Metformin promotes survivin degradation through AMPK/PKA/GSK-3β-axis in non–small cell lung cancer

Metformin, a first-line antidiabetic drug, has been reported with anticancer activities in many types of cancer. However, its molecular mechanisms remain largely unknown. As a member of inhibitor of apoptosis proteins, survivin plays an important role in the regulation of cell death. In the present study, we investigated the role of survivin in metformin-induced anticancer activity in non–small cell lung cancer in vitro. Metformin mainly induced apoptotic cell death in A549 and H460 cell lines. It remarkably suppressed the expression of survivin, decreased the stability of this protein, then promoted its proteasomal degradation. Moreover, metformin greatly suppressed protein kinase A (PKA) activity and induced its downstream glycogen synthase kinase 3β (GSK-3β) activation. PKA activators, both 8-Br-cAMP and forskolin, significantly increased the expression of survivin. Consistently both GSK-3β inhibitor LiCl and siRNA restored the expression of survivin in lung cancer cells. Furthermore, metformin induced adenosine 5′-monophosphate-activated protein kinase (AMPK) activation. Suppression of the activity of AMPK with Compound C reversed the degradation of survivin induced by metformin, and meanwhile, restored the activity of PKA and GSK-3β. These results suggest that metformin kills lung cancer cells through AMPK/PKA/GSK-3β-axis–mediated survivin degradation, providing novel insights into the anticancer effects of metformin.     

β-arrestin 2 attenuates cardiac dysfunction in polymicrobial sepsis through gp130 and p38

Sepsis is an exaggerated systemic inflammatory response to persistent bacteria infection with high morbidity and mortality rate clinically. β-arrestin 2 modulates cell survival and cell death in different systems. However, the effect of β-arrestin 2 on sepsis-induced cardiac dysfunction is not yet known. Here, we show that β-arrestin 2 overexpression significantly enhances animal survival following cecal ligation and puncture (CLP)-induced sepsis. Importantly, overexpression of β-arrestin 2 in mice prevents CLP-induced cardiac dysfunction. Also, β-arrestin 2 overexpression dramatically attenuates CLP-induced myocardial gp130 and p38 mitogen-activated protein kinase (MAPK) phosphorylation levels following CLP. Therefore, β-arrestin 2 prevents CLP-induced cardiac dysfunction through gp130 and p38. These results suggest that modulation of β-arrestin 2 might provide a novel therapeutic approach to prevent cardiac dysfunction in patients with sepsis.     

Resonance Raman and FTIR spectroscopic characterization of the closed and open states of channelrhodopsin-1

Channelrhodopsin-1 from Chlamydomonas augustae (CaChR1) is a light-activated cation channel, which is a promising optogenetic tool. We show by resonance Raman spectroscopy and retinal extraction followed by high pressure liquid chromatography (HPLC) that the isomeric ratio of all-trans to 13-cis of solubilized channelrhodopsin-1 is with 70:30 identical to channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2). Critical frequency shifts in the retinal vibrations are identified in the Raman spectrum upon transition to the open (conductive P₂³⁸⁰) state. Fourier transform infrared spectroscopy (FTIR) spectra indicate different structures of the open states in the two channelrhodopsins as reflected by the amide I bands and the protonation pattern of acidic amino acids.     

Expression characterization of testicular DMRT1 in both Sertoli cells and spermatogenic cells of polyploid gibel carp

Dmrt1 has been suggested to play significant roles in sex determination and differentiation, but various expression patterns and cell types have been observed in the testis of vertebrates. Polyploid gibel carp, because of the multiple modes of unisexual gynogenesis and sexual reproduction, has become a unique case to explore the evolution of sex determination and differentiation. However, the sex-determination related genes in gibel carp have remained unknown. In this study, we identified and characterized 4 cDNAs of Dmrt1 genes. Subsequently, a polyclonal antibody specific to CagDMRT1 was prepared to examine its expression and distribution patterns at protein level. Significantly, both relative real-time PCR and Western blot detection confirmed predominant expression of CagDmrt1 in the adult testis of gibel carp. Moreover, the intensive expression of CagDMRT1 around spermatogenic cysts was revealed during spermatogenesis. And, following immunofluorescence co-localization of CagDMRT1 and CagVASA, a prominent CagDMRT1 expression in Sertoli cells and a mild CagDMRT1 expression in spermatogenic cells including spermatogonia and primary spermatocytes were clearly characterized. The CagDMRT1 signal in Sertoli cells is extensively distributed in both nuclei and cytoplasm, while the CagDMRT1 in spermatogonia and primary spermatocytes is mainly expressed in nuclei, and there is only the remained CagDMRT1 signal in the cytoplasm of secondary spermatocytes. These findings suggest that DMRT1 should be related to testis differentiation and spermatogenesis in gibel carp.     

Membrane fusogenic lysine type lipid assemblies possess enhanced NLRP3 inflammasome activation potency

Lysine (K) type cationic lipid with a propyl spacer and ditetradecyl hydrophobic moieties composing liposomes, K3C14, previously studied for gene delivery, were reported to activate the NLRP3 inflammasomes in human macrophages via the conventional phagolysosomal pathway. In this study, K3C16, a propyl spacer bearing lysine type lipids with dihexadecyl moieties (an extension of two hydrocarbon tail length) were compared with K3C14 as liposomes. Such a small change in tail length did not alter the physical properties such as size distribution, zeta potential and polydispersity index (PDI). The NLRP3 activation potency of K3C16 was shown to be 1.5-fold higher. Yet, the toxicity was minimal, whereas K3C14 has shown to cause significant cell death after 24 h incubation. Even in the presence of endocytosis inhibitors, cytochalasin D or dynasore, K3C16 continued to activate the NLRP3 inflammasomes and to induce IL-1β release. To our surprise, K3C16 liposomes were confirmed to fuse with the plasma membrane of human macrophages and CHO-K1 cells. It is demonstrated that the change in hydrophobic tail length by two hydrocarbons drastically changed a cellular entry route and potency in activating the NLRP3 inflammasomes.     

