Bile salt-induced apoptosis involves NADPH oxidase isoform activation

BACKGROUND & AIMS: Hydrophobic bile salts trigger a rapid oxidative stress response as an upstream event of CD95 activation and hepatocyte apoptosis. METHODS: The underlying mechanisms were studied by Western blot, immunocytochemistry, protein knockdown, and fluorescence resonance energy transfer microscopy in rat hepatocytes and human hepatoma cell line 7 (Huh7). RESULTS: The rapid oxidative stress formation in response to taurolithocholate-3-sulfate (TLCS) was inhibited by diphenyleneiodonium, apocynin, and neopterin, suggestive for the involvement of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases. TLCS induced a rapid serine phosphorylation of the regulatory subunit p47phox, which was sensitive to inhibition of sphingomyelinase and protein kinase Czeta (PKCzeta). Inhibitors of p47phox phosphorylation and p47phox protein knockdown abolished the TLCS-induced oxidative stress response and blunted subsequent CD95 activation. Consequences of TLCS-induced oxidative stress were c-Jun-N-terminal kinase activation and Yes-dependent activation of the epidermal growth factor receptor (EGFR), followed by EGFR-catalyzed CD95 tyrosine phosphorylation, formation of the death-inducing signaling complex, and execution of apoptosis. As shown by fluorescence resonance energy transfer experiments in Huh7 cells, TLCS induced a c-Jun-N-terminal kinase-dependent EGFR/CD95 association in the cytosol and trafficking of this protein complex to the plasma membrane. Inhibition of EGFR tyrosine kinase activity by AG1478 allowed for cytosolic EGFR/CD95 association, but prevented targeting of the EGFR/CD95 complex to the plasma membrane. Both processes, and TLCS-induced Yes and EGFR activation, were sensitive to inhibition of sphingomyelinase, PKCzeta, or NADPH oxidases. CONCLUSIONS: The data suggest that hydrophobic bile salts activate NADPH oxidase isoforms with the resulting oxidative stress response triggering activation of the CD95 system and apoptosis.     

表达H9N2禽流感病毒HA1蛋白的转基因和缓艾美耳球虫的构建

H9N2亚型禽流感病毒已经被证实可以传播到人类,对它存在的潜在危害已受到越来越多的关注。因此,研究安全而有效的H9N2禽流感病毒疫苗已迫在眉睫。为研究和缓艾美耳球虫作为活载体表达及运输禽流感病毒抗原的潜能,本研究构建了可稳定表达H9N2禽流感病毒HA1蛋白的转基因和缓艾美耳球虫。利用黄色荧光蛋白（YFP）及融合的乙胺嘧啶抗性基因（DHFR-TSm2m3）作为报告基因及筛选基因,我们将表达H9N2禽流感病毒HA1抗原的质粒载体核转球虫子孢子,接种鸡后在乙胺嘧啶药物的选择压力下进行筛选并连续传代获得转基因和缓艾美耳球虫系。利用染色体步移和免疫印迹证实了HA1基因的成功插入和表达;利用间接免疫荧光证实HA1蛋白定位于球虫子孢子细胞膜表面和头部。研究进一步发现,转基因球虫的繁殖力与野生球虫相当,感染后亦于第6d达到排卵囊高峰。该转基因和缓艾美耳球虫株具有作为疫苗活载体的潜能。     

Perchlorate transport and inhibition of the sodium iodide symporter measured with the yellow fluorescent protein variant YFP-H148Q/I152L

