miR-488 mediates negative regulation of the AKT/NF-κB pathway by targeting Rac1 in LPS-induced inflammation

Endometritis is an inflammatory change in the structure of the endometrium due to various causes and is a common cause of infertility. Studies have confirmed that microRNAs (miRNAs) play a key regulatory role in various inflammatory diseases. However, the miRNA-mediated mechanism of endometrial inflammation induced by lipopolysaccharides (LPS) remains unclear. In this study, real-time quantitative polymerase chain reaction, Western blot analysis, immunofluorescence and Rac family small GTPase 1 (Rac1) interference were used to reveal the overexpression of miR-488 in the LPS-induced bovine uterus, and the effect of protein kinase B κ-light chain enhancement of the nuclear factor-activated B cells (AKT/NF-κB) pathway in intimal epithelial cells. The results showed that the expression of inflammatory cytokines such as interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α in the experimental group was significantly lower than that in the control group when miR-488 was overexpressed. Similar results were observed in the expression levels of p-AKT, p-IKK, and p-p65 proteins. In addition, the dual-luciferase reporter system confirmed that miRNA-488 may directly target the 3′-untranslated region of Rac1. In turn, the expression of Rac1 was inhibited. Moreover, the nuclear translocation of NF-κB was inhibited, and meanwhile, the accumulation of reactive oxygen species (ROS) in the cells was reduced. Thus, we provide basic data for the negative regulation of miR-488 in LPS-induced inflammation by inhibiting ROS production and the AKT/NF-kB pathway in intimal epithelial cells.     

Opposing Effects of Nitric Oxide on Different Connexins Expressed in the Vascular System

Gap junctions—clusters of intercellular channels built by connexins (Cx)—are thought to be important for vascular cell functions such as differentiation, control of tone, or growth. In the vascular system, gap junctions can be formed by four different connexins (Cx37, Cx40, Cx43 and Cx45). The permeability of these connexin-formed gap junctions determines the amount of intercellular coupling and can be modulated by several vasoactive substances such as prostacyclin or nitric oxide (NO). We demonstrate here that NO has specific effects on certain connexins. Using two different techniques—injection of a fluorescent dye in single cells as well as detection of the de novoformation of gap junctions by a flow cytometry based technique—we found that NO decreases the functional coupling in Cx37 containing gap junctions whereas it increases the de novoformation of gap junctions containing Cx40. We conclude that NO, in addition to its known vasomotor effects, has a novel role in controlling intercellular coupling resulting in opposing effects depending on the specific connexin expressed in the cells.     

Mitochondrial membrane permeabilisation by amyloid aggregates and protection by polyphenols

Alzheimer's disease and Parkinson's disease are neurodegenerative disorders characterised by the misfolding of proteins into soluble prefibrillar aggregates. These aggregate complexes disrupt mitochondrial function, initiating a pathophysiological cascade leading to synaptic and neuronal degeneration. In order to explore the interaction of amyloid aggregates with mitochondrial membranes, we made use of two in vitro model systems, namely: (i) lipid vesicles with defined membrane compositions that mimic those of mitochondrial membranes, and (ii) respiring mitochondria isolated from neuronal SH-SY5Y cells. External application of soluble prefibrillar forms, but not monomers, of amyloid-beta (Aβ₄₂ peptide), wild-type α-synuclein (α-syn), mutant α-syn (A30P and A53T) and tau-441 proteins induced a robust permeabilisation of mitochondrial-like vesicles, and triggered cytochrome c release (CCR) from isolated mitochondrial organelles. Importantly, the effect on mitochondria was shown to be dependent upon cardiolipin, an anionic phospholipid unique to mitochondria and a well-known key player in mitochondrial apoptosis. Pharmacological modulators of mitochondrial ion channels failed to inhibit CCR. Thus, we propose a generic mechanism of thrilling mitochondria in which soluble amyloid aggregates have the intrinsic capacity to permeabilise mitochondrial membranes, without the need of any other protein. Finally, six small-molecule compounds and black tea extract were tested for their ability to inhibit permeation of mitochondrial membranes by Aβ₄₂, α-syn and tau aggregate complexes. We found that black tea extract and rosmarinic acid were the most potent mito-protectants, and may thus represent important drug leads to alleviate mitochondrial dysfunction in neurodegenerative diseases.     

