A1 Treatment Research Articles

The Erv41-Erv46 complex serves as a retrograde receptor to retrieve escaped ER proteins.

Signal-dependent sorting of proteins in the early secretory pathway is required for dynamic retention of endoplasmic reticulum (ER) and Golgi components. In this study, we identify the Erv41-Erv46 complex as a new retrograde receptor for retrieval of non-HDEL-bearing ER resident proteins. In cells lacking Erv41-Erv46 function, the ER enzyme glucosidase I (Gls1) was mislocalized and degraded in the vacuole. Biochemical experiments demonstrated that the luminal domain of Gls1 bound to the Erv41-Erv46 complex in a pH-dependent manner. Moreover, in vivo disturbance of the pH gradient across membranes by bafilomycin A1 treatment caused Gls1 mislocalization. Whole cell proteomic analyses of deletion strains using stable isotope labeling by amino acids in culture identified other ER resident proteins that depended on the Erv41-Erv46 complex for efficient localization. Our results support a model in which pH-dependent receptor binding of specific cargo by the Erv41-Erv46 complex in Golgi compartments identifies escaped ER resident proteins for retrieval to the ER in coat protein complex I-formed transport carriers.

길경 추출물에 의한 HCT-116 대장암 세포주에서의 autophagy와 apoptosis 유발 효과

Platycodon grandiflorum (PG) has been known to possess many biological effects, including anti-inflammatory and anti-allergy activity and anti-obesity and hyperlipidemia effects. However, little research has been conducted regarding its anticancer effects, with the exception of its ability to stimulate apoptosis in skin cells. There has also been no study regarding PG-induced autophagy. The modulation of autophagy is recognized as one of the hallmarks of cancer cells. Depending on the type of cancer and the context, autophagy can suppress or help cancer cells to overcome metabolic stress and the cytotoxicity of chemotherapy. Therefore, the present study was designed to investigate whether or not extracts from PG-induced cell death were connected with autophagy and apoptosis in HCT-116 human colon cancer cells. PG stimulation decreased cell proliferation in a dose- and time-dependent manner and induced apoptosis, which was partially dependent on the activation of caspases. PG treatment also resulted in the formation of autophagic vacuoles simultaneously with regulation of autophagy-related genes. Interestingly, a PG-mediated apoptotic effect was further triggered by pretreatment with the autophagy inhibitors 3-methyladenin and bafilomycin A1. However, cell viability recovered quite well with bafilomycin A1 treatment. These findings show that PG treatment promotes both autophagy and apoptosis and that PG-induced autophagic response might play a role in the autophagic cell death of HCT-116 cells.

Abstract 15689: Activation of PKR-Like Endoplasmic Reticulum Kinase is Required for Autophagy Stimulation and Cardiomyocyte Survival During Acute Myocardial Ischemia

PKR-like endoplasmic reticulum (ER) kinase (PERK) is an ER-resident serine/threonine kinase that regulates the cellular response to ER stress and energy deprivation. Under stress, PERK activation can be either protective or detrimental. We studied for the first time whether PERK regulates cardiomyocyte (CM) survival and death during myocardial ischemia, which is characterized by both ER and energy stress. We found that PERK is significantly activated after 30 minutes of myocardial ischemia (2.4 ± 0.04-fold, p<0.001), as shown by its increased phosphorylation status. After 3 hours of ischemia, mice with cardiac-specific overexpression of dominant-negative PERK (Tg-DN-PERK) displayed a significant increase in infarct size/area at risk with respect to non-transgenic (NTg) mice (66.4 ± 4.1% vs. 38.5 ± 6.8%, p<0.05), indicating that ischemia-induced PERK activation is cardioprotective. PERK signaling regulates autophagy in cancer cells. Autophagy is an intracellular degradation mechanism that promotes CM survival during ischemia by relieving both energy and ER stress. We found that autophagy activation during ischemia is abrogated in the hearts of Tg-DN-PERK with respect to NTg, as indicated by reduced LC3-II accumulation (0.71 ± 0.05-fold, p<0.01). Consistently, CMs with adenovirus-mediated overexpression of DN-PERK displayed reduced LC3-II accumulation (0.69 ± 0.1-fold vs. control CMs, p<0.05) after 4 hours of glucose deprivation (GD) in vitro, a condition that mimics ischemia-induced energy stress. A similar result was also observed after bafilomycin A1 treatment to test autophagic flux. These data indicate that PERK is required for autophagy activation and flux during CM ischemia and GD. Finally, we saw that DN-PERK overexpression significantly promotes CM death after GD, as shown by propidium iodide assay (2.01 ± 0.25-fold vs. control CMs, p<0.05). However, co-overexpression of Atg7, which activates autophagy, significantly reduced cell death induced by DN-PERK (-61%, p<0.05). These data suggest that autophagy inhibition partly mediates the detrimental effects of PERK inhibition during energy stress. In conclusion, our study demonstrates that PERK activation is required for autophagy activation and CM survival during ischemia.