Perchlorate is an environmental contaminant that impairs thyroid function by interacting with the sodium iodide symporter (NIS), the transporter responsible for iodide uptake in the thyroid gland. Perchlorate is well known as a competitive inhibitor of iodide transport by NIS, and recent evidence demonstrates that NIS can also transport perchlorate. In this study, we evaluated the yellow fluorescent protein (YFP) variant YFP-H148Q/I152L, as a genetically encodable biosensor of intracellular perchlorate concentration monitored by real-time fluorescence microscopy. Fluorescence of recombinant YFP-H148Q/I152L was suppressed by perchlorate and iodide with similar affinities of 1.2 mM and 1.6 mM, respectively. Perchlorate suppressed YFP-H148Q/I152L fluorescence in FRTL-5 thyroid cells and NIS-expressing COS-7 cells, but had no effect on COS-7 cells lacking NIS. Fluorescence changes in FRTL-5 cells were Na⁺-dependent, consistent with the Na⁺-dependence of NIS activity. Perchlorate uptake in FRTL-5 cells resulted in 10-fold lower intracellular concentrations than iodide uptake, and was characterized by a higher affinity (K_m 4.6 μM for perchlorate and 34.8 μM for iodide) and lower maximal velocity (V_max 6.8 μM/s for perchlorate and 39.5 μM/s for iodide). Perchlorate also prevented iodide-induced changes in YFP-H148Q/I152L fluorescence in FRTL-5 cells, with half-maximal inhibition occurring at 1.1-1.6 μM. In conclusion, YFP-H148Q/I152L detects perchlorate accumulation by thyroid and other NIS-expressing cells, and reveals differences in the kinetics of perchlorate versus iodide transport by NIS.     

In vivo two‐photon imaging of structural dynamics in the spinal dorsal horn in an inflammatory pain model

Two‐photon microscopy imaging has recently been applied to the brain to clarify functional and structural synaptic plasticity in adult neural circuits. Whereas the pain system in the spinal cord is phylogenetically primitive and easily exhibits behavioral changes such as hyperalgesia in response to inflammation, the structural dynamics of dendrites has not been analysed in the spinal cord mainly due to tissue movements associated with breathing and heart beats. Here we present experimental procedures to prepare the spinal cord sufficiently to follow morphological changes of neuronal processes in vivo by using two‐photon microscopy and transgenic mice expressing fluorescent protein specific to the nervous system. Structural changes such as the formation of spine‐like structures and swelling of dendrites were observed in the spinal dorsal horn within 30 min after the multiple‐site injections of complete Freund's adjuvant (a chemical irritant) to a leg, and these changes continued for 5 h. Both AMPA and N‐methyl‐D‐aspartate receptor antagonists, and gabapentin, a presynaptic Ca²⁺ channel blocker, completely suppressed the inflammation‐induced structural changes in the dendrites in the spinal dorsal horn. The present study first demonstrated by in vivo two‐photon microscopy imaging that structural synaptic plasticity occurred in the spinal dorsal horn immediately after the injection of complete Freund's adjuvant and may be involved in inflammatory pain. Furthermore, acute inflammation‐associated structural changes in the spinal dorsal horn were shown to be mediated by glutamate receptor activation.     

Interaction between the insulin receptor and Grb14: a dynamic study in living cells using BRET

Grb14 is a molecular adaptor that binds to the activated insulin receptor (IR) and negatively regulates insulin signaling. We have studied the dynamics of interaction of the IR with Grb14, in real time, in living HEK cells, using bioluminescence resonance energy transfer (BRET). Insulin rapidly and dose-dependently stimulated this interaction. Removing insulin from the incubation medium only resulted in a modest decrease in BRET signal, indicating that the interaction between the IR and Grb14 can remain long after insulin stimulus has disappeared. BRET saturation experiments indicated that insulin markedly increases the affinity between IR and Grb14, resulting in recruitment of the adaptor to the activated IR. In addition, using both BRET and co-immunoprecipitation experiments, we demonstrated that insulin induced the dimerization of Grb14, most likely as a result of simultaneous binding of two Grb14 molecules on the activated IR. We also investigated the relationships between IR, Grb14 and the protein tyrosine phosphatase PTP1B. We observed that insulin-induced BRET between the IR and PTP1B was markedly reduced by Grb14, suggesting that Grb14 regulated this interaction in living cells. Using site-specific antibodies against phosphorylated tyrosines of the insulin receptor, we showed that Grb14 protected the three tyrosines of the kinase loop from dephosphorylation by PTP1B, while favouring dephosphorylation of tyrosine 972. This resulted in decreased IRS-1 binding to the IR and decreased activation of the ERK pathway. Our work suggests that Grb14 may regulate signalling through the insulin receptor by controlling its tyrosine-dephosphorylation in a site-specific manner.     