Mutational specificities of brominated DNA adducts catalyzed by human DNA polymerases

Chronic inflammation is known to lead to an increased risk for the development of cancer. Under inflammatory condition, cellular DNA is damaged by hypobromous acid, which is generated by myeloperoxidase and eosinophil peroxidase. The reactive brominating species induced brominated DNA adducts such as 8-bromo-2′-deoxyguanosine (8-Br-dG), 8-bromo-2′-deoxyadenosine (8-Br-dA), and 5-bromo-2′-deoxycytidine (5-Br-dC). These DNA lesions may be implicated in carcinogenesis. In this study, we analyzed the miscoding properties of the brominated DNA adducts generated by human DNA polymerases (pols). Site-specifically modified oligodeoxynucleotides containing a single 8-Br-dG, 8-Br-dA, or 5-Br-dC were used as a template in primer extension reactions catalyzed by human pols α, κ, and η. When 8-Br-dG-modified template was used, pol α primarily incorporated dCMP, the correct base, opposite the lesion, along with a small amount of one-base deletion (4.8%). Pol κ also promoted one-base deletion (14.2%), accompanied by misincorporation of dGMP (9.5%), dAMP (8.0%), and dTMP (6.1%) opposite the lesion. Pol η, on the other hand, readily bypassed the 8-Br-dG lesion in an error-free manner. As for 8-Br-dA and 5-Br-dC, all the pols bypassed the lesions and no miscoding events were observed. These results indicate that only 8-Br-dG, and not 5-Br-dC and 8-Br-dA, is a mutagenic lesion; the miscoding frequency and specificity vary depending on the DNA pol used. Thus, hypobromous acid-induced 8-Br-dG adduct may increase mutagenic potential at the site of inflammation.     

Transgenic Rodent Assay for Quantifying Male Germ Cell Mutant Frequency

De novo mutations arise mostly in the male germline and may contribute to adverse health outcomes in subsequent generations. Traditional methods for assessing the induction of germ cell mutations require the use of large numbers of animals, making them impractical. As such, germ cell mutagenicity is rarely assessed during chemical testing and risk assessment. Herein, we describe an in vivo male germ cell mutation assay using a transgenic rodent model that is based on a recently approved Organisation for Economic Co-operation and Development (OECD) test guideline. This method uses an in vitro positive selection assay to measure in vivo mutations induced in a transgenic λgt10 vector bearing a reporter gene directly in the germ cells of exposed males. We further describe how the detection of mutations in the transgene recovered from germ cells can be used to characterize the stage-specific sensitivity of the various spermatogenic cell types to mutagen exposure by controlling three experimental parameters: the duration of exposure (administration time), the time between exposure and sample collection (sampling time), and the cell population collected for analysis. Because a large number of germ cells can be assayed from a single male, this method has superior sensitivity compared with traditional methods, requires fewer animals and therefore much less time and resources.     

Tunneling nanotube (TNT)-like structures facilitate a constitutive, actomyosin-dependent exchange of endocytic organelles between normal rat kidney cells

Tunneling nanotube (TNT)-like structures are intercellular membranous bridges that mediate the transfer of various cellular components including endocytic organelles. To gain further insight into the magnitude and mechanism of organelle transfer, we performed quantitative studies on the exchange of fluorescently labeled endocytic structures between normal rat kidney (NRK) cells. This revealed a linear increase in both the number of cells receiving organelles and the amount of transferred organelles per cell over time. The intercellular transfer of organelles was unidirectional, independent of extracellular diffusion, and sensitive to shearing force. In addition, during a block of endocytosis, a significant amount of transfer sustained. Fluorescence microscopy revealed TNT-like bridges between NRK cells containing F-actin but no microtubules. Depolymerization of F-actin led to the disappearance of TNT and a strong inhibition of organelle exchange. Partial ATP depletion did not affect the number of TNT but strongly reduced organelle transfer. Interestingly, the myosin II specific inhibitor S-(−)-blebbistatin strongly induced both organelle transfer and the number of TNT, while the general myosin inhibitor 2,3-butanedione monoxime induced the number of TNT but significantly inhibited transfer. Taken together, our data indicate a frequent and continuous exchange of endocytic organelles between cells via TNT by an actomyosin-dependent mechanism.     