Autophagy modulates amino acid signaling network in myotubes: differential effects on mTORC1 pathway and the integrated stress response.

Induction of autophagy and the integrated stress response is important for amino acid homeostasis. It remains unknown whether the autophagy coregulates both mechanistic target of rapamycin complex 1 (mTORC1) signaling and the integrated stress response. In mouse C2C12 myotubes, we found that amino acid limitation induced autophagy and that the subsequent release of amino acid is required to sustain mTORC1 signaling. Inhibition of autophagy by bafilomycin A1 or chloroquine treatment during amino acid scarcity abolished mTORC1 signaling, an effect that could be rescued by inhibiting protein synthesis or amino acid supplementation, respectively. Autophagy is required to sustain the balance of both essential and nonessential amino acids during amino acid starvation, and it has a predominant role over the ubiquitin-proteasome system in the regulation of mTORC1. Inhibition of autophagy was found to activate the integrated stress response, as well as eukaryotic initiation factor 2α (eIF2α) and its target genes independent of amino acid availability. Conversely, autophagy induction via mTOR inhibition is sufficient to reduce eIF2α phosphorylation. Thus, autophagy protects the eIF2α-mediated stress response independent of amino acid supply in cultured myotubes. Our results showed that autophagy uniquely modulates mTORC1 and the integrated stress response in an amino acid-dependent and -independent manner, respectively.

STANOVENÍ KVALITY FERMENTACE PIVOVARSKÉHO MLÁTA SILÁŽOVANÉHO V KOMBINACI S PŘÍDAVKEM SLADOVÉHO KVĚTU A CHEMICKÉHO SILÁŽNÍHO ADITIVA

The aim of the work was to evaluate the effect of addition of humidity absorbent (malt sprouts) and chemical conservation additive on fermentation process quality of brewer grains’ silage. Chemical conservation additive was based on formic acid, propionic acid, benzoic acid and ammonium formate content. In a model experiment the fresh brewer grains were used. A dry matter (DM) content of brewer grains was 187.4 g / kg. Six treatments with three repetitions per treatment were prepared. The treatments A1, A2 and A3 were not supplied by humidity absorbent. Treatment A1 was a control treatment without any additive. The treatments A2 and A3 were supplied by chemical conservation additive in a dose of 3 L per tonne and 6 L per tonne, respectively. The treatments B1, B2 and B3 were supplied by malt sprouts to reach DM content of conserved matter on level 320–350 g / kg. Moreover the treatments B2 and B3 were supplied by chemical additive with its dose 3 and 6 L per tonne. Model silages were evaluated after 8 months of conservation at average laboratory temperature 26–28 °C, from each treatment were the final laboratory samples taken and analyzed. During conservation of treatments B1, B2 and B3 were no drain recognized. From A1 treatment drained 1300 ml of waste fluid that is 145 L per tonne of conserved matter. That was significant (P < 0.01) the malt sprouts addition support the lactic acid production and eliminate acetic acid production. There was no propionic acid or butyric acid detected in silages with malt sprouts event in these silages were analyzed higher (P < 0.01) concentration of ammoniac. Chemical additive supplementation improved (P < 0.01) the pH value and water leach acidity. The results show the malt sprout addition eliminates waste fluid drain and improves fermentation process. The higher concentration of chemical additive (6 l / t) inhibited the fermentation process in our model experiment.

Separate cellular localizations of human T-lymphotropic virus 1 (HTLV-1) Env and glucose transporter type 1 (GLUT1) are required for HTLV-1 Env-mediated fusion and infection.

