Abstract:Alzheimer's disease (AD) is one of the commonest neurodegenerative diseases affected mainly the elderly. AD is characterized by the formation of neuritic plaque in brain, which is composed mainly of extracellular β amyloid deposion, the Aβ. Aβ is deprived from serial hydrolysis of amyloid precursor protein (APP) by two secretases, the β and γ-secretase respectively. Alternatively, APP can also be sequential processed by α-secretase and γ-secretase, which not only preclude the formation of Aβ, but also generate a large ectodomain (sAPPα) who has several neuroprotective properties. Thus the secondary processing pathway has become the focus of AD research. Many results have indicated that members of the adamalysin family of proteins, mainly the ADAM 10, ADAM 17 and ADAM 9, fulfill some of the criteria required of α-secretase. Here the biological characteristics of α-secretase, its activity regulation and its potential function as targets for the treatment of AD were summerized.

Abstract:Botulinum neurotoxin(BoNT) is the most lethal biotoxin known to mankind. It inhibits acetylcholine release from the cholinergic nerve ending by cleavage of SNARE proteins, followed by neuromuscular blockade and paralysis. Gangliosides are considered to act as a first receptor of BoNT with low affinity.Then the membrane bound gangliosides-BoNT complex moves laterally to reach and bind the toxin specific protein receptor, synaptotagmin, with a high affinity constant. At last the gangliosides-BoNT-synaptotagmin complex undergoes receptor-mediated endocytosis. This double-receptors theory is widely accepted. The research data are summarized and reviewed.

Abstract:Cell cycle checkpoints are protective mechanism responding to DNA damages originated from external or internal factors. When cells are exposed to genotoxic stress or when nutrition crisis occurs, cell cycle progression is usually stopped or slowed down by cell cycle checkpoints to allow for DNA repair or for handling the crisis. Besides, recent studies suggest that some cell cycle checkpoint proteins are also involved in regulating physiological DNA replication via controlling the rate of DNA replication. Cell cycle checkpoint proteins ATR, 9-1-1 complex, Chk1, Cdc25A and CDK2 may participate in this process. This kind of regulation is supposed to be very important for ensuring accurate DNA replication and maintaining genomic stability.

Abstract:A full-length Beet black scorch virus (BBSV) cDNA clone (pUBF52) was constructed by RT-PCR. The clone contains an upstream T7 RNA polymerase promoter designed for in vitro transcription of infectious RNAs from the linearized plasmid that faithfully represent the viral cDNA. Leaves of Chenopodium amaranticolor inoculated with in vitro transcripts developed the same symptoms and disease phenotype as the wild type virus. The presence of BBSV RNA and coat protein in the leaves was confirmed respectively by Northern blotting and Western blotting. Comparisons of specific immunoreactions between the expression product of the BBSV p24 gene in E. coli and antiserum against purified BBSV virions demonstrated that the p24 gene encodes the coat protein. Based on the sequence of the pUBF52 cDNA, a frame-shift mutant and two deletion mutants were generated. One of the deletions encompasses the entire CP ORF and the other truncates 174 amino acids from the central region of the protein. Transcripts derived from the frame-shift CP mutant, which terminates the CP after the first 23 amino acids, elicited the same symptom phenotype and levels of RNA accumulation as the wild type virus, but the leaves infected with the CP deletion mutants exhibited greatly reduced RNA accumulation. In addition, leaves inoculated with in vitro transcripts of the mutant in which the entire CP gene was deleted had lower local lesions than wild type virus transcripts. Two expression vectors, pBGFP and pBGUS, were constructed by fusing the GFP and GUS genes to the 23 N-terminal amino acids of the CP gene, respectively. Leaves infected with in vitro transcripts of pBGFP and pBGUS exhibited expression of GFP and GUS proteins as assessed by laser confocal microscopy and histochemical staining, respectively. The high levels of expression of the GFP and GUS proteins provide tools that can be used for studies of replication and movement of the virus, and indicate that BBSV has considerable biotechnology potential as a plant virus expression vehicle.

Abstract:NG-nitro-D-arginine (D-NNA) produced pressor responses in rats by acting via chiral inversion into NG-nitro-L-arginine (L-NNA), an inhibitor of nitro oxide synthase. The present investigation aimed to study the role of the D-amino acid oxidase (DAAO) in chiral inversion of D-NNA and the relationship between DAAO activities on various D-amino acids and their inversion rate. Benzoate (400 mg/kg) or creatinine (400 mg/kg), two inhibitors of DAAO, blocked D-NNA-induced pressor responses in rats. Furthermore, the addition of the pure DAAO significantly potentiates L-NNA production rate in kidney homogenates by approximately 2-folds. The in vivo and in vitro results suggested that DAAO plays an essential role in the pressor responses elicited by D-NNA. Moreover, crude DAAO solution from the kidney showed marked selection (the maximal ratio of Kcat/Km was nearly 15 times) on different D-amino acids that exhibited similar chiral inversion rate in vivo, suggesting that other enzymes, such as transaminase, are also required for the entire process of D-NNA chiral inversion.

