Antioxidative and Prooxidative Activity of Caffeic Acid toward H2O2-induced DNA Strand Breakage Dependent on the State of the Fe Ion in the Medium

Hen egg lysozyme–carboxymethyl dextran (HEL–CMD) conjugate was prepared by using water-soluble carbodiimide as a model protein-acidic polysaccharide conjugate for improving the protein function. An acid-amide bond between HEL and CMD was confirmed by SDS-PAGE, isoelectric focusing and IR spectra. The molar ratio of CMD to HEL in the conjugate was 1:1. The isoelectric point of the conjugate was 5.5–6.0, which is much lower than that of HEL. Spectroscopic studies suggested that the conformation around the Trp residue had not changed but the α-helix content had decreased to about 1/3 that for native HEL. The conjugate maintained about 60% of the enzymatic activity of native HEL at 40–60°C, while it was about 1.4 times as active as native HEL at 4°C and 80°C. The conjugate was more stable to proteolysis than native HEL. The denaturation temperature of the conjugate was about 73°C, which is almost the same as that of native HEL. However, the enthalpy for denaturation of the conjugate was about 1/3 that of native HEL, which corresponds to the decrease in α-helix content.     

Optimization of Exopolysaccharide Overproduction by Lactobacillus confusus in Solid State Fermentation under High Salinity Stress

It is believed that high concentrations of sodium chloride (NaCl) suppress the biosynthesis of exopolysaccharide (EPS) in lactic acid bacteria (LAB). Nevertheless, overproduction of EPSs due to high salinity stress in solid state fermentation performed on an agar surface was demonstrated in this study using a response surface methodology via a central composite design (CCD). Under optimized conditions with NaCl 4.97% and sucrose 136.5 g/L at 40.79 h of incubation, the EPS yield was 259% (86.36 g/L of EPS), higher than the maximum yield produced with the modified MRS medium containing only 120 g/L of sucrose without NaCl (33.4 g/L of EPS). Biosynthesis of EPS by Lactobacillus confusus TISTR 1498 was independent of biomass production. Our results indicated that high salinity stress can enhance EPS production in solid state fermentation.     

植物间正相互作用对种群动态和群落结构的影响:基于个体模型的研究进展

植物间的相互作用对种群动态和群落结构有着重要的影响。大量的野外实验已经揭示了正相互作用(互利)在群落中的普遍存在及其重要性。为了弥补野外实验方法的不足, 模型方法被越来越多地应用于正相互作用及其生态学效应的研究中。该文基于个体模型研究, 探讨了植物间正相互作用对种群动态和群落结构的影响。介绍了植物间正相互作用的定义和发生机制、植物间相互作用与环境梯度的关系。正相互作用是指发生在相邻的植物个体之间, 至少对其中一个个体有益的相互作用。植物通过直接(生境改善或资源富集)或间接(协同防御等)作用使局部环境有利于邻体而发生正相互作用。胁迫梯度假说认为互利的强度或重要性随着环境胁迫度的增加而增加, 但是越来越多的经验研究认为胁迫梯度假说需要改进。以网格模型和影响域模型为例, 介绍了基于个体的植物间相互作用模型方法。基于个体模型, 对近年来国内外正相互作用对种群时间动态(如生物量-密度关系)、空间分布格局和群落结构(如群落生物量-物种丰富度关系)影响的研究进行了总结。指出未来的研究应集中在对正相互作用概念和机制的理解, 新的模型, 新的种群、群落, 甚至生态系统问题, 以及在全球变化背景下进行相关的研究。     

HIV gp120- and methamphetamine-mediated oxidative stress induces astrocyte apoptosis via cytochrome P450 2E1

HIV-1 glycoprotein 120 (gp120) is known to cause neurotoxicity via several mechanisms including production of proinflammatory cytokines/chemokines and oxidative stress. Likewise, drug abuse is thought to have a direct impact on the pathology of HIV-associated neuroinflammation through the induction of proinflammatory cytokines/chemokines and oxidative stress. In the present study, we demonstrate that gp120 and methamphetamine (MA) causes apoptotic cell death by inducing oxidative stress through the cytochrome P450 (CYP) and NADPH oxidase (NOX) pathways. The results showed that both MA and gp120 induced reactive oxygen species (ROS) production in concentration- and time-dependent manners. The combination of gp120 and MA also induced CYP2E1 expression at both mRNA (1.7±0.2- and 2.8±0.3-fold in SVGA and primary astrocytes, respectively) and protein (1.3±0.1-fold in SVGA and 1.4±0.03-fold in primary astrocytes) levels, suggesting the involvement of CYP2E1 in ROS production. This was further confirmed by using a selective inhibitor of CYP2E1, diallylsulfide (DAS), and CYP2E1 knockdown using siRNA, which significantly reduced ROS production (30–60%). As the CYP pathway is known to be coupled with the NOX pathway, including Fenton–Weiss–Haber (FWH) reaction, we examined whether the NOX pathway is also involved in ROS production induced by either gp120 or MA. Our results showed that selective inhibitors of NOX, diphenyleneiodonium (DPI), and FWH reaction, deferoxamine (DFO), also significantly reduced ROS production. These findings were further confirmed using specific siRNAs against NOX2 and NOX4 (NADPH oxidase family). We then showed that gp120 and MA both induced apoptosis (caspase-3 activity and DNA lesion using TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling) assay) and cell death. Furthermore, we showed that DAS, DPI, and DFO completely abolished apoptosis and cell death, suggesting the involvement of CYP and NOX pathways in ROS-mediated apoptotic cell death. In conclusion, this is the first report on the involvement of CYP and NOX pathways in gp120/MA-induced oxidative stress and apoptotic cell death in astrocytes, which has clinical implications in neurodegenerative diseases, including neuroAIDS.     