Interaction of the envelope glycoprotein (Env) of human T-lymphotropic virus 1 (HTLV-1) with the glucose transporter type 1 (GLUT1) expressed in target cells is essential for viral entry. This study found that the expression level of GLUT1 in virus-producing 293T cells was inversely correlated with HTLV-1 Env-mediated fusion activity and infectivity. Chimeric studies between GLUT1 and GLUT3 indicated that the extracellular loop 6 (ECL6) of GLUT1 is important for the inhibition of cell-cell fusion mediated by Env. When GLUT1 was translocated into the plasma membrane from intracellular storage sites by bafilomycin A1 (BFLA1) treatment in 293T cells, HTLV-1 Env-mediated cell fusion and infection also were inhibited without the overexpression of GLUT1, indicating that the localization of GLUT1 in intracellular compartments rather than in the plasma membrane is crucial for the fusion activity of HTLV-1 Env. Immunoprecipitation and laser scanning confocal microscopic analyses indicated that under normal conditions, HTLV-1 Env and GLUT1 do not colocalize or interact. BFLA1 treatment induced this colocalization and interaction, indicating that GLUT1 normally accumulates in intracellular compartments separate from that of Env. Western blot analyses of FLAG-tagged HTLV-1 Env in virus-producing cells and the incorporation of HTLV-1 Env in virus-like particles (VLPs) indicate that the processing of Env is inhibited by either overexpression of GLUT1 or BFLA1 treatment in virus-producing 293T cells. This inhibition probably is due to the interaction of the Env with GLUT1 in intracellular compartments. Taken together, separate intracellular localizations of GLUT1 and HTLV-1 Env are required for the fusion activity and infectivity of HTLV-1 Env. The deltaretrovirus HTLV-1 is a causative agent of adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Although HTLV-1 is a complex retrovirus that has accessory genes, no HTLV-1 gene product has yet been shown to regulate its receptor GLUT1 in virus-producing cells. In this study, we found that a large amount of GLUT1 or translocation of GLUT1 to the plasma membrane from intracellular compartments in virus-producing cells enhances the colocalization and interaction of GLUT1 with HTLV-1 Env, leading to the inhibition of cell fusion activity and infectivity. The results of our study suggest that GLUT1 normally accumulates in separate intracellular compartments from Env, which is indeed required for the proper processing of Env.

MEMPELAJARI CITARASA CUKO PEMPEK BUBUK DENGAN PENAMBAHAN ASAM SITRAT

This research aims to study the effect of the addition of citric acid powder to taste cuko pempek and get the best treatment . This research was conducted in the laboratory of the Faculty of Agriculture, University of Muhammadiyah Palembang in July 2013 to December 2013. This study used a randomized block design (RBD ) are arranged in non- factor factorial with the addition of citric acid treatment consisting of five levels of treatment and repeated four times. Parameters observed in this study is the chemical analysis includes analysis of citric acid and pH levels , for the physical test is soluble viscosity and velocity , while for organoleptic test includes flavor, color and aroma cuko pempek powder by using multiple comparison test. Lowest levels of citric acid present in the A1 treatment (addition of citric acid 3g) with an average value of 0.469% , the highest pH found in the A1 treatment (addition of citric acid 3g) with an average value of 5.70. The rate was highest viscosity at A5 treatment (addition of citric acid 15g) with an average value of 2,271 cPs and speed pempek vinegar powder dissolves. Velocity is highest late in the A1 treatment (addition of citric acid 3 g) with an average value of 6.29 seconds. Test multiple comparison test sensory adding citric acid to the taste, color and aroma cuko pempek powder , found that treatment A1 has the taste, color and aroma of different unreal with cuko pempek comparator (cuko pempek standard) with the lowest average value of 28.45, 37.9%, 32.76%, which is almost the same as cuko pempek comparator (cuko pempek standard). cuko pempek To obtain powder authors suggest you should use the A1 treatment is the treatment of the addition of 3g of citric acid in powder 1000g pempek cuko materials.

조방형 옥상녹화에서 노랑조팝나무의 활착에 미치는 토심별 유기질 토양개량제의 시용 효과

This study investigated the effects of soil depths and soil organic fertilizer application on the growth characteristics of Spiraea bumalda ‘Gold Mound’ in a extensive green roof system. The treatments were 3 soil depths (10, 15 and 25 cm) and 5 soil types in mixture of artificial soil and organic fertilizer. We measured plant height, leaf width, leaf length, number of flowers, visual quality and survival rate from March to October in 2011. The growing medium of 10 cm soil depth showed the highest plant growth in A1 (amended soil 100%), and the lowest plant growth in O1A4 (organic fertilizer 20% + amended soil 80%) treatment. In case of 15 cm soil depth, Spiraea bumalda ‘Gold Mound’ showed a high leaf length and visual quality in O1A2(organic fertilizer 33% + amended soil 67%) treatment and high leaf width and number of flowers in O1 (organic fertilizer 100%) treatment. A1 treatment without organic fertilizer showed the lowest leaf length and poorest visual quality, and O1A4 treatment showed the lowest plant height and lowest number of flowers. At soil depth 25 cm, O1A1 (organic fertilizer 50% + amended soil 50%) treatment showed greater plant height, visual quality and number of flowers than other treatments. The leaf length and leaf width were more effective in O1 treatment. A1 treatment showed a relatively low leaf length, leaf width and visual quality. The higher the organic conditioner, the better the plant growth. And, survival rates of Spiraea bumalda ‘Gold Mound’ showed 92%, 88% and 76% at soil depths of 25 cm, 15 cm and 10 cm, respectively, in this a extensive green roof system. Therefore, the results showed that the growth of Spiraea bumalda ‘Gold Mound’ was affected by both soil quality and soil depth. Different optimal mixtures of organic fertilizer and amended soil were determined, depending upon soil depth.

