Proteasome Contributes to the α-Secretase Pathway of Amyloid Precursor Protein in Human Cells

Abstract: A major histopathological hallmark in Alzheimer's disease consists of the extracellular deposition of the amyloid β-peptide (Aβ) that is proteolytically derived from the β-amyloid precursor protein (βAPP). An alternative, nonamyloidogenic cleavage, elicited by a protease called α-secretase, occurs inside the Aβ sequence and gives rise to APPα, a major secreted C-terminal-truncated form of βAPP. Here, we demonstrate that human embryonic kidney 293 (HK293) cells contain a chymotryptic-like activity that can be ascribed to the proteasome and that selective inhibitors of this enzyme reduce the phorbol 12,13-dibutyrate-sensitive APPα secretion by these cells. Furthermore, we establish that a specific proteasome blocker, lactacystin, also induces increased secretion of Aβ peptide in stably transfected HK293 cells overexpressing wild-type βAPP751. Altogether, this study represents the first identification of a proteolytic activity, namely, the proteasome, contributing likely through yet unknown intracellular relays, to the α-secretase pathway in human cells.     

Nitric oxide reacts with the ferryl-oxo catalytic intermediate of the CuB-lacking cytochrome bd terminal oxidase

Cytochrome bd is a bacterial respiratory oxidase carrying three hemes but no copper. We show that nitric oxide (NO) reacts with the intermediate F of cytochrome bd from Azotobacter vinelandii: (i) with a 1:1 stoichiometry, (ii) rapidly (k=1.2 +/- 0.1 x 10(5)M(-1)s(-1) at 20 degrees C), and (iii) yielding the oxidized enzyme with nitrite bound to heme d at the active site. Unexpectedly, the NO reaction mechanism of this catalytic intermediate in the Cu(B)-lacking cytochrome bd appears similar to that of beef heart cytochrome c oxidase, where Cu(B) was proposed to play a key role.     

TRPV1 receptors and nasal trigeminal chemesthesis

The trigeminal nerve responds to a wide variety of irritants. Trigeminal nerve fibers express several receptors that respond to chemicals, including TRPV1 (vanilloid) receptors, acid-sensing ion channels, P2X (purinergic) receptors, and nicotinic acetylcholine receptors. In order to assess whether TRPV1 plays a role in responses to a broad array of substances, TRPV1 (along with green fluorescent protein) was expressed in human embyonic kidney cells (HEK) 293t cells which were then stimulated with diverse trigeminal irritants. Calcium imaging was used to measure responses to capsaicin, amyl acetate, cyclohexanone, acetic acid, toluene, benzaldehyde, (-)-nicotine, (R)-(+)-limonene, (R)-(-)-carvone, and (S)-(+)-carvone. Three irritants (acetic acid and the 2 carvones) stimulated nontransfected controls. Two irritants (capsaicin and cyclohexanone) stimulated only transfected cells. The response could be eliminated with capsazepine, a TRPV1 blocker. The 5 remaining irritants were nonstimulatory in both nontransfected and transfected cells. Because all the compounds tested on HEK cells elicited neural responses from the ethmoid branch of the trigeminal nerve in rats, the 5 nonstimulatory compounds must do so by a non-TRPV1 receptor. These results suggest that TRPV1 serves as a receptor for both cyclohexanone and capsaicin in trigeminal nerve endings.     

Identification and expression analysis of miRNAs during batch culture of HEK-293 cells

MicroRNAs (miRNAs) are small non-coding RNAs of about 20–24 nucleotides in length. They regulate gene expression negatively and have been implicated in a wide variety of biological processes. To identify potential miRNAs that may influence the growth and proliferation of mammalian cells cultured in bioreactors, we applied miRNA microarray expression profiling technology to batch cultures of HEK293 cells in protein free media. In our study, we identified miRNAs that were differentially expressed during the exponential and stationary phases, 13 of these showed distinct up regulation trends while 1 exhibited down regulation. These miRNAs have been implicated in cellular differentiation, growth arrest and apoptosis. Specifically, miR-16 and let-7b are potentially useful in the enhancement of bioreactor cell cultures.     

Real-time monitoring of odorant-induced cellular reactions using surface plasmon resonance

Surface plasmon resonance (SPR) is a powerful technique for measuring molecular interaction in real-time. SPR can be used to detect molecule to cell interactions as well as molecule to molecule interactions. In this study, the SPR-based biosensing technique was applied to real-time monitoring of odorant-induced cellular reactions. An olfactory receptor, OR I7, was fused with a rho-tag import sequence at the N-terminus of OR I7, and expressed on the surface of human embryonic kidney (HEK)-293 cells. These cells were then immobilized on a SPR sensor chip. The intensity of the SPR response was linearly dependent on the amount of injected odorant. Among all the aldehyde containing odorants tested, the SPR response was specifically high for octanal, which is the known cognate odorant for the OR I7. This SPR response is believed to have resulted from intracellular signaling triggered by the binding of odorant molecules to the olfactory receptors expressed on the cell surface. This SPR system combined with olfactory receptor-expressed cells provides a new olfactory biosensor system for selective and quantitative detection of volatile compounds.     

