Inhibition of PrPSc formation in scrapie infected N2a cells by 5,7,8-trimethyl-3,4-dihydro-2H-1,4-benzoxazine derivatives

Prion diseases are fatal, neurodegenerative diseases characterized by the structural conversion of the normal, cellular prion protein, PrP^C into an abnormally structured, aggregated and partially protease-resistant isoform, termed PrP^Sc. Although substantial research has been directed toward development of therapeutics targeting prions, there is still no curative treatment for the disease. Benzoxazines are bicyclic heterocyclic compounds possessing several pharmaceutically important properties, including neuroprotection and reactive oxygen species scavenging. In an effort to identify novel inhibitors of prion formation, several 5,7,8-trimethyl-1,4-benzoxazine derivatives were evaluated in vitro for their effectiveness on the expression levels of normal PrP^C and its conversion to the abnormal isoforms of PrP^Sc in a scrapie-infected cell culture model. The most potent compound was 2-(4-methoxyphenyl)-5,7,8-trimethyl-3,4-dihydro-2H-1,4-benzoxazine, with a diminishing effect on the formation of PrP^Sc, thus establishing a class of compounds with a promising therapeutic use against prion diseases.     

Exposure of sheep scrapie brain homogenate to rumen-simulating conditions does not result in a reduction of PrP(Sc) levels

AIMS: Experiments were designed to evaluate the potential of rumen-simulating conditions to reduce PrP(Sc) levels. METHODS AND RESULTS: Scrapie-positive brain material was incubated under rumen-simulating conditions. Time points were taken over a 24-h period and PrP(Sc) levels were analysed by Western blot. No loss of PrP(Sc) was observed over a 24-h time period. CONCLUSIONS: Our results indicate that a fully developed rumen fermentation does not provide significant protection against prion infection via the oral route. Developmental changes including senescence of immune system function or other developmental changes in the gastrointestinal tract are potential mechanisms by which relative bovine spongiform encephalopathy (BSE) susceptibility might vary with age. SIGNIFICANCE AND IMPACT OF THE STUDY: Epidemiology of the BSE outbreak in the United Kingdom indicates that younger animals were at higher risk of infection. The rumen undergoes pronounced developmental changes early in life, coinciding with the introduction of fibre into the diet. The timeframe of highest risk of infection overlaps the time in life prior to full rumen development. This work indicates that a fully developed rumen does not provide significant protection against prion infection via the oral route of infection. This result implicates other developmental changes that are responsible for the age-dependent susceptibility of cattle to BSE.     

Structural characterization of a neurotoxic threonine‐rich peptide corresponding to the human prion protein α2‐helical 180–195 segment,and comparison with full‐length α2‐helix‐derived peptides

The 173–195 segment corresponding to the helix 2 of the globular PrP domain is a good candidate to be one of the several ‘spots’ of intrinsic structural flexibility, which might induce local destabilization and concur to protein transformation, leading to aggregation‐prone conformations. Here, we report CD and NMR studies on the α2‐helix‐derived peptide of maximal length (hPrP[180–195]) that is able to exhibit a regular structure different from the prevalently random arrangement of other α2‐helix‐derived peptides. This peptide, which has previously been shown to be affected by buffer composition via the ion charge density dependence typical of Hofmeister effects, corresponds to the C‐terminal sequence of the PrP^C full‐length α2‐helix and includes the highly conserved threonine‐rich 188–195 segment. At neutral pH, its conformation is dominated by β‐type contributions, which only very strong environmental modifications are able to modify. On TFE addition, an increase of α‐helical content can be observed, but a fully helical conformation is only obtained in neat TFE. However, linking of the 173–179 segment, as occurring in wild‐type and mutant peptides corresponding to the full‐length α2‐helix, perturbs these intrinsic structural propensities in a manner that depends on whether the environment is water or TFE. Overall, these results confirm that the 180–195 parental region in hPrP^C makes a strong contribution to the chameleon conformational behavior of the segment corresponding to the full‐length α2‐helix, and could play a role in determining structural rearrangements of the entire globular domain. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Protection from MPTP-induced neurotoxicity in differentiating mouse N2a neuroblastoma cells

