胞内抗体及其研究进展

胞内抗体是指在细胞内表达并被定位于亚细胞区室的一类新的工程抗体。目前胞内抗体的研究主要集中于ScFv,ScFv基因结构简单,易导入细胞内表达且便于体外重组操作。胞内抗体作为一种新的基因治疗工具,在肿瘤基因治疗、人艾滋病基因治疗的实验研究及潜在的临床治疗方面展示了广泛的应用前景。同时,胞内抗体可以用作分析靶蛋白功能的研究工具,是对传统的“基因剔除”转基因动物的一种有效补充。现从胞内抗体的设计及载体选择、肿瘤基因治疗、人艾滋病基因治疗等方面对胞内抗体的研究进展进行综述。     

Knockdown of phosphotyrosyl phosphatase activator induces apoptosis via mitochondrial pathway and the attenuation by simultaneous tau hyperphosphorylation

Phosphotyrosyl phosphatase activator (PTPA) is decreased in the brains of Alzheimer's disease (AD) and the AD transgenic mouse models. Here, we investigated whether down‐regulation of PTPA affects cell viability and the underlying mechanisms. We found that PTPA was located in the integral membrane of mitochondria, and knockdown of PTPA induced cell apoptosis in HEK293 and N2a cell lines. PTPA knockdown decreased mitochondrial membrane potential and induced Bax translocation into the mitochondria with a simultaneous release of Cyt C, activation of caspase‐3, cleavage of poly (DNA ribose) polymerase (PARP), and decrease in Bcl‐xl and Bcl‐2 protein levels. Over‐expression of Protein phosphatase 2A (PP2A) catalytic subunit (PP2A_C) did not rescue the apoptosis induced by PTPA knockdown, and PTPA knockdown did not affect the level of and their phosphorylation of mitogen‐activated protein kinases (MAPKs), indicating that PP2A and MAPKs were not involved in the apoptosis induced by PTPA knockdown. In the cells with over‐expression of tau, PTPA knockdown induced PP2A inhibition and tau hyperphosphorylation but did not cause significant cell death. These data suggest that PTPA deficit causes apoptotic cell death through mitochondrial pathway and simultaneous tau hyperphosphorylation attenuates the PTPA‐induced cell death.

     

Site-specific phosphorylation of tau accompanied by activation of mitogen-activated protein kinase (MAPK) in brains of Niemann-Pick type C mice

Niemann-Pick type C (NPC) disease is characterized by an accumulation of cholesterol in most tissues and progressive neurodegeneration with the formation of neurofibrillary tangles. Neurofibrillary tangles are composed of paired helical filaments (PHF), a major component of which is the hyperphosphorylated tau. In this study we used NPC heterozygous and NPC homozygous mouse brains to investigate the molecular mechanism responsible for tauopathy in NPC. Immunoblot analysis using anti-tau antibodies (Tau-1, PHF-1, AT-180, and AT-100) revealed site-specific phosphorylation of tau at Ser-396 and Ser-404 in the brains of NPC homozygous mice. Mitogen-activated protein kinase, a potential serine kinase known to phosphorylate tau, was activated, whereas other serine kinases such as glycogen synthase kinase-3beta and cyclin-dependent kinase 5 were inactive. Morphological examination demonstrated that a number of neurons, the perikarya of which strongly immunostained with PHF-1, exhibited polymorphorous cytoplasmic inclusion bodies and multi-concentric lamellar-like bodies. Importantly, the accumulation of intracellular cholesterol in NPC mouse brains was determined to be a function of age. From these results we conclude that abnormal cholesterol metabolism due to the genetic mutation in NPC1 may be responsible for activation of the mitogen-activated protein kinase-signaling pathway and site-specific phosphorylation of tau in vivo, leading to tauopathy in NPC.     

Assessment of Pollution-Induced Microbial Community Tolerance to Heavy Metals in Soil Using Ammonia-Oxidizing Bacteria and Biolog Assay

This study attempted to investigate if the tolerance of soil bacterial communities in general, and autotrophic ammonia-oxidizing bacteria (AOB) in particular, evolved as a result of prolonged exposure to metals, and could be used as an indigenous bioindicator for soil metal pollution. A soil contaminated with copper, chromium, and arsenic (CCA) was mixed with an uncontaminated garden soil (GS3) to make five test soils with different metal concentrations. A modified potential ammonium oxidation assay was used to determine the metal tolerance of the AOB community. Tolerance to Cr, Cu, and As was tested at the beginning and after up to 13 months of incubation. Compared with the reference GS3 soil, the five CCA soils showed significantly higher tolerance to Cr no matter which form of Cr (Cr³⁺, CrO₄ ^2?, or Cr₂O₇ ^2?) was tested, and the Cr tolerance correlated with the total soil Cr concentration. However, the tolerance to Cu²⁺, As³⁺, and As⁵⁺ did not differ significantly between the GS3 soil and the five CCA soils. Community level physiological profiles using Biolog microtiter plates were also used to examine the chromate tolerance of the bacterial communities extracted after six months of exposure. Our results showed that the bacterial community tolerance was altered and increased as the soil Cr concentration was increased, indicating that the culturable microbial community and the AOB community responded in a similar manner.     