Drebrin controls neuronal migration through the formation and alignment of the leading process

Formation of a functional nervous system requires neurons to migrate to the correct place within the developing brain. Tangentially migrating neurons are guided by a leading process which extends towards the target and is followed by the cell body. How environmental cues are coupled to specific cytoskeletal changes to produce and guide leading process growth is unknown. One such cytoskeletal modulator is drebrin, an actin-binding protein known to induce protrusions in many cell types and be important for regulating neuronal morphology.Using the migration of oculomotor neurons as a model, we have shown that drebrin is necessary for the generation and guidance of the leading process. In the absence of drebrin, leading processes are not formed and cells fail to migrate although axon growth and pathfinding appear grossly unaffected. Conversely, when levels of drebrin are elevated the leading processes turn away from their target and as a result the motor neuron cell bodies move along abnormal paths within the brain. The aberrant trajectories were highly reproducible suggesting that drebrin is required to interpret specific guidance cues. The axons and growth cones of these neurons display morphological changes, particularly increased branching and filopodial number but despite this they extend along normal developmental pathways.Collectively these results show that drebrin is initially necessary for the formation of a leading process and subsequently for this to respond to navigational signals and grow in the correct direction. Furthermore, we have shown that the actions of drebrin can be segregated within individual motor neurons to direct their migration independently of axon guidance.     

Flow cytometric analysis of CFP-YFP FRET as a marker for in vivo protein-protein interaction

The technique of observing fluorescence resonance energy transfer (FRET) between a cyan fluorescent protein (CFP) fusion protein and a yellow fluorescent protein (YFP) fusion protein has been used in numerous studies as a reliable indicator of protein-protein interaction. Moreover, because CFP and YFP fusions generally retain activity and maintain their normal subcellular localizations, the detection of CFP-YFP FRET in live cells typically reflects the steady-state association of functional proteins in their native states. Although CFP-YFP FRET can also be monitored by fluorimetry and fluorescence microscopy, the ability of flow cytometry to rapidly acquire multiparameter fluorescence data on a large number of individual cells provides several important advantages. The analysis of CFP-YFP FRET on a cell-by-cell basis allows for a sensitive and highly rigorous assessment of protein interaction, and the large number of cells that can be examined by flow cytometry provides for a high degree of statistical confidence. In addition, simple, yet stringent, gating-based analyses of FRET can be performed on a flow cytometer simultaneously with data collection, allowing FRET-based cell sorting that can be used in high-throughput screens to identify interacting proteins. As a brief review of flow cytometric CFP-YFP FRET analysis, this article outlines a typical instrument configuration, describes the required controls, explains how gating is performed, and discusses the basic physical principles behind FRET as they relate to the successful design and interpretation of protein interaction experiments.     

Identification of recombinant antibodies against multiple distinct toll-like receptors by homolog mining a single immune scFv phage library