Proteomic analysis of Bacillus thuringiensis ΔphaC mutant BMB171/PHB(-1) reveals that the PHB synthetic pathway warrants normal carbon metabolism

A phaC knockout mutant from Bacillus thuringiensis (Bt) strain BMB171, named BMB171/PHB(-1), was constructed. A physiological and metabolic investigation and a proteomic analysis were conducted for both ΔphaC mutant and its parent strain. Grown in peptone medium with 5 gram glucose per liter as sole carbon source, BMB171/PHB(-1) produced various organic acids. Here the excreted pyruvate, citrate, lactate, acetate and glutamate were quantitatively analyzed. Deletion of phaC gene from the BMB171 strain resulted in 1) growth delay; 2) higher consumption of dioxigen but lower cell yield; 3) stagnation of pH movement; 4) overproduction of organic acids; 5) rapid descent of cell density in the stationary phase; and 6) a sporulation-deficient phenotype. Our proteomic study with qPCR reconfirmation reveals that the absence of PhaC led to a metabolic turmoil which showed repressed glycolysis, and over-expressed TCA cycle, various futile pathways and amino acid synthesis during vegetative growth. It is thus thought that B. thuringiensis BMB171 effectively regulated its carbon metabolism upon the presence of the functional PHB synthetic pathway. The presence of this pathway warrants a PHB-producing bacterium better surviving under different environmental conditions.     

多烯磷脂酰胆碱对β-淀粉样蛋白（Aβ 1—40）致阿尔茨海默病模型大鼠的治疗作用

目的：研究多烯磷脂酰胆碱（Polyene Phosphatiayl choline,PPC）对β-淀粉样蛋白（Apl-40）致阿尔茨海默病（Alzheimer＇s Dis．ease,AD）模型大鼠的治疗作用。方法：32只SD大鼠随机分为正常组、假手术组、模型组和处理组,海马内注射淀粉样蛋白（Aβ1—40）,制作大鼠阿尔茨海默病模型,处理组给予多烯磷脂酰胆碱。通过Moms水迷宫实验验检测各组大鼠认知行为学改变,海马组织学及免疫组化染色,观察多烯磷脂酰胆碱对阿尔茨海默病模型大鼠的影响,并进行统计学分析。结果：①Moms水迷宫实验,模型组与正常组、假手术组比较,学习和记忆潜伏期显著增加;处理组与模型组比较,学习和记忆潜伏期显著减少;组间比较差异有统计学意义（P〈0．05）,提示多烯磷脂酰胆碱使AD模型大鼠的认知行为学功能改善。（2）Nissl染色,模型组与正常对照组、假手术组组织学染色结果有显著性差异（P〈0．05）,PPC处理组与模型组比较有显著性差异（P〈0．05）,PPC处理组与正常组及假手术组比较也呈现出显著性差异（P〈0．05）;提示多烯磷脂酰胆碱可以减少神经元的凋亡。③β-淀粉样蛋白免疫组化染色,模型组与正常对照组、假手术组比较,Aβ沉积明显增加;PPC处理组与模型组比较,Aβ沉积范围明显缩小,PPC处理组与正常对照组、假手术组也呈现出显著性差。结论：多烯磷脂酰胆碱对淀粉样蛋白（Aβ1—40）致阿尔茨海默病模型大鼠有治疗作用。     

Hypoxia induces beta-amyloid in association with death of RGC-5 cells in culture

Beta-amyloid (Aβ) derived from amyloid precursor protein (APP) has been associated with retinal degeneration in Alzheimer’s disease (AD) and glaucoma. This study examined whether hypoxia exposure induces Aβ accumulation in RGC-5 cells. While levels of APP mRNA and protein significantly increased in the cells, elevated abundance of Aβ was also observed in cells and culture medium between 12 or 24 and 48 h after 5% O₂ hypoxia treatment. Additionally, there is a close relationship between induction of APP and Aβ and intracellular accumulation of ROS along with loss of mitochondrial membrane potential followed by the death of RGC-5 cells in culture under hypoxia. These results suggest a possible involvement of APP and Aβ in the death of RGCs challenged by hypoxia.     

Endogenous generation of sulfur dioxide in rat tissues

While sulfur dioxide (SO₂) has been previously known for its toxicological effects, it is now known to be produced endogenously in mammals from sulfur-containing amino acid l-cysteine. l-cysteine is catalyzed by cysteine dioxygenase (CDO) to l-cysteinesulfinate, which converts to β-sulfinylpyruvate through transamination by aspartate aminotransferase (AAT), and finally spontaneously decomposes to pyruvate and SO₂. The present study explored endogenous SO₂ production, and AAT and CDO distribution in different rat tissue. SO₂ content was highest in stomach, followed by tissues in the right ventricle, left ventricle, cerebral gray matter, pancreas, lung, cerebral white matter, renal medulla, spleen, renal cortex and liver. AAT activity and AAT1 mRNA expression were highest in the left ventricle, while AAT1 protein expression was highest in the right ventricle. AAT2 and CDO mRNA expressions were both highest in liver tissue. AAT2 protein expression was highest in the renal medulla, but CDO protein expression was highest in liver tissue. In all tissues, AAT1 and AAT2 were mainly distributed in the cytoplasm rather than the nucleus. These observed differences among tissues endogenously generating SO₂ and associated enzymes are important in implicating the discovery of SO₂ as a novel endogenous signaling molecule.