Gain in 1q is a common abnormality in phyllodes tumours of the breast.

We studied DNA copy number changes by CGH and allelic imbalance (AI) on 3p by LOH analysis on 22 phyllodes tumours (PT) of the breast in order to gain insight into the genetic basis of tumour progression in PT. Copy number changes were observed in 14 cases (63%). Gain in 1q with 1q21-23 as the minimal overlapping area was seen in 12 cases (55%). The gain was observed both in benign and malignant tumours. Our study did not reveal any DNA copy number changes or allelic loss on 3p. The results suggest that DNA copy number changes are not associated with the histological grade or clinical behaviour of PT and the chromosomal changes on 3p appear to be rare. Colour figure can be viewed on http://www.esacp.org/acp/2003/25-2/jee.htm     

The ubiquinone-binding site in NADH:ubiquinone oxidoreductase from Escherichia coli

An azido-ubiquinone derivative, 3-azido-2-methyl-5-methoxy[3H]-6-decyl-1,4-benzoquinone ([3H]azido-Q), was used to study the ubiquinone/protein interaction and to identify the ubiquinone-binding site in Escherichia coli NADH:ubiquinone oxidoreductase (complex I). The purified complex I showed no loss of activity after incubation with a 20-fold molar excess of [3H]azido-Q in the dark. Illumination of the incubated sample with long wavelength UV light for 10 min at 0 degrees C caused a 40% decrease of NADH:ubiquinone oxidoreductase activity. SDS-PAGE of the complex labeled with [3H]azido-Q followed by analysis of the radioactivity distribution among the subunits revealed that subunit NuoM was heavily labeled, suggesting that this protein houses the Q-binding site. When the [3H]azido-Q-labeled NuoM was purified from the labeled reductase by means of preparative SDS-PAGE, a 3-azido-2-methyl-5-methoxy-6-decyl-1,4-benzoquinone-linked peptide, with a retention time of 41.4 min, was obtained by high performance liquid chromatography of the protease K digest of the labeled subunit. This peptide had a partial NH2-terminal amino acid sequence of NH2-VMLIAILALV-, which corresponds to amino acid residues 184-193 of NuoM. The secondary structure prediction of NuoM using the Toppred hydropathy analysis showed that the Q-binding peptide overlaps with a proposed Q-binding motif located in the middle of the transmembrane helix 5 toward the cytoplasmic side of the membrane. Using the PHDhtm hydropathy plot, the labeled peptide is located in the transmembrane helix 4 toward the periplasmic side of the membrane.     

枯草芽孢杆菌壳聚糖酶水解制备低脱乙酰度壳寡糖及其组分分析

对来源于枯草芽孢杆菌菌株168(Bacillus subtilis 168)的壳聚糖酶编码基因进行了序列优化及全合成,并在毕赤酵母(Pichia pastoris)中实现了分泌表达,表达产物的蛋白质浓度达到0.30mg/ml。表达的壳聚糖酶最适p H为5.6,最适温度为55℃,比酶活达84.54U/ml。该酶在50℃及以下较稳定。利用该酶水解低脱乙酰度壳聚糖并使用超高效液相色谱-四极杆飞行时间质谱(ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry,UPLC-QTOF MS)对产物的组分进行了分离及鉴定。根据一级质谱信息,推测酶解产物中包含至少37种聚合度2~18,不同脱乙酰度的壳寡糖组分。综上,利用毕赤酵母分泌表达了来源于枯草芽孢杆菌菌株168的壳聚糖酶基因,利用表达产物水解制备了低脱乙酰度壳寡糖并对其组分进行了分析,可为后续壳寡糖结构与功能关系的研究提供参考。     

Response of osteoblasts to low fluid shear stress is time dependent

The process of mechanotransduction of bone, the conversion of a mechanical stimulus into a biochemical response, is known to occur in osteoblasts in response to fluid shear stress. In order to understand the reaction of osteoblasts to various times of flow perfusion, osteoblasts were seeded on three-dimensional scaffolds, and cultured in the following conditions: continuous flow perfusion, intermittent flow perfusion, and static condition. We collected samples on day 4, 8 and 12 for analysis. Osteoblast proliferation was demonstrated by cell proliferation and scanning electron microscopy assay. Additionally, the expression of known markers of differentiation, including alkaline phosphatase and osteocalcin, were tested by qRT-PCR and alkaline phosphatase activity assay, and the deposition of calcium was used as an indicator of mineralization demonstrated by calcium content assay. The results supported that low fluid shear stress plays an important role in the activation of osteoblasts: enhance cell proliferation, increase calcium deposition, and promote the expression of osteoblastic markers. Furthermore, the continuous flow perfusion is a more favorable environment for the initiation of osteoblast activity compared with intermittent flow perfusion. Therefore, the force and time of fluid shear stress are important parameters for osteoblast activation.