recombinant system is a central step towards deciphering the activation mechanisms of the γ-TuRC and the function of individual γ-TuRC core components.