Ubiquitination and Degradation of CFTR by the E3 Ubiquitin Ligase MARCH2 through Its Association with Adaptor Proteins CAL and STX6

Golgi-localized cystic fibrosis transmembrane conductance regulator (CFTR)-associated ligand (CAL) and syntaxin 6 (STX6) regulate the abundance of mature, post-ER CFTR by forming a CAL/STX6/CFTR complex (CAL complex) that promotes CFTR degradation in lysosomes. However, the molecular mechanism underlying this degradation is unknown. Here we investigated the interaction of a Golgi-localized, membrane-associated RING-CH E3 ubiquitin ligase, MARCH2, with the CAL complex and the consequent binding, ubiquitination, and degradation of mature CFTR. We found that MARCH2 not only co-immunoprecipitated and co-localized with CAL and STX6, but its binding to CAL was also enhanced by STX6, suggesting a synergistic interaction. In vivo ubiquitination assays demonstrated the ubiquitination of CFTR by MARCH2, and overexpression of MARCH2, like that of CAL and STX6, led to a dose-dependent degradation of mature CFTR that was blocked by bafilomycin A1 treatment. A catalytically dead MARCH2 RING mutant was unable to promote CFTR degradation. In addition, MARCH2 had no effect on a CFTR mutant lacking the PDZ motif, suggesting that binding to the PDZ domain of CAL is required for MARCH2-mediated degradation of CFTR. Indeed, silencing of endogenous CAL ablated the effect of MARCH2 on CFTR. Consistent with its Golgi localization, MARCH2 had no effect on ER-localized ΔF508-CFTR. Finally, siRNA-mediated silencing of endogenous MARCH2 in the CF epithelial cell line CFBE-CFTR increased the abundance of mature CFTR. Taken together, these data suggest that the recruitment of the E3 ubiquitin ligase MARCH2 to the CAL complex and subsequent ubiquitination of CFTR are responsible for the CAL-mediated lysosomal degradation of mature CFTR.