Manumycin A inhibits triple-negative breast cancer growth through LC3-mediated cytoplasmic vacuolation death

Therapy resistance can be attributed to acquisition of anti-apoptotic mechanisms by the cancer cells. Therefore, developing approaches that trigger non-apoptotic cell death in cancer cells to compensate for apoptosis resistance will help to treat cancer effectively. Triple-negative breast cancers (TNBC) are among the most aggressive and therapy resistant to breast tumors. Here we report that manumycin A (Man A), an inhibitor of farnesyl protein transferase, reduces cancer cell viability through induction of non-apoptotic, non-autophagic cytoplasmic vacuolation death in TNBC cells. Man A persistently induced cytoplasmic vacuolation and cell death through the expression of microtubule-associated protein 1 light chain 3 (LC3) and p62 proteins along with endoplasmic reticulum (ER) stress markers, Bip and CHOP, and accumulation of ubiquitinated proteins. As inhibitors of apoptosis and autophagy failed to block cytoplasmic vacuolation and its associated protein expression or cell death, it appears that these processes are not involved in the death induced by Man A. Ability of thiol antioxidant, NAC in blocking Man A-induced vacuolation, death and its related protein expression suggests that sulfhydryl homeostasis may be the target of Man A. Surprisingly, normal human mammary epithelial cells failed to undergo cytoplasmic vacuolation and cell death, and grew normally in presence of Man A. In conjunction with its in vitro effects, Man A also reduced tumor burden in vivo in xenograft models that showed extensive cytoplasmic vacuoles and condensed nuclei with remarkable increase in the vacuolation-associated protein expression together with increase of p21, p27, PTEN and decrease of pAkt. Interestingly, Man A-mediated upregulation of p21, p27 and PTEN and downregulation of pAkt and tumor growth suppression were also mimicked by LC3 knockdown in MDA-MB-231 cells. Overall, these results suggest novel therapeutic actions by Man A through the induction of non-apoptotic and non-autophagic cytoplasmic vacuolation death by probably affecting ER stress, LC3 and p62 pathways in TNBC but not in normal mammary epithelial cells.     

Endoplasmic reticulum stress sensitizes pancreatic beta cells to interleukin-1β-induced apoptosis via Bim/A1 imbalance

We have recently shown that the crosstalk between mild endoplasmic reticulum (ER) stress and low concentrations of the pro-inflammatory cytokine interleukin (IL)-1β exacerbates beta cell inflammatory responses via the IRE1α/XBP1 pathway. We presently investigated whether mild ER stress also sensitizes beta cells to cytokine-induced apoptosis. Cyclopiazonic acid (CPA)-induced ER stress enhanced the IL-1β apoptosis in INS-1E and primary rat beta cells. This was not prevented by XBP1 knockdown (KD), indicating the dissociation between the pathways leading to inflammation and cell death. Analysis of the role of pro- and anti-apoptotic proteins in cytokine-induced apoptosis indicated a central role for the pro-apoptotic BH3 (Bcl-2 homology 3)-only protein Bim (Bcl-2-interacting mediator of cell death), which was counteracted by four anti-apoptotic Bcl-2 (B-cell lymphoma-2) proteins, namely Bcl-2, Bcl-XL, Mcl-1 and A1. CPA+IL-1β-induced beta cell apoptosis was accompanied by increased expression of Bim, particularly the most pro-apoptotic variant, small isoform of Bim (Bim_S), and decreased expression of A1. Bim silencing protected against CPA+IL-1β-induced apoptosis, whereas A1 KD aggravated cell death. Bim inhibition protected against cell death caused by A1 silencing under all conditions studied. In conclusion, mild ER stress predisposes beta cells to the pro-apoptotic effects of IL-1β by disrupting the balance between pro- and anti-apoptotic Bcl-2 proteins. These findings link ER stress to exacerbated apoptosis during islet inflammation and provide potential mechanistic targets for beta cell protection, namely downregulation of Bim and upregulation of A1.     