We have shown previously that subcytotoxic concentrations of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) inhibit axon outgrowth and are associated with increased neurofilament heavy chain (NF-H) phosphorylation in differentiating mouse N2a neuroblastoma cells while higher doses (> 100 microM) cause cell death. In this work we assessed the ability of potential neuroprotective agents to alleviate both MPTP-induced cell death (cytotoxicity) and MPTP-induced NF-H phosphorylation/reduction in axon outgrowth (neurotoxicity) in N2a cells induced to differentiate by dbcAMP. The neurotoxic effects of MPTP occurred in the absence of significant alterations in energy status or mitochondrial membrane potential. The hormone oestradiol (100 microM) reduced the cytotoxic effect of MPTP, but blocked di-butyryl cyclic AMP (dbcAMP)-induced differentiation, i.e. axon outgrowth. Both the cytotoxic and neurotoxic effects of MPTP were reduced by the monoamine oxidase (MAO) inhibitors deprenyl and, to a lesser extent, clorgyline. Alleviation of both neurotoxicity and cytotoxicity was also achieved by conditioned medium derived from rat C6 glioma cells. In contrast, whilst the p38 MAP kinase inhibitor, SB202190, protected cells against MPTP-induced neurotoxicity, it could not maintain cell viability at high MPTP exposures. In each case neuroprotection involved maintenance of the differentiating phenotype linked with attenuation of NF-H hyper-phosphorylation; the latter may represent a mechanism by which neuronal cells can moderate MPTP-induced neurotoxicity. The use of a simplified neuronal cell model, which expresses subtle biochemical changes following neurotoxic insult, could therefore provide a valuable tool for the identification of potential neuroprotective agents.     

Effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine on differentiating mouse N2a neuroblastoma cells

The effect of the neurotoxin 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine (MPTP) was investigated in mouse N2a neuroblastoma cells, induced to differentiate by serum withdrawal and addition of dibutyryl cyclic AMP, over a 24-h period. Addition of MPTP (10 microM) during differentiation caused a change in cell morphology characterised by an inhibition of axon outgrowth, in the absence of cell death. Biochemical characterisation by western blotting revealed that MPTP had no significant effects on the levels of actin, alpha-tubulin, or total heavy-chain neurofilament (NF-H). However, NF-H phosphorylation appeared to increase following MPTP treatment when blots were probed with the phosphorylation state-specific antibodies RMd09 and Ta51. In addition, indirect immunofluorescence analysis revealed an accumulation of phosphorylated NF-H in the cell perikaryon, suggesting that altered NF-H distribution was associated with the observed effects of MPTP on cell morphology. These changes may represent a useful in vitro marker of MPTP neurotoxicity within a simple differentiating neuronal cell model system.     

Identification of unusual truncated forms of nucleocapsid protein in MDCK cells infected by Avian influenza virus (H9N2)

Unusual truncated forms of nucleocapsid protein (NP) were identified in the lysate of MDCK cells infected by Avian influenza virus (H9N2) using MS‐based proteomics approach. Moreover, O‐sulfonation that was considered as an unusual modification was identified in one of the tryptic peptides from the truncated NP. The findings might have implications on better understanding on the role of nucleoprotein in Avian influenza virus–host interaction.     

Cytochalasin D enhances the accumulation of a protease‐resistant form of prion protein in ScN2a cells: involvement of PI3 kinase/Akt signalling pathway

The conversion of a host‐encoded PrPsen (protease‐sensitive cellular prion protein) into a PrPres (protease‐resistant pathogenic form) is a key process in the pathogenesis of prion diseases, but the intracellular mechanisms underlying PrPres amplification in prion‐infected cells remain elusive. To assess the role of cytoskeletal proteins in the regulation of PrPres amplification, the effects of cytoskeletal disruptors on PrPres accumulation in ScN2a cells that were persistently infected with the scrapie Chandler strain have been examined. Actin microfilament disruption with cytochalasin D enhanced PrPres accumulation in ScN2a cells. In contrast, the microtubule‐disrupting agents, colchicine, nocodazole and paclitaxel, had no effect on PrPres accumulation. In addition, a PI3K (phosphoinositide 3‐kinase) inhibitor, wortmannin and an Akt kinase inhibitor prevented the cytochalasin D‐induced enhancement of PrPres accumulation. Cytochalasin D‐induced extension of neurite‐like processes might correlate with enhanced accumulation of PrPres. The results suggest that the actin cytoskeleton and PI3K/Akt pathway are involved in the regulation of PrPres accumulation in prion‐infected cells.     