土霉素对堆肥过程中酶活性和微生物群落代谢的影响

以猪粪和秸秆为试验材料，研究了土霉素对堆肥温度、种子发芽指数、C/N、纤维素酶、脲酶、过氧化氢酶以及微生物群落代谢的影响。结果表明，0 mg/kg（对照处理）的堆肥在第2天上升到 50 ℃以上，维持了 5 d，达到了无害化处理的要求。35 mg/kg土霉素处理（A1 处理）第4天升到 51.0 ℃，其高温期维持了 1 d。70 mg/kg 土霉素处理（A2 处理）第3天升到 50 ℃以上，高温期维持了 2 d。105 mg/kg 土霉素处理（A3 处理）和 140 mg/kg 土霉素处理（A4 处理）的温度在整个堆肥期间均未达到 50 ℃。在堆肥结束时各个处理的种子发芽指数均达到 80% 以上。CK（对照处理）、A1、A2、A3 和 A4 处理的 C/N 由 34.50 分别降为 16.64、16.07、19.48、18.45 和 19.83。堆肥的第1天，A1、A2、A3 和 A4 处理对纤维素酶活性的抑制率分别为 60.30%、21.30%、48.81% 和 76.05%，第3天，土霉素对纤维素酶活性起促进作用，在 4-30 d，A3、A4 处理对纤维素酶活性有抑制作用。在堆肥的前期（1-3 d），土霉素刺激脲酶活性，随着堆肥时间的延长，土霉素对脲酶活性由刺激变为抑制作用，在第18-30天 A3、A4 处理与 CK 相比有着显著的抑制作用。在堆肥第1天到第18天，土霉素基本上促进过氧化氢酶的活性，堆肥 18 d 之后土霉素对过氧化氢酶起着是抑制作用。用 Biolog（ECO Microplate）方法研究了土霉素对堆肥过程中微生物群落代谢的影响，结果表明，在升温期 CK 处理的平均颜色变化率AWCD（Average Well Color Development）在培养 60 h 之后大于其他处理，高温期 A2 处理的 AWCD 值最高，在降温期 CK 的 AWCD 值一直是最高的。对 Shannon 指数进行分析显示，堆肥的初始阶段土霉素降低微生物群落的功能多样性，随着时间的延长，土霉素增加微生物群落的功能多样性。对微生物利用六大类碳源分析表明，140 mg/kg 的土霉素浓度能够改变微生物利用碳源的种类。;An outdoor experiment was conducted to study the effect of oxytetraeyeline addition on composting temperature, seed germination index, ratio of carbon to nitrogen, cellulose, urease, catalase and microbial community metabolism during composting of pig manure mixed with wheat straw. The results showed that composting temperature was reached to 50 ℃ in the second day of composting and lasted for 5 days on treatment without addition of oxytetraeyeline (Control check,CK), which could meet the need of the requirement of harmlessness. The composting temperature was above 51.0 ℃ in the fourth day of composting with addition of 35 mg/kg oxytetracycline (A1 treatment), and the high temperature period maintained for 1 day. The temperature was above 50 ℃ in the third day of composting where 70 mg/kg oxytetracycline was added (A2 treatment), and the high temperature period only hold on for 2 days. The temperature could not reach to 50 ℃ over the whole time of composting on both A3 and A4 treatments where 105 mg/kg and 140 mg/kg of oxytetracycline were spiked respectively. The germination indices (<em>GI</em>) of seeds, which contained in the compost materials, were higher than 80% for all treatments at the end of the composting. The C/N ratios for all treatments were decreased from an initial value of 34.50 to 16.64, 16.07, 19.48, 18.45 and 19.83, respectively, when composting completed. Activities of cellulose on treatments those receiving oxytetracycline were inhibited by 60.30%, 21.30%, 48.81% and 76.05%, respectively, over CK treatment in the first day of the composting, and they were increased in the third day. Nevertheless, activities of cellulase were inhibited under A3 and A4 treatments compared with CK from day 4 to day 30. The activity of urease was increased by addition of oxytetracycline in the first 3 days, since then on, it was decreased. The higher concentrations of oxytetracycline (A3 and A4) had a significant inhibitory effect on activity of urease from day 18 to day 30. The catalase activity was enhanced generally by the addition of oxytetraeyeline from day 1 to day 18 compared to CK, from then on it was decreased over CK. The effect of oxytetracycline addition on microbial community metabolic profiles during composting was assayed with Biolog (ECO Microplate) method. The results showed that AWCD (Average Well Color Development) values of CK treatment were the highest after 60 h incubation during temperature raising period of the compost. The highest AWCD values were observed on A2 treatment during the high temperature period. AWCD values under CK were greater than other treatments over cooling period. Shannon index results suggested that the oxytetracycline addition could reduce the functional diversity of microbial communities in the initial stage of the composting, in contrast, it could increase the functional diversity of microbial communities at other stages of composting. The carbon source utilization data showed that the addition of 140 mg/kg of oxytetracycline could significantly change the types of carbon sources used by microorganisms.

The Wnt Coreceptor Ryk Regulates Wnt/Planar Cell Polarity by Modulating the Degradation of the Core Planar Cell Polarity Component Vangl2

The Wnt signaling pathways control many critical developmental and adult physiological processes. In vertebrates, one fundamentally important function of Wnts is to provide directional information by regulating the evolutionarily conserved planar cell polarity (PCP) pathway during embryonic morphogenesis. However, despite the critical roles of Wnts and PCP in vertebrate development and disease, little is known about the molecular mechanisms underlying Wnt regulation of PCP. Here, we have found that the receptor-like tyrosine kinase (Ryk), a Wnt5a-binding protein required in axon guidance, regulates PCP signaling. We show that Ryk interacts with Vangl2 genetically and biochemically, and such interaction is potentiated by Wnt5a. Loss of Ryk in a Vangl2(+/-) background results in classic PCP defects, including open neural tube, misalignment of sensory hair cells in the inner ear, and shortened long bones in the limbs. Complete loss of both Ryk and Vangl2 results in more severe phenotypes that resemble the Wnt5a(-/-) mutant in many aspects such as shortened anterior-posterior body axis, limb, and frontonasal process. Our data identify the Wnt5a-binding protein Ryk as a general regulator of the mammalian Wnt/PCP signaling pathway. We show that Ryk transduces Wnt5a signaling by forming a complex with Vangl2 and that Ryk regulates PCP by at least in part promoting Vangl2 stability. As human mutations in WNT5A and VANGL2 are found to cause Robinow syndrome and neural tube defects, respectively, our results further suggest that human mutations in RYK may also be involved in these diseases.