Interactions of fluorinated surfactants with diphtheria toxin T-domain: testing new media for studies of membrane proteins

The principal difficulty in experimental exploration of the folding and stability of membrane proteins (MPs) is their aggregation outside of the native environment of the lipid bilayer. To circumvent this problem, we recently applied fluorinated nondetergent surfactants that act as chemical chaperones. The ideal chaperone surfactant would 1), maintain the MP in solution; 2), minimally perturb the MP's structure; 3), dissociate from the MP during membrane insertion; and 4), not partition into the lipid bilayer. Here, we compare how surfactants with hemifluorinated (HFTAC) and completely fluorinated (FTAC) hydrophobic chains of different length compare to this ideal. Using fluorescence correlation spectroscopy of dye-labeled FTAC and HFTAC, we demonstrate that neither type of surfactant will bind lipid vesicles. Thus, unlike detergents, fluorinated surfactants do not compromise vesicle integrity even at concentrations far in excess of their critical micelle concentration. We examined the interaction of surfactants with a model MP, DTT, using a variety of spectroscopic techniques. Site-selective labeling of DTT with fluorescent dyes indicates that the surfactants do not interact with DTT uniformly, instead concentrating in the most hydrophobic patches. Circular dichroism measurements suggest that the presence of surfactants does not alter the structure of DTT. However, the cooperativity of the thermal unfolding transition is reduced by the presence of surfactants, especially above the critical micelle concentration (a feature of regular detergents, too). The linear dependence of DTT's enthalpy of unfolding on the surfactant concentration is encouraging for future application of (H)FTACs to determine the stability of the membrane-competent conformations of other MPs. The observed reduction in the efficiency of Förster resonance energy transfer between donor-labeled (H)FTACs and acceptor-labeled DTT upon addition of lipid vesicles indicates that the protein sheds the layer of surfactant during its bilayer insertion. We discuss the advantages of fluorinated surfactants over other types of solubilizing agents, with a specific emphasis on their possible applications in thermodynamic measurements.     

κ阿片肽受体激活对培养心肌细胞生长抑制的影响

目的 :通过体外培养乳鼠心肌细胞 ,观测κ阿片肽受体激活对心肌细胞生长的影响。方法 :对培养乳鼠心肌细胞分组给药 4 8h后 ,用结晶紫染色法测心肌细胞的增殖程度 ,用Lowry’s法测心肌细胞的蛋白质含量。 结果 :κ阿片肽受体激动剂U5 0 ,4 88H(0 .1μmol/L～ 10 μmol/L)对培养心肌细胞的增殖程度和蛋白质含量均有抑制作用并呈剂量依赖性 ;κ阿片肽受体拮抗剂nor binaltorphimine (nor BNI) (1μmol/L)对U5 0 ,4 88H(1μmol/L)的这些抑制效应具有相应的拮抗作用。结论 :阿片肽对心肌细胞生长具有负性调节作用 ,这种作用是通过激活κ阿片肽受体发挥的     

红外荧光素Alexa Fluor 647标记的2型腺相关病毒保持天然的感染活性

近年来红外荧光素的研发及相应的标记技术的进展在生物医学领域的研究中备受关注,通过与尖端仪器的结合在一系列生物样本的检测中得到广泛的应用. 然而,红外荧光标记技术在病毒感染生物学研究中的应用却少见报道.本文建立了Alexa Fluor 647（AF647）红外荧光素标记重组2型腺相关病毒（AAV2）的方法,并通过凝胶电泳的银染、红外荧光成像和透射电镜分析等手段,对标记病毒的结构和形态与未标记的病毒进行了细致的比较,同时进一步鉴定了其感染293T细胞后的转导活性.结果发现,AAV2在标记后的理化性质和感染活性均未发生改变. 应用狭缝印迹和激光共聚焦显微成像技术,分别对标记病毒的细胞摄取效率和在宿主细胞内的分布进行描述,为红外标记AAV2在研究中的应用提供了范例.AF647标记的 AAV2为腺相关病毒的感染动力学研究提供了新的手段,具有敏感度高、抗荧光漂白和低非特异干扰等优点,并且对直接有效地结合形态学分析和生化分子生物学检测,具有重要的应用价值.