An efficient and scalable process for helper-dependent adenoviral vector production using polyethylenimine-adenofection

Safety requirements for adenoviral gene therapy protocols have led to the development of the third generation of vectors commonly called helper-dependent adenoviral vectors (HDVs). HDVs have demonstrated a high therapeutic potential; however, the poor efficiency and reliability of the actual production process hampers further large-scale clinical evaluation of this new vector. The current HDV production methods involve a preliminary rescue step through transfection of adherent cell cultures by an HDV plasmid followed by a helper adenovirus (HV) infection. Amplification by serial co-infection of complementary cells allows an increase in the HDV titer. Using a HEK293 FLP/frt cell system in suspension culture, an alternative protocol to the current transfection/infection procedure was evaluated. In this work, the adenofection uses the HDV plasmid linked to the HV with the help of polyethylenimine (PEI) and has shown to outperform standard protocols by producing higher HDV yield. The influence of complex composition on the HDV production was examined by a statistical design. The optimized adenofection and amplification conditions were successively performed to generate HDV at the 3 L bioreactor scale. Following only two serial co-infection passages, up to 1.44 x 10(8) HDV infectious units/mL of culture were generated, which corresponded to 26% of the total particles produced. This production strategy, realized in cell suspension culture, reduced process duration and therefore the probability of vector recombination by introducing a cost-effective transfection protocol, ensuring production of high-quality vector stock.     

磁性多肽纳米探针的新法制备及其在细胞分离中的应用

通过静电自组装方法,在磁性纳米颗粒表面依次修饰带正电的聚二甲基二烯丙基氯化胺(Poly(diallyldimethylammoniumchloride),PDADMAC)和带负电的聚丙烯酸(Poly(acrylicacid),PAA),获得羧基化的磁性纳米复合粒子。进而将PAA上的羧基在1-乙基-3(3-二甲基胺基丙基)碳二亚胺盐酸盐(1-ethyl-3(3-dimethylaminopropyl)-carbodiimide,EDC)活化下与多肽LyP-1(CGNKRTRGC)上的氨基发生反应,从而使多肽LyP-1成功地连接到磁性颗粒表面,获得的磁性多肽纳米颗粒可靶向分离肺腺癌SPCA-1细胞,但不识别正常人胚肾293细胞。     

Ryanodine receptor mutations in arrhythmia: The continuing mystery of channel dysfunction

Mutations in RyR2 are causative of an inherited disorder which often results in sudden cardiac death. Dysfunctional channel behaviour has been the subject of many investigations varying from single channel analysis through to complex animal models. This review discusses recent advances in the field, describes the controversy surrounding the exact consequences of RyR2 mutation and how the disparate data may be reconciled. This heterogeneity of function with respect to the effects of polymorphisms, phosphorylation, cytosolic and luminal Ca²⁺ as well as inter-domain interactions may have important implications for the recent pharmaceutical therapies which have been put forward. We surmise that a comprehensive characterisation of mutations on a case-by-case basis may be beneficial for the development of specifically targeted therapies.     

人源性抗体在HEK293T细胞中瞬时表达条件的优化

以HEK293T细胞为宿主对抗体基因的转染和表达条件进行优化。以GFP(green fluorescence protein)和人源性抗b FGF抗体1A2为报告基因,考察了3种转染试剂磷酸钙,PEI,FuGene HD的转染效率,确定FuGene HD的转染效率最高后,对DNA∶FuGene HD的比例,加入氯喹的浓度,转染后的孵育时间,最佳的低温培养温度以及加入组蛋白抑制剂的浓度进行了优化。结果显示,FuGene HD的转染效率最高,达到56.7%。当转染的DNA=2μg/4×106细胞时,DNA∶FuGene HD的最佳比例为1∶4(W/V);加入氯喹能够增强FuGene HD的转染效率,最佳浓度为100μmol/L,转染后的最佳孵育时间为6h;低温诱导能够提高293T细胞表达抗体的能力,最佳的温度为33℃;加入组蛋白抑制剂丁酸钠或丙戊酸能够明显提高抗体的表达量,丙戊酸的效果优于丁酸钠。通过对转染试剂,转染方法,培养温度,组蛋白抑制剂的加入等条件进行优化使人源性抗bFGF抗体的表达量从1.5mg/L提高到了15.1mg/L。     

Prolonged activation of ERK1,2 induces FADD-independent caspase 8 activation and cell death

Prolonged ERK/MAPK activation has been implicated in neuronal cell death in vitro and in vivo. We found that HEK293 cells, recently reported to express neuronal markers, are exquisitely sensitive to long term ERK stimulation. Activation of an inducible form of Raf-1 (Raf-1:ER) in HEK293 cells induced massive apoptosis characterized by DNA degradation, loss of plasma membrane integrity and PARP cleavage. Cell death required MEK activity and protein synthesis and occurred via the death receptor pathway independently of the mitochondrial pathway. Accordingly, prolonged ERK stimulation activated caspase 8 and strongly potentiated Fas signaling. The death receptor adaptator FADD was found to be rapidly induced upon ERK activation. However using RNA interference and ectopic expression, we demonstrated that neither FADD nor Fas were necessary for caspase 8 activation and cell death. These findings reveal that prolonged ERK/MAPK stimulation results in caspase 8 activation and cell death. This work was supported by grant from Association pour la Recherche sur le Cancer (CNRS6543/ARC). S. Cagnol is supported by a fellowship from the Ligue Nationale contre le Cancer.