蝙蝠葛碱拮抗缓激肽引起的钙稳态改变 及细胞骨架蛋白的异常磷酸

为研究蝙蝠葛碱 (dauricine , Dau) 拮抗缓激肽 (bradykinin , BK) 诱导的 Alzheimer 样钙稳态失衡及细胞骨架蛋白异常磷酸化的作用,采用双波长荧光分光光度计测定细胞内钙离子浓度 ([Ca2+] i) ,用 MTT 法检测细胞代谢水平,用免疫组织化学方法观察 tau 蛋白表达和磷酸化 . 结果表明,Dau (3 μmol/L , 6 μmol/L) 可抑制 BK 诱导的 [Ca2+]i 升高,保护 BK 引起的神经元代谢降低,拮抗 BK 引起的 tau 蛋白异常磷酸化和聚集 . 结果提示： Dau 可拮抗 BK 诱导的 Alzheimer 样钙稳态失衡及细胞骨架蛋白异常磷酸化的作用 .     

Prolonged Alzheimer-like tau hyperphosphorylation induced by simultaneous inhibition of phosphoinositol-3 kinase and protein kinase C in N2a cells

Co-injection of wortmannin (inhibitor of phosphatidylinositol-3 kinase, PI3K) and GF109203X(inhibitor of protein kinase C, PKC) into the rat brain was found to induce spatial memory deficiency and enhance tau hyperphosphorylation in the hippocampus of rat brain. To establish a cell model with durative Alzheimer-like tau hyperphosphorylation in this study, we treated N2a neuroblastoma cells with wortmannin and GF109203X separately and simultaneously, and measured the glycogen synthase kinase 3 (GSK-3)activity by y-32p-labeling and the level of tau phosphorylation by Western blotting. It was found that the application of wortmannin alone only transitorily increased the activity of GSK-3 (about 1 h) and the level of tau hyperphosphorylation at Ser^396/Ser^404 and Ser^199/Ser^202 sites (no longer than 3 h); however, a prolonged and intense activation of GSK-3 (over 12 h) and enhanced tau hyperphosphorylation (about 24 h) were observed when these two selective kinase inhibitors were applied together. We conclude that the simultaneous inhibition of PI3K and PKC can induce GSK-3 overactivation, and further strengthen and prolong the Alzheimerlike tau hyperphosphorylation in N2a cells, suggesting the establishment of a cell model with early pathological events of Alzheimer‘s disease.     

Uptake of 3,4-methylenedioxymethamphetamine and its related compounds by a proton-coupled transport system in Caco-2 cells

3,4-Methylenedioxymethamphetamine (MDMA) is an illegal amphetamine-type stimulant (ATS) that is abused orally in the form of tablets for recreational purposes. The aim of this work is to investigate the absorption mechanism of MDMA and other related compounds that often occur together in ATS tablets, and to determine whether such tablet components interact with each other in intestinal absorption. The characteristics of MDMA uptake by the human intestinal epithelial Caco-2 cell line were investigated. The Michaelis constant and the maximal uptake velocity at pH 6.0 were 1.11 mM and 13.79 nmol/min/mg protein, respectively, and the transport was electroneutral. The initial uptake rate was regulated by both intra- and extracellular pH. MDMA permeation from the apical to the basolateral side was inferior to that in the reverse direction, and a decrease in apical pH enhanced MDMA permeation from the basolateral to the apical side. These facts indicate that this transport system may be an antiporter of H⁺. However, under physiological conditions, the proton gradient cannot drive the MDMA uptake because it is inwardly directed. Large concentration differences of MDMA itself drive this antiporter. Various compounds with similar amine moieties inhibited the uptake, but substrates of organic cation transporters (OCT1-3) and an H⁺-coupled efflux antiporter, MATE, were not recognized.     