Drosophila TRPML Is Required for TORC1 Activation

Loss-of-function mutations in TRPML1 (transient receptor potential mucolipin 1) cause the lysosomal storage disorder, mucolipidosis type IV (MLIV). Here, we report that flies lacking the TRPML1 homolog displayed incomplete autophagy and reduced viability during the pupal period--a phase when animals rely on autophagy for nutrients. We show that TRPML was required for fusion of amphisomes with lysosomes, and its absence led to accumulation of vesicles of significantly larger volume and higher luminal Ca(2+). We also found that trpml(1) mutant cells showed decreased TORC1 (target of rapamycin complex 1) signaling and a concomitant upregulation of autophagy induction. Both of these defects in the mutants were reversed by genetically activating TORC1 or by feeding the larvae a high-protein diet. The high-protein diet also reduced the pupal lethality and the increased volume of acidic vesicles. Conversely, further inhibition of TORC1 activity by rapamycin exacerbated the mutant phenotypes. Finally, TORC1 exerted reciprocal control on TRPML function. A high-protein diet caused cortical localization of TRPML, and this effect was blocked by rapamycin. Our findings delineate the interrelationship between the TRPML and TORC1 pathways and raise the intriguing possibility that a high-protein diet might reduce the severity of MLIV.

Endocytosis and trafficking of human lactoferrin in macrophage-like human THP-1 cells1This article is part of a Special Issue entitled Lactoferrin and has undergone the Journal’s usual peer review process.

Different cell types have been reported to internalize lactoferrin (Lf) by specific or nonspecific receptors. Our studies focused on the endocytic pathway of human Lf in macrophage-like THP-1 cells. Lactoferrin was found to be internalized by THP-1 cells differentiated with phorbol myristate acetate. Incubation of cells with chlorpromazine and dansylcadaverine, inhibitors of clathrin-dependent endocytosis, led to a 50% inhibition of Lf internalization compared with untreated cells. Bafilomycin A1 and NH4Cl treatment also resulted in 40%–60% inhibition, respectively, suggesting that the internalization of Lf may partly be mediated by acidic endosome-like organelles. Endocytic uptake of Lf was also cholesterol-dependent, as shown by methyl-β-cyclodextrin or nystatin treatment of the cells prior to internalization. Partial colocalization of Lf and EEA-1, a marker specific for early endosomes, could be observed. Colocalization of Lf with a specific endoplasmic reticulum marker was also detected. Our results suggest that Lf is internalized mainly by the clathrin-dependent pathway in THP-1 cells and targets the ER. The physiological consequences of this intracellular trafficking will be the subject of future investigations.

LPS-induced Pellino3 degradation is mediated by p62-dependent autophagy

Background: In macrophages Toll-like receptor 4 (TLR4) is activated in response to lipopolysaccharide (LPS) and induces proinflammatory cytokine expression. Therefore, mechanisms terminating proinflammatory gene expression are important. Autophagy plays a central role in controlling innate immune responses by lysosomal degradation of signaling proteins, thus contributing to the resolution of inflammation. Autophagic proteins like p62 directly interact with molecules involved in the TLR4-signaling pathway, but a correlation with the IRAK E3 ligase and scaffold protein Pellino3 remains obscure. Hence, we are interested in elucidating the function of Pellino3 to prove our hypothesis that it is a key regulator in the TLR4-signaling cascade. Methods: We used the cecal ligation and puncture (CLP) mouse model causing polymicrobial sepsis to analyze Pellino3 protein and mRNA expression. Furthermore, we induced endotoxemia in RAW264.7 mouse macrophages by LPS treatment to verify in vivo experiments. Lentiviral Pellino3 knockdown in RAW264.7 macrophages was used for cytokine measurements at mRNA level. To analyze potential Pellino3 binding partners in TLR4-signaling by mass spectrometry (MS), we overexpressed FLAG-tagged Pellino3 in RAW264.7 macrophages, treated cells for 3, 6 and 24 hours with LPS and immunoprecipitated Pellino3 via its FLAG-tag. To consider Pellino3 degradation as a result of p62-mediated autophagy, we transiently knocked down p62 by siRNA in RAW264.7 macrophages and also pharmacologically blocked LPS-induced autophagy by Bafilomycin A1. Results: We demonstrated Pellino3 protein degradation in primary CD11b+ splenocytes after 24 hours following CLP operation and confirmed this in RAW264.7 macrophages after 24-hour LPS stimulation. Knockdown of Pellino3 attenuates proinflammatory cytokines, for example IL-6 mRNA, after 6 hours of LPS. Furthermore, we found by MS and verifying immunoprecipitation experiments that p62 is a Pellino3 binding partner, thus targeting Pellino3 for degradation. In line, both p62 knockdown and Bafilomycin A1 treatment prevent Pellino3 degradation, supporting an autophagic mechanism. Conclusion: Our observations highlight a regulatory role of Pellino3 on TLR4 signaling. Thus, antagonism of Pellino3 in the hyperinflammatory phase of sepsis may counteract the cytokine storm. Furthermore, stabilization of Pellino3 by inhibition of autophagy in the hypoinflammatory phase of sepsis may improve immunity. In consideration of these two conflictive sepsis phases, modulation of Pellino3 may provide a new strategy for the development of a therapy approach in sepsis.

