Proteomics study on the protective mechanism of soybean isoflavone against inflammation injury of bovine mammary epithelial cells induced by Streptococcus agalactiae

This study aimed to verify the anti-inflammatory effect of soybean isoflavones (SI) on the inflammatory response induced by Streptococcus agalactiae (S. agalactiae) of bovine mammary epithelial cells (bMECs) and to elucidate its possible mechanism. BMECs were pretreated with SI of different concentrations (20, 40, 60, 80, 100 μg/mL) for 0.5, 3, 6, 9, 12, 15, 18, 24 h. And then, S. agalactiae was used to infect bMECs for 6 h (MOI = 50:1) to establish the inflammation model. Cell viability, growth curves of S. agalactiae, cytotoxicity, and S. agalactiae invasion rate were determined. A proteomics technique was used to further detect differential proteins and enrichment pathways. SI (40 μg/mL) improved the viability of bMECs at 12 h (p < 0.05) and 60 and 80 μg/mL of SI greater (p < 0.01). Moreover, 60 μg/mL of SI protects cells from bacterial damage (p < 0.05). SI could inhibit S. agalactiae growth and internalization into bMECs in a time- and dose-dependent manner. In addition, proteomics results showed that 133 proteins were up-regulated and 89 proteins were down-regulated significantly. The differentially significantly expressed proteins (DSEPs) were mainly related to cell proliferation, differentiation, apoptosis, and migration. GO annotation showed that 222 DSEPs were divided into 23 biological processes (BP) terms, 14 cell components (CC) terms, and 12 molecular functions (MF) terms. DSEPs were significantly enriched in 10 pathways, of which the immune pathway was the main enrichment pathway.     

Sodium benzoate exposure downregulates the expression of tyrosine hydroxylase and dopamine transporter in dopaminergic neuronsin developing zebrafish

BACKGROUND: Recent data have demonstrated that treatment with sodium benzoate (SB) leads to significant developmental defects in motor neuron axons and neuromuscular junctions in zebrafish larvae, thereby implying that SB can be neurotoxic. This study examined whether SB affects the development of dopaminergic neurons in the zebrafish brain. METHODS: Zebrafish embryos were exposed to different concentrations of SB for various durations, during which the survival rates were recorded, the expression of tyrosine hydroxylase (TH) and dopamine transporter (DAT) in the neurons in the ventral diencephalon were detected by in situ hybridization and immunofluorescence, and the locomotor activity of larval zebrafish was measured. RESULTS: The survival rates were significantly decreased with the increase of duration and dose of SB-treatment. Compared to untreated clutch mates (untreated controls), treatment with SB significantly downregulated expression of TH and DAT in neurons in the ventral diencephalon of 3-day post-fertilization (dpf) zebrafish embryos in a dose-dependent manner. Furthermore, there was a marked decrease in locomotor activity in zebrafish larvae at 6dpf in response to SB treatment. CONCLUSIONS: The results suggest that SB exposure can cause significantly decreased survival rates of zebrafish embryos in a time- and dose-dependent manner and downregulated expression of TH and DAT in dopaminergic neurons in the zebrafish ventral diencephalon, which results in decreased locomotor activity of zebrafish larvae. This study may provide some important information for further elucidating the mechanism underlying SB-induced developmental neurotoxicity. Birth Defects Res (Part B)86: 85-91, 2009. © 2009 Wiley-Liss, Inc.     

Formation of fluorescent carbon nanodots from kitchen wastes and their application for detection of Fe3+

This work reports a scalable synthesis of water‐dispersible fluorescent carbon nanodots based on the simple hydrothermal method (180 °C for 6 h) of kitchen wastes (grape peel for example). We discuss the feasibility of synthesis from kitchen wastes both experimentally and theoretically, and the as‐prepared nanodots have high selectivity for Fe³⁺ ions based on fluorescence quenching which is due to the complexes between nanodots and metal ions. Copyright © 2014 John Wiley & Sons, Ltd.     

