Amyloid-beta (Aβ) D7H mutation increases oligomeric Aβ42 and alters properties of Aβ-zinc/copper assemblies

Amyloid precursor protein (APP) mutations associated with familial Alzheimer's disease (AD) usually lead to increases in amyloid β-protein (Aβ) levels or aggregation. Here, we identified a novel APP mutation, located within the Aβ sequence (Aβ(D7H)), in a Taiwanese family with early onset AD and explored the pathogenicity of this mutation. Cellular and biochemical analysis reveal that this mutation increased Aβ production, Aβ42/40 ratio and prolonged Aβ42 oligomer state with higher neurotoxicity. Because the D7H mutant Aβ has an additional metal ion-coordinating residue, histidine, we speculate that this mutation may promote susceptibility of Aβ to ion. When co-incubated with Zn(2+) or Cu(2+), Aβ(D7H) aggregated into low molecular weight oligomers. Together, the D7H mutation could contribute to AD pathology through a "double punch" effect on elevating both Aβ production and oligomerization. Although the pathogenic nature of this mutation needs further confirmation, our findings suggest that the Aβ N-terminal region potentially modulates APP processing and Aβ aggregation, and further provides a genetic indication of the importance of Zn(2+) and Cu(2+) in the etiology of AD.     

Epithelial Mesenchymal Transition Is Required for Acquisition of Anoikis Resistance and Metastatic Potential in Adenoid Cystic Carcinoma

Human adenoid cystic carcinoma (ACC) is characterized by diffused invasion of the tumor into adjacent organs and early distant metastasis. Anoikis resistance and epithelial mesenchymal transition (EMT) are considered prerequisites for cancer cells to metastasize. Exploring the relationship between these processes and their underlying mechanism of action is a promising way to better understand ACC tumors. We initially established anoikis-resistant sublines of ACC cells; the variant cells revealed a mesenchymal phenotype through Slug-mediated EMT-like transformation and displayed enhanced metastatic potential both in vitro and in vivo. Suppression of EMT by knockdown of Slug significantly impaired anoikis resistance, migration, and invasion of the variant cells. With overexpression of Slug and Twist, we determined that induction of EMT in normal ACC cells could prevent anoikis, albeit partially. These findings strongly suggest that EMT is indispensable in anoikis resistance, at least in ACC cells. Furthermore, we found that the EGFR/PI3K/Akt pathway acts as the common regulator for EMT-like transformation and anoikis resistance, as confirmed by their specific inhibitors. Gefitinib and {"type":"entrez-nucleotide","attrs":{"text":"LY294003","term_id":"1257998347","term_text":"LY294003"}}LY294003 restored the sensibilities of anoikis-resistant cells to anoikis and simultaneously impaired their metastatic potential. In addition, the results from our in vivo model of metastasis suggest that pretreatment with gefitinib promotes mouse survival by alleviating pulmonary metastasis. Most importantly, immunohistochemistry of human ACC specimens showed a correlation between the overexpression of Slug and EGFR staining. This study has demonstrated that Slug-mediated EMT-like transformation is required by human ACC cells to achieve anoikis resistance and their metastatic potential. Targeting the EGFR/PI3K/Akt pathway holds potential as a preventive strategy against distant metastasis of ACC.     

Autophagy regulates hypoxia-induced osteoclastogenesis through the HIF-1α/BNIP3 signaling pathway

Previous studies have implicated that hypoxic stress could enhance osteoclast differentiation; however, the underlying mechanism remains poorly understood. Autophagy is a dynamic lysosomal degradation process that has emerged as an important regulator under hypoxic environment. In the present study, we demonstrate for the first time that autophagy regulates hypoxia-induced osteoclastogenesis in vitro. We found that exposure of RAW264.7 cells to hypoxia (0.2% oxygen) resulted in enhanced osteoclast differentiation, accompanied by the observation of several specific features of autophagy, including appearance of membranous vacuoles, formation of acidic vesicular organelles, cleavage and recruitment of microtubule-associated protein 1 light chain 3 (LC3) to autophagosomes, increase in autophagic flux, as well as up-regulation of autophagy-related gene (Atg) expression. Moreover, suppression of autophagy with DN-Atg5(K130R) or 3-methyladenine (3-MA) significantly attenuated the osteoclast differentiation under hypoxic conditions, indicating the functional significance of autophagy in hypoxia-induced osteoclastogenesis. The data also showed that the activation of autophagy under hypoxic conditions was caused by up-regulated expression of hypoxia-inducible factor-1α (HIF-1α)-dependent Bcl-2 adenovirus E1a 19 kDa interacting protein 3 (BNIP3). Importantly, knockdown of HIF-1α or BNIP3 obviously abrogated hypoxia-induced autophagy activation and osteoclastogenesis enhancement. Collectively, our results highlight the fact that autophagy is a pivotal regulator for hypoxia-induced osteoclast differentiation, which may provide new insight into the pathological processes of osteoclastogenesis under hypoxic stress and help develop new therapeutic strategies for abnormal osteoclastogenesis.     