Abstract 3463: The role of Atg13 in response to radiotherapy in cervical cancer cells

Abstract Introduction The role of autophagy protecting cancer cells against anti-cancer therapy, especially via the Beclin-1 axis, has become more clear. In cervical cancer, functionality of the Beclin-1 axis can be questioned, since promoter methylation and silencing of two components of this axis, C13ORF18 (Rubicon-like) and DAPK, is frequently observed. The involvement of another autophagy pathway, the ULK1 axis is less well studied in this tumor type. The ULK1 axis, consisting of ULK1, Atg13 and FIP200 is under control of mTOR. Here, we investigated the role of the essential component of the ULK1 complex, Atg13 in the response to (chemo)radiation in cervical cancer. Methods and Results Using immunohistochemistry we first analyzed expression of Atg13 in a large cohort of 375 pretreatment tissue samples of advantaged stage cervical cancer patients primarily treated with (chemo)radiation. Positive expression of Atg13 was more frequently observed in adenocarcinoma, known to have a poor response to chemoradiation, compared to squamous carcinoma (75% versus 56 % positive tumors, p&amp;lt; 0.03). Transient overexpression of GFP-tagged Atg13 in HEK293T kidney cells increased radiotherapy resistance (1.5-2 fold). Western blot showed that Atg13 was expressed in several cervical cancer cell lines, including the adenocarcinoma model HeLa. Downregulation of Atg13 expression with siRNA, resulted in a 2-fold increase in radiotherapy sensitivity in HeLa cells as determined with clonogenic assays. In HeLa-GFP-LC3 cells, we found that irradiation caused a strong increase in LC-3 containing autophagosomes, which co-localized with Atg13. The autophagy inhibitor bafilomycin A1 blocks the conversion of autophagosomes into autolysosomes. Bafilomycin A1 treatment indeed resulted in an accumulation of radiation-induced autophagosomes, demonstrating that autophagy was occurring after irradiation. In addition, HeLa cells were sensitized to irradiation, when autophagy was inhibited with bafilomycin A1. These results were confirmed using the less specific autophagy inhibitor 3-methyladenine and the autophagy inducer rapamycin (an mTOR inhibitor). Conclusion We showed that the Atg13 expression is related to the presence of cervical adenocarcinoma. Our in-vitro results indicate that the autophagic ULK1 pathway plays an important role in protecting cervical cancer cells to irradiation. Thus, inhibition of autophagy may be an interesting strategy to sensitize cervical adenocarcinoma to radiotherapy. 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 3463. doi:1538-7445.AM2012-3463

Effect of heavy metal Cu on compost materials property and oxidoreductase activity during pig waste composting

以猪粪和秸秆为主要试验材料, 添加不同浓度重金属Cu, 采取发酵罐处理方法, 在好氧高温条件下研究了重金属Cu对猪粪堆肥过程中多酚氧化酶、脱氢酶活性的变化, 以及堆腐过程堆体温度、堆料pH、胡敏酸E4/E6值变化的影响。结果表明: 不添加外源重金属Cu的CK处理在降温期堆温高于Cu含量为100 mg·kg^-1、500 mg·kg^-1的堆料; CK堆料pH最低点为5.94, E4/E6平均值为3.88。Cu含量为500 mg·kg^-1的堆料pH最高点为8.85, E4/E6平均值为3.68。Cu含量为100 mg·kg^-1的堆料升温快, 高温期持续时间长, 最高温度高于CK和Cu含量为500 mg·kg^-1的堆料, 能有效杀灭病原微生物, 达到无害化处理要求; 堆料pH控制在7~8之间, E4/E6大多情况低于CK和Cu含量为500 mg·kg^-1的堆料, 多酚氧化酶平均活性、脱氢酶平均活性均最高, 但在脱氢酶活性测定中表现出“抗性酶活性现象”。

Effect of serum amyloid A1 treatment on global gene expression in THP-1-derived macrophages

To investigate the effect of serum amyloid A1 (SAA1) on global gene expression in macrophages derived from THP-1 monocytes. Global genetic expression in THP-1-derived macrophages was determined using Illumina HT-12 microarray chips and the results were validated by real-time PCR. Cytokine levels in cellular supernatant were quantified by ELISA. In total, 55 genes were upregulated with fold difference greater than two when THP-1-derived macrophages were incubated with SAA1 for 8h. SAA1 is a strong cytokine inducer with significant upregulation of chemokines CCL1, CCL3, and CCL4 and this was confirmed by both real-time PCR and ELISA quantification. SAA1 also promotes the upregulation of genes involved in phagocytosis, anti-apoptosis, and tissue remodeling. SAA1 appears to play an important role during the immune response and in chronic inflammatory diseases through the stimulation of genes involved in cytokine production, phagocytosis, and anti-apoptosis.