Doublecortin facilitates the elongation of the somatic Golgi apparatus into proximal dendrites

Mutations in the doublecortin (DCX) gene, which encodes a microtubule (MT)-binding protein, cause human cortical malformations, including lissencephaly and subcortical band heterotopia. A deficiency in DCX and DCX-like kinase 1 (DCLK1), a functionally redundant and structurally similar cognate of DCX, decreases neurite length and increases the number of primary neurites directly arising from the soma. The underlying mechanism is not completely understood. In this study, the elongation of the somatic Golgi apparatus into proximal dendrites, which have been implicated in dendrite patterning, was significantly decreased in the absence of DCX/DCLK1. Phosphorylation of DCX at S47 or S327 was involved in this process. DCX deficiency shifted the distribution of CLASP2 proteins to the soma from the dendrites. In addition to CLASP2, dynein and its cofactor JIP3 were abnormally distributed in DCX-deficient neurons. The association between JIP3 and dynein was significantly increased in the absence of DCX. Down-regulation of CLASP2 or JIP3 expression with specific shRNAs rescued the Golgi phenotype observed in DCX-deficient neurons. We conclude that DCX regulates the elongation of the Golgi apparatus into proximal dendrites through MT-associated proteins and motors.     

A suite of new Dre recombinase drivers markedly expands the ability to perform intersectional genetic targeting

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Diacylglycerol Kinase �� and Protein Kinase C�� Modulate Epidermal Growth Factor Receptor Abundance and Degradation through Ubiquitin-specific Protease 8

Many human epithelial cancers are characterized by abnormal activation of the epidermal growth factor receptor (EGFR), which is often caused by its excessive expression in tumor cells. The abundance of EGFR is modulated, in part, by its ubiquitination, which targets it for degradation. The components responsible for adding ubiquitin to EGFR are well characterized, but this is a reversible process, and the mechanisms that modulate the removal of ubiquitin from the EGFR are not well known. We found that de-ubiquitination of EGFR was regulated by diacylglycerol kinase δ (DGKδ), a lipid kinase that terminates diacylglycerol signaling. In DGKδ-deficient cells, ubiquitination of EGFR was enhanced, which attenuated the steady-state levels of EGFR and promoted its ligand-induced degradation. These effects were not caused by changes in the ubiquitinating apparatus, but instead were due to reduced expression of the de-ubiquitinase, ubiquitin-specific protease 8 (USP8). Depletion of protein kinase Cα (PKCα), a target of diacylglycerol, rescued the levels of USP8 and normalized EGFR degradation in DGKδ-deficient cells. Moreover, the effects of PKCα were caused by its inhibition of Akt, which stabilizes USP8. Our data indicate a novel mechanism where DGKδ and PKCα modulate the levels of ubiquitinated EGFR through Akt and USP8.     

Proteomics Analysis Identifies IRSp53 and Fascin as Critical for PRV Egress and Direct Cell–Cell Transmission

Pseudorabies virus (PRV) has been widely used as a live trans‐synaptic tracer for mapping neuronal circuits. Systematically identifying mature PRV virion proteomes and defining co‐purified host proteins are necessary to fully understand the detailed mechanism underlying PRV transmission processes. Here, a PRV virion purification strategy based on sorting with flow cytometry is developed and the mature extracellular and intracellular PRV virion proteomes using LC coupled with MS/MS are characterized. In addition to viral proteins, a large number of host proteins are also identified, including proteins related to actin cytoskeletal dynamics and membrane protrusion. How many of these host proteins are true virion components are unknown and the majority of these may not be. Through functional analysis, it is found that IRSp53 and fascin are critical for the egress process and play a role in direct cell–cell transmission. Moreover, it is shown that CDC42 and Rac1 are also involved in the production of mature extracellular virions. The results suggest that the formation of the filopodia‐like cytoskeleton and the rearrangement of the membrane, which are both associated with IRSp53 and fascin, may be important for the transmission of viruses used in neuronal tracing.     

FASN基因的原核表达及生物信息学分析

脂肪酸合成酶(FASN)在生物体内起着重要的作用,主要参与恶性肿瘤的数量调控。本研究旨在构建pET28a-FASN原核表达载体,并表达重组His-FASN蛋白,对该基因进行结构与功能的生物信息学分析。设计FASN基因特异性引物,通过PCR扩增获得的目的基因与原核表达载体pET28a连接,经IPTG诱导表达His-FASN蛋白。获得基因片段大小为1 320 bp,编码440个氨基酸;成功构建至pET28a原核表达载体,通过优化表达,确定在温度为35℃、IPTG浓度0.5 mmol/L、诱导时间为6 h的条件下融合蛋白表达量较高,获得蛋白大小约为53 kD;生物信息学分析结果表明FASN基因编码的蛋白是一个不稳定且具有亲水性的蛋白,不存在信号肽及跨膜区,可成为蛋白激酶磷酸化位点有12个Ser、5个Thr、3个Tyr。此外,从蛋白相互作用网络中发现,相互作用的蛋白包括主要酰基辅酶A合成酶长链家族成员及乙酰辅酶A羧化酶家族成员,为开发抑制剂药物提供了理论依据。