The generation of recombinant single-chain antibodies from either non-immune or immune phage display antibody libraries is an effective means to obtain high affinity antibodies against a specific target. Non-immune libraries contain a wide variety of antibodies but these are often low affinity. Immune libraries contain a high frequency of high affinity antibodies, but are typically limited to a single antigen. Due to the V_H and V_L recombination that occurs during antibody library construction, we hypothesized that an immune antibody library produced against one member of a protein family would contain antibodies specific for other members of the same protein family. Here, we tested this hypothesis by mining an existing anti-human Toll-like receptor-2 (hTLR2) antibody library for antibodies specific for other members of the TLR family. This procedure, referred to as homolog mining, proved to be effective. Using a cell-based system to pan and screen the anti-TLR2 library, we identified single chain antibodies specific for three of the four hTLR2 homologs we targeted. The antibodies identified, anti-murine TLR2, anti-hTLR5, and anti-hTLR6, bind specifically to their target, with no cross-reactivity to hTLR2 or other TLRs tested. These results demonstrate that combinatorial re-assortment of V_H and V_L fragments from multiple sources during Ab library construction increases Ab repertoire complexity, allowing antibody libraries produced by immunization with one antigen to be used to obtain antibodies specific to related antigens. The principle of homolog mining may be extended to other protein families and will facilitate and accelerate antibody production processes.     

Vav-Cre介导的YFP报告基因系统标记小鼠NK细胞的

目的 探索Vav-Cre介导的YFP报告基因系统标记小鼠体内自然杀伤（NK）细胞的特异性和效率。方法 通过基因型分型筛选ROSA26R-YFP与Vav-Cre小鼠杂交后代中双阳性基因型小鼠,流式细胞术分析免疫器官淋巴结、脾、胸腺以及骨髓细胞的黄色荧光蛋白（YFP）表达效率,通过细胞表面抗体标记淋巴结、脾及骨髓的NK细胞,流式细胞术分析NK细胞群体中YFP阳性细胞百分比。结果 ROSA26R-YFP与Vav-Cre小鼠杂交后代共17只,双阳性基因型小鼠（ROSA26R-YFP（+/-）Vav-Cre）11只;流式细胞术分析淋巴结、脾、胸腺、骨髓细胞中YFP阳性细胞的百分比（%）分别为73.87±1.51、56.07±1.47、86.17±1.74、53.60±3.56,与阴性对照组相应器官分别为0.27±0.01、1.33±0.91、0.11±0.01,0.29±0.03相比,差异有统计学意义（均P<0.01）,两种类型小鼠非免疫器官肾中均无明显YFP 表达（双阳性鼠0.72%±0.43,对照鼠0.92%±0.27,P>0.05）;淋巴结、脾和骨髓中NK细胞YFP阳性百分比（%）76.94±0.84、81.66±1.18、88.92±0.77,与阴性对照组比较均明显增高（均P<0.01）。 结论 Vav-Cre介导的YFP报告基因系统标记小鼠体内NK细胞具有特异性及高效性。     

Organ of Corti explants direct tonotopically graded morphology of spiral ganglion neurons in vitro

The spiral ganglion is a compelling model system to examine how morphological form contributes to sensory function. While the ganglion is composed mainly of a single class of type I neurons that make simple one‐to‐one connections with inner hair cell sensory receptors, it has an elaborate overall morphological design. Specific features, such as soma size and axon outgrowth, are graded along the spiral contour of the cochlea. To begin to understand the interplay between different regulators of neuronal morphology, we cocultured neuron explants with peripheral target tissues removed from distinct cochlear locations. Interestingly, these “hair cell microisolates” were capable of both increasing and decreasing neuronal somata size, without adversely affecting survival. Moreover, axon characteristics elaborated de novo by the primary afferents in culture were systematically regulated by the sensory endorgan. Apparent peripheral nervous system (PNS)‐like and central nervous system (CNS)‐like axonal profiles were established in our cocultures allowing an analysis of putative PNS/CNS axon length ratios. As predicted from the in vivo organization, PNS‐like axon bundles elaborated by apical cocultures were longer than their basal counterparts and this phenotype was methodically altered when neuron explants were cocultured with microisolates from disparate cochlear regions. Thus, location‐dependent signals within the organ of Corti may set the “address” of neurons within the spiral ganglion, allowing them to elaborate the appropriate tonotopically associated morphological features in order to carry out their signaling function. J. Comp. Neurol. 524:2182–2207, 2016. © 2015 Wiley Periodicals, Inc.