Characterization of Fasciola samples by ITS of rDNA sequences revealed the existence of Fasciola hepatica and Fasciola gigantica in Yunnan Province, China

On mainland China, liver flukes of Fasciola spp. (Digenea: Fasciolidae) can cause serious acute and chronic morbidity in numerous species of mammals such as sheep, goats, cattle, and humans. The objective of the present study was to examine the taxonomic identity of Fasciola species in Yunnan province by sequences of the first and second internal transcribed spacers (ITS-1 and ITS-2) of nuclear ribosomal DNA (rDNA). The ITS rDNA was amplified from 10 samples representing Fasciola species in cattle from 2 geographical locations in Yunnan Province, by polymerase chain reaction (PCR), and the products were sequenced directly. The lengths of the ITS-1 and ITS-2 sequences were 422 and 361-362 base pairs, respectively, for all samples sequenced. Using ITS sequences, 2 Fasciola species were revealed, namely Fasciola hepatica and Fasciola gigantica. This is the first demonstration of F. gigantica in cattle in Yunnan Province, China using a molecular approach; our findings have implications for studying the population genetic characterization of the Chinese Fasciola species and for the prevention and control of Fasciola spp. in this province.     

Structure of the Alkalohyperthermophilic Archaeoglobus fulgidus Lipase Contains a Unique C-Terminal Domain Essential for Long-Chain Substrate Binding

Several crystal structures of AFL, a novel lipase from the archaeon Archaeoglobus fulgidus, complexed with various ligands, have been determined at about 1.8 Å resolution. This enzyme has optimal activity in the temperature range of 70-90 °C and pH 10-11. AFL consists of an N-terminal α/β-hydrolase fold domain, a small lid domain, and a C-terminal β-barrel domain. The N-terminal catalytic domain consists of a 6-stranded β-sheet flanked by seven α-helices, four on one side and three on the other side. The C-terminal lipid binding domain consists of a β-sheet of 14 strands and a substrate covering motif on top of the highly hydrophobic substrate binding site. The catalytic triad residues (Ser136, Asp163, and His210) and the residues forming the oxyanion hole (Leu31 and Met137) are in positions similar to those of other lipases. Long-chain lipid is located across the two domains in the AFL-substrate complex. Structural comparison of the catalytic domain of AFL with a homologous lipase from Bacillus subtilis reveals an opposite substrate binding orientation in the two enzymes. AFL has a higher preference toward long-chain substrates whose binding site is provided by a hydrophobic tunnel in the C-terminal domain. The unusually large interacting surface area between the two domains may contribute to thermostability of the enzyme. Two amino acids, Asp61 and Lys101, are identified as hinge residues regulating movement of the lid domain. The hydrogen-bonding pattern associated with these two residues is pH dependent, which may account for the optimal enzyme activity at high pH. Further engineering of this novel lipase with high temperature and alkaline stability will find its use in industrial applications.     

同型半胱氨酸对人脐静脉血管内皮细胞tPA 及PAI-1基因表达的影响

目的探讨同型半胱氨酸(Hcy)对纤溶系统的影响,观察Hcy在转录水平对人脐静脉血管内皮细胞(HUVEC)表达组织型纤溶酶原激活物(tPA)和纤溶酶原激活物抑制剂1(PAI1)的影响。方法将体外培养的HUVEC分为生理浓度(10μmol/LHcy)组,病理浓度(50、200、500μmol/L)Hcy组及单纯培养基组(0μmol/LHcy),培养24h后,提取RNA,反转录聚合酶链反应分析(RTPCR)法分析各组tPA及PAI1基因表达水平。结果500μmol/LHcy组与10μmol/LHcy组相比,tPAmRNA基因表达明显下调(P<0.05),PAI1mRNA表达则明显上调(P<0.05)。而与单纯培养基组相比,10μmol/LHcy组tPAmRNA表达明显增高(P<0.05)。结论生理浓度Hcy可以增加纤溶系统活性,减少血栓性疾病的发生。高Hcy(病理浓度)则抑制纤溶系统活性,促进缺血性心脑血管疾病的发生。     

胚胎干细胞向肝细胞诱导分化

胚胎干细胞具有分化成三胚层细胞的潜能。它已被视为治疗多种疾痛的一种新兴策略。在现阶段,通过不同的诱导途径可将胚胎干细胞诱导成为肝细胞：体外诱导、体内诱导以及体外和体内相结合诱导分化。然而从体内实验结果来看,其嵌合率及分化率不高,这是一个亟需解决的问题,否则就无法成功地将其应用于临床治疗。     

利用脂肪酸生物标记对尖镰孢寄主专化型的快速鉴定

尖镰孢寄主范围广、遗传差异大,其种下存在多种寄主专化型。对尖镰孢寄主专化型的快速鉴定可为科学制定植物病害防控策略提供依据。利用Sherlock MIS脂肪酸鉴定系统对分离自番茄、棉花、黄瓜、茄子等4种寄主专化型的18株尖镰孢进行脂肪酸成分测定,共检测到10种脂肪酸。运用SPSS软件中的PCA法对被检测到的脂肪酸进行主成分分析,确定了18:1CIS9（W9）[X1],18:2 CIS 9,12/18:0a[X2]和18:00[X3]等3个脂肪酸为其主成分。利用Bayes逐步判别法建立了尖镰孢4种不同寄主专化型判别模型为Y1=-157.750+2.809X1+3.391X2+8.099X3;Y2=-178.343+0.586X1+7.587X2- 0.214X3;Y3=-129.132+2.749X1+4.163X2+4.476X3;Y4=-201.307+2.016X1+7.345X2+2.400X3。通过对43株未知寄主专化型菌株主成分脂肪酸的测定,利用判别法对尖镰孢进行判定,结果发现有40株与原寄主来源一致,判对率达93%。表明脂肪酸生物标记法可用于尖镰孢寄主专化型的快速鉴定。