Choline kinase inhibition induces exacerbated endoplasmic reticulum stress and triggers apoptosis via CHOP in cancer cells

Endoplasmic reticulum (ER) is a central organelle in eukaryotic cells that regulates protein synthesis and maturation. Perturbation of ER functions leads to ER stress, which has been previously associated with a broad variety of diseases. ER stress is generally regarded as compensatory, but prolonged ER stress has been involved in apoptosis induced by several cytotoxic agents. Choline kinase α (ChoKα), the first enzyme in the Kennedy pathway, is responsible for the generation of phosphorylcholine (PCho) that ultimately renders phosphatidylcholine. ChoKα overexpression and high PCho levels have been detected in several cancer types. Inhibition of ChoKα has demonstrated antiproliferative and antitumor properties; however, the mechanisms underlying these activities remain poorly understood. Here, we demonstrate that ChoKα inhibitors (ChoKIs), MN58b and RSM932A, induce cell death in cancer cells (T47D, MCF7, MDA-MB231, SW620 and H460), through the prolonged activation of ER stress response. Evidence of ChoKIs-induced ER stress includes enhanced production of glucose-regulated protein, 78 kDa (GRP78), protein disulfide isomerase, IRE1α, CHOP, CCAAT/enhancer-binding protein beta (C/EBPβ) and TRB3. Although partial reduction of ChoKα levels by small interfering RNA was not sufficient to increase the production of ER stress proteins, silencing of ChoKα levels also show a decrease in CHOP overproduction induced by ChoKIs, which suggests that ER stress induction is due to a change in ChoKα protein folding after binding to ChoKIs. Silencing of CHOP expression leads to a reduction in C/EBPβ, ATF3 and GRP78 protein levels and abrogates apoptosis in tumor cells after treatment with ChoKIs, suggesting that CHOP maintains ER stress responses and triggers the pro-apoptotic signal. Consistent with the differential effect of ChoKIs in cancer and primary cells previously described, ChoKIs only promoted a transient and moderated ER stress response in the non-tumorogenic cells MCF10A. In conclusion, pharmacological inhibition of ChoKα induces cancer cell death through a mechanism that involves the activation of exaggerated and persistent ER stress supported by CHOP overproduction.     

Drosophila ref(2)P is required for the parkin-mediated suppression of mitochondrial dysfunction in pink1 mutants

Autophagy is a critical regulator of organellar homeostasis, particularly of mitochondria. Upon the loss of membrane potential, dysfunctional mitochondria are selectively removed by autophagy through recruitment of the E3 ligase Parkin by the PTEN-induced kinase 1 (PINK1) and subsequent ubiquitination of mitochondrial membrane proteins. Mammalian sequestrome-1 (p62/SQSTM1) is an autophagy adaptor, which has been proposed to shuttle ubiquitinated cargo for autophagic degradation downstream of Parkin. Here, we show that loss of ref(2)P, the Drosophila orthologue of mammalian P62, results in abnormalities, including mitochondrial defects and an accumulation of mitochondrial DNA with heteroplasmic mutations, correlated with locomotor defects. Furthermore, we show that expression of Ref(2)P is able to ameliorate the defects caused by loss of Pink1 and that this depends on the presence of functional Parkin. Finally, we show that both the PB1 and UBA domains of Ref(2)P are crucial for mitochondrial clustering. We conclude that Ref(2)P is a crucial downstream effector of a pathway involving Pink1 and Parkin and is responsible for the maintenance of a viable pool of cellular mitochondria by promoting their aggregation and autophagic clearance.     

Embryonic response to long‐term exposure of the marine crustacean Nephrops norvegicus to ocean acidification and elevated temperature

Due to anthropogenic CO₂ emissions, our oceans have gradually become warmer and more acidic. To better understand the consequences of this, there is a need for long‐term (months) and multistressor experiments. Earlier research demonstrates that the effects of global climate change are specific to species and life stages. We exposed berried Norway lobsters (Nephrops norvegicus), during 4 months to the combination of six ecologically relevant temperatures (5–18°C) and reduced pH (by 0.4 units). Embryonic responses were investigated by quantifying proxies for development rate and fitness including: % yolk consumption, mean heart rate, rate of oxygen consumption, and oxidative stress. We found no interactions between temperature and pH, and reduced pH only affected the level of oxidative stress significantly, with a higher level of oxidative stress in the controls. Increased temperature and % yolk consumed had positive effects on all parameters except on oxidative stress, which did not change in response to temperature. There was a difference in development rate between the ranges of 5–10°C (Q₁₀: 5.4) and 10–18°C (Q₁₀: 2.9), implicating a thermal break point at 10°C or below. No thermal limit to a further increased development rate was found. The insensitivity of N. norvegicus embryos to low pH might be explained by adaptation to a pH‐reduced external habitat and/or internal hypercapnia during incubation. Our results thus indicate that this species would benefit from global warming and be able to withstand the predicted decrease in ocean pH in the next century during their earliest life stages. However, future studies need to combine low pH and elevated temperature treatments with hypoxia as hypoxic events are frequently and increasingly occurring in the habitat of benthic species.