Inhibition of D1, D2, and a cyclin expression in HL-60 cells by the lipid peroxydation product 4-hydroxynonenal

4-Hydroxynonenal (HNE), a product of lipid peroxidation, is an highly reactive aldehyde that, at concentration similar to those found in normal cells, blocks proliferation and induces a granulocytic-like differentiation in HL-60 cells. These effects are accompained by a marked increase in the proportion G0/G1 cells. The mechanisms of HNE action were investigated by analyzing the expression of the cyclins and cyclin-dependent protein kinases (CDKs), controlling the cell cycle progression. Data obtained by exposing cells to dimethyl sulfoxide (DMSO) were used for comparison. 4-Hydroxynonenal downregulated both mRNA and protein contents of cyclins D1, D2, and A until 24 h from the treatments, whereas DMSO inhibited cyclin D1 and D2 expression until the end of experiment (2 days) and induces an increase of cyclin A until 1 day. Cyclins B and E, and protein kinase CDK2 and CDK4 expressions were not affected by HNE, whereas DMSO induced an increase of cyclin E, B, and CDK2 from 8 h to 1 day. These data are in agreement with previous results indicating a different time-course of accumulation in G0/G1 phases of cells treated with HNE and DMSO and suggest that the HNE inhibitory effect on proliferation and cell cycle progression may depend by the downregulation of D1, D2, and A cyclin expression.     

Inhibition of protease activity by antisense RNA improves recombinant protein production in Nicotiana tabacum cv. Bright Yellow 2 (BY‐2) suspension cells

Recombinant proteins produced in plant suspension cultures are often degraded by endogenous plant proteases when secreted into the medium, resulting in low yields. To generate protease‐deficient tobacco BY‐2 cell lines and to retrieve the sequence information, we cloned four different protease cDNAs from tobacco BY‐2 cells (NtAP, NtCP, NtMMP1, and NtSP), which represent the major catalytic classes. The simultaneous expression of antisense RNAs against these endogenous proteases led to the establishment of cell lines with reduced levels of endogenous protease expression and activity at late stages of the cultivation cycle. One of the cell lines showing reduced proteolytic activity in the culture medium was selected for the expression of the recombinant full‐length IgG1(κ) antibody 2F5, recognizing the gp41 surface protein of HIV‐1. This cell line showed significantly reduced degradation of the 2F5 heavy chain, resulting in four‐fold higher accumulation of the intact antibody heavy chain when compared to transformed wild type cells expressing the same antibody. N‐terminal sequencing data revealed that the antibody has two cleavage sites within the CDR‐H3 and one site at the end of the H4‐framework region. These cleavage sites are found to be vulnerable to serine proteases. The data provide a basis for further improvement of plant cells for the production of recombinant proteins in plant cell suspension cultures.     

Inhibition of Voltage-Sensitive Calcium Channels by the A2A Adenosine Receptor in PC12 cells

Abstract: The role of the A_2A adenosine receptor in regulating voltage-sensitive calcium channels (VSCCs) was investigated in PC12 cells. Ca²⁺ influx induced by membrane depolarization with 70 m M K⁺ could be inhibited with CGS21680, an A_2A receptor-specific agonist. Both L- and N-type VSCCs were inhibited by CGS21680 treatment. Effects of adenosine receptor agonists and antagonists indicate that the typical A_2A receptor mediates inhibition of VSCCs. Cholera toxin (CTX) treatment for 24 h completely eliminated the CGS21680 potency. Similar inhibitory effects on VSCCs were obtained by membrane-permeable activators of protein kinase A (PKA). These effects were blocked by Rp -adenosine-3',5'-cyclic monophosphothioate, a PKA inhibitor. The data suggest that activation of the A_2A receptor leads to inhibition of VSCCs via a CTX-sensitive G protein and PKA. ATP pretreatment caused a reduction in subsequent rise in cytosolic free Ca²⁺ concentration induced by 70 m M K⁺, presumably by inactivation of VSCCs. Simultaneous treatment with ATP and CGS21680 produced significantly greater inhibition of VSCCs than treatment with CGS21680 or ATP alone. Furthermore, the CGS21680-induced inhibition of VSCCs was not affected by the presence of reactive blue 2. CGS21680 still significantly inhibited ATP-evoked Ca²⁺ influx without VSCC activity after cobalt or 70 m M K⁺ pretreatment. These data suggest that the A_2A receptor-sensitive VSCCs differ from those activated by ATP treatment. Although A_2A receptors induce inhibition of VSCCs as well as ATP-induced Ca²⁺ influx, the two inhibitory effects are clearly distinct from each other.     