A Novel Role for α-Tocopherol Transfer Protein (α-TTP) in Protecting against Chloroquine Toxicity

Chloroquine (CQ) is a widely prescribed anti-malarial agent and is also prescribed to treat autoimmune diseases. Clinical treatment with CQ is often accompanied by serious side effects such as hepatitis and retinopathy. As a weak base, CQ accumulates in intracellular acidic organelles, raises the pH, and induces osmotic swelling and permeabilization of acidic organelles, which account for CQ-induced cytotoxicity. We reported previously that CQ treatment caused α-tocopherol transfer protein (α-TTP), a gene product of familial vitamin E deficiency, to change its location from the cytosol to the surface of acidic organelles. Here we show that α-TTP plays a novel role in protecting against CQ toxicity both in vitro and in vivo. In the presence of CQ, rat hepatoma McARH7777 cells, which do not express α-TTP endogenously, showed more severe cytotoxicity, such as larger vacuolation of acidic organelles and caspase activation, than α-TTP transfectant cells. Similarly, α-TTP knockout mice showed more severe CQ toxicity, such as hepatotoxicity and retinopathy, than wild-type mice. These effects were not ameliorated by vitamin E supplementation. In contrast to bafilomycin A1 treatment, which prevents CQ accumulation in cells by raising the pH of acidic organelles, α-TTP expression prevented CQ accumulation without affecting the pH of acidic organelles. Taken together, our data suggest that α-TTP protects against CQ toxicity by preventing CQ accumulation in acidic organelles through a mechanism distinct from vitamin E transport.

EFFECT OF PHOSPHOLIPASE A1ON THE PHYSICOCHEMICAL AND FUNCTIONAL PROPERTIES OF HEN'S EGG YOLK, PLASMA AND GRANULES

ABSTRACT Fresh egg yolk was separated into plasma and granules. Then, the egg yolk and its fractions were enzymatically modified with phospholipase A1 (PLA1). Changes in physicochemical and functional properties were investigated. We observed that enzymatic treatment showed a significant impact on the properties of egg yolk and its fractions with regard to emulsifying properties, protein solubility and foaming properties. Meanwhile, the color changes and the microstructure and the thermal behavior of egg yolk, plasma and granules before and after enzymatic treatment were also studied. This study indicated that the existence of lysophospholipids and structural changes in egg yolk granules could be responsible for significant differences in the functional properties of untreated and PLA1-modified egg yolk, plasma and granules. PRACTICAL APPLICATIONS This study aimed to investigate the physicochemical and functional properties of egg yolk, including its fractions (plasma and granules), before and after phospholipase A1 (PLA1) treatment. Such modified egg yolk would have higher protein solubility, thermal stability and emulsifying properties, and present high resistance to extreme temperatures. As these properties of egg yolk powder were improved through PLA1 treatment, the application of PLA1 could make egg yolk have a good practical effect and extend the scope of application.

Polyphenolic compounds from Clusiaceae plants modulating angiogenesis and vascular endothelium

Polyphenolic compounds have created an increasing interest for their potency about cardiovascular diseases for several years1-2. Nevertheless, most of this research had been focused on polyphenolic compound such as flavanols (e.g. catechine from tea), anthocyanin (e.g. delphinidin from blueberry) and stilbenoides (e.g. resveratrol from grape).The present study was designed to screen the potent effect of polyphenolic compounds isolated from plants belonging to Clusiaceae family on endothelium. A huge number of polyphenols such as xanthones and coumarines have been identified from those species and some of them exhibiting various biological activities such as anti-inflammatory and antioxidant properties3-4. Their effect on endothelium, more particularly on angiogenesis, is not yet well-known. Firstly, we assessed the capacity of six molecules to induce endothelium-dependent relaxation in mice aortic rings involving nitric oxide production. Isocalolongic acid (A1) and 2-deprenylrheediaxanthone (A2) are able to increase NO production on endothelial cells and to induce endothelium-dependant relaxation. Then, we investigated the effects of these compounds on in vitro and ex vivo angiogenesis. We showed that A1 treatment promoted the formation of capillary-like network contrary to A2. Endothelial cell adhesion, migration and proliferation were decreased in presence of A2 whereas endothelial migration and proliferation were improved with A1 treatment. We could explain these results with the capacity of A1 to increase VEGF expression and for A2, to decrease ICAM-1 expression. Thus, the strategy used for the screening allows the detection of active molecules from Clusiaceae family that might be of therapeutic benefit in cardiovascular diseases5.