Proteome-wide study of endoplasmic reticulum stress induced by thapsigargin in N2a neuroblastoma cells

Disturbances in intraluminal endoplasmic reticulum (ER) Ca²⁺ concentration leads to the accumulation of unfolded proteins and perturbation of intracellular Ca²⁺ homeostasis, which has a huge impact on mitochondrial functioning under normal and stress conditions and can trigger cell death. Thapsigargin (TG) is widely used to model cellular ER stress as it is a selective and powerful inhibitor of sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPases. Here we provide a representative proteome-wide picture of ER stress induced by TG in N2a neuroblastoma cells. Our proteomics study revealed numerous significant protein expression changes in TG-treated N2a cell lysates analysed by two-dimensional electrophoresis followed by mass spectrometric protein identification. The proteomic signature supports the evidence of increased bioenergetic activity of mitochondria as several mitochondrial enzymes with roles in ATP-production, tricarboxylic acid cycle and other mitochondrial metabolic processes were upregulated. In addition, the upregulation of the main ER resident proteins confirmed the onset of ER stress during TG treatment. It has become widely accepted that metabolic activity of mitochondria is induced in the early phases in ER stress, which can trigger mitochondrial collapse and subsequent cell death. Further investigations of this cellular stress response in different neuronal model systems like N2a cells could help to elucidate several neurodegenerative disorders in which ER stress is implicated.     

Activation of a Microtubule-Associated Protein-2 Kinase by Insulin-Like Growth Factor-I in Bovine Chromaffin Cells

Treatment of bovine chromaffin cells with insulin-like growth factor-I (IGF-I) caused the activation of a protein kinase that phosphorylates microtubule-associated protein-2 (MAP-2) in vitro. Activation of MAP-2 kinase by IGF-I varied with the time of treatment (maximal at 10-15 min) and the concentration of IGF-I (maximal at 10 nM). The IGF-I-activated MAP-2 kinase was localized to the soluble fraction of chromaffin cell extracts and required Mg2+ for activity. The IGF-I-activated kinase also phosphorylated myelin basic protein, but had little or no activity toward histones or ribosomal S6 protein. To examine the role of protein tyrosine phosphorylation in the activation of the MAP-2 kinase, we isolated phosphotyrosine (PTyr)-containing proteins from chromaffin cells by immunoaffinity adsorption on anti-PTyr-Sepharose beads. Anti-PTyr-Sepharose eluates from IGF-I-treated cells showed increased MAP-2 kinase activity; thus, the MAP-2 kinase (or a closely associated protein) appears to be a PTyr-containing protein. Treatment of anti-PTyr-Sepharose eluates or crude chromaffin cell extracts with alkaline phosphatase significantly decreased kinase activity toward myelin basic protein, indicating that phosphorylation of the IGF-I-activated kinase is required for its activity.     

Analysis of a silkworm F1 hybrid with yellow cocoon generated by crossing two white-cocoon strains: Further evidences for the roles of Cameo2 and CBP in formation of yellow cocoon

In this report, we examined the gene expression related to carotenoid transport for a silkworm F₁ hybrid with yellow cocoon generated by crossing two white-cocoon strains, Qiubai and 12-260. Our results showed that, in Qiubai, Cameo2, a transmembrane protein gene belonging to the CD36 family genes, was expressed normally in the silk gland, but no intact carotenoid-binding protein (CBP) mRNA (only the truncated CBP mRNA) was detected in the midgut. In 12-260, we detected the intact CBP mRNA expression in the midgut, but no Cameo2 expression in the silk gland. Regarding the F₁ hybrid from crossing Qiubai and 12-260, both Cameo2 and intact CBP mRNA expressed normally in the silk gland and midgut. HPLC detection confirmed that in the F₁ hybrid the carotenoids could be absorbed from dietary mulberry leaves through the midgut and transferred to silk gland via the hemolymph, which eventually colored cocoons into yellow. We also identified four CBP mRNA isoforms expressed in the midgut of the F₁ hybrid, subsequently named as variants 5–8. Our results provide further evidences for the roles of Cameo2 and CBP in the formation of yellow cocoon of silkworm.