Abstract:Overexpression of the neutrophil gelatinase-associated lipocalin (NGAL) gene is related to malignant transformation of human immortalized esophageal epithelial cell induced by 12-O-tetradecanoyl phorbol 13-acetate (TPA). However, the mechanisms of the transcriptional activation of this gene remain unclear. The recent study indicated that there might be TPA response elements in the 5′flanking promoter region and the sequences surrounding it. In order to locate the position of TPA response elements, the NGAL fragments for －152～+84, －140～+84, －78～+84, －59～+84, －50～+84, －41～+84, －37～+84, －29～+84 and －10～+84 were obtained from esophageal cancer cells by PCR and nested deletion and cloned into pGL3-Basic (pGLB), pGL3-Enhancer (pGLE) and pGL3-promoter (pGLP) vector which are luciferase reportors, respectively. The constructed recombinant expression plasmids pGLB-152, pGLP-152, pGLE-152, pGLB-140, pGLB-78, pGLB-59, pGLB-50, pGLB-41, pGLB-37, pGLB-29 and pGLB-10 were adopted to cotransfect with pRL-TK into EC109 esophageal cancer cells stimulated with TPA (5 μg/L) respectively or not. Relative luciferase activity of whole cell extracts from cells was measured with Dual Luciferase Report Gene Assay System (DLR) to verify the position of TPA response elements. Results showed that the TPA response elements of NGAL are located on －152～－60 region and have a strong response to TPA stimulation in the esophageal cancer cells. The analysis by bioinformatics showed that the potential TPA responsive element in －152～－60 position of NGAL is a new sequence. This suggested that there is the structural character responding to TPA induction in the gene. These results will help to clarify the molecular mechanisms of NGAL overexpression in malignant transformation of human immortalized esophageal epithelial cell induced by TPA.

Abstract:NF-κB has been reported to play an important role in immunity, inflammation and carcinogenesis by regulating expression of its target genes. Thus, NF-κB was recognized as a promising target for the prevention and treatment drugs against cancer and inflammation. NF-κB sequence, reporter gene SEAP and selection gene for Zeocine resistance in plasmid pNiFty-SEAP were transfected into HEK293 cells. Therefore, NF-κB activity was able to be evaluated by SEAP activity in the transfected cells. Based on pNiFtySEAP/HEK293 cells, a screening assay was established, by which known NF-κB stimulators TNFα and PMA were demonstrated to activate the NF-κB in dose and time dependent manners. In contrast, NF-κB inhibitors PDTC and NAC showed inhibitory effects on the NF-κB activity in indicated doses and times. Except several characteristics such as stability, sensitivity, sepecifity, the assay was capable to screen both agents inducing and inhibiting NF-κB activity at the same experiment, which will be useful for the discovery of compounds targeting NF-κB signal pathway.

Abstract:One of the important approaches to structure analysis is protein fold recognition, which is often applied when there is no significant sequence similarity between structurally similar proteins. A framework with a three-layer support vector machines fusion network (SFN) is presented. The framework is applied to 27-class protein fold recognition from primary structure of proteins. SFN uses support vector machines as member classifiers, and adopts All-Versus-All as multi-class categorization. Six groups of features are divided into major and minor ones by SFN, and several diversity fusion schemes are correspondingly built. The final decision is made by dynamic selection of the results of all fusion schemes. When it is still difficult to know what kind of fusion of feature groups can achieve good prediction，SFN is a dependable solution by selecting the optimal fusion of feature groups automatically, which can ensure the best recognition. Overall recognition system achieves 61.04% fold prediction accuracy on the independent test dataset. The results and the comparison with other approaches demonstrate the effectiveness of SFN, and thus encourage its further exploration.

Abstract:HEK293 or HeLa cells were transfected by NIRF and, or P53, whole cell extracts and immunoprecipitates were subjected to SDS-PAGE followed by Western blotting. GST pull-down was carried out to identify the interactions between NIRF and P53. In vitro ubiquitination reaction was carried out to identify P53 ubquitinate by NIRF. The results suggested that NIRF could interact with P53 in vivo and in vitro. The results also showed that NIRF could ubiquitinate P53 in vivo and in vitro. The results indicated that NIRF would be a new negative regulator of P53.

Abstract:The rice Rim2/Hipa is a stress-induced transposon superfamily recently identified in Oryza genomes. Genomic variation was found in the Rim2 core region among rice genetic resources/genomes, indicative of high genomic divergence accumulated during the Rim2 evolution. Based on the divergence and quiescent state of the Rim2 elements, a Rim2 paralog display-based fingerprinting approach was developed to effectively identify rice genetic resources and explore their genetic relationships within a set of rice germplasm including 45 accessions of O. sativa and 8 accessions of its wild relatives O. rufipogon. A dendrogram showed not only clear genetic diversity of rice germplasm, but also considerable genetic differentiation among wild rice resources. The wild rice relatives were either clustered as an independent group, or among the japonica varieties. This Rim2-based fingerprinting approach could also serve as a sensitive tool to identify rice hybrids from their parents, and variety stability, demonstrating its great potential in evolution study of rice genomes and in rice breeding and seed production.

Abstract:Acutely isolated rat hippocampal CA3 pyramidal neurons were irradiated with a semiconductor laser for wavelength 670 nm and power 5 mW. And properties of voltage-gated Na+ channel were studied using the whole-cell patch clamp technique. The experiment revealed that activation voltage and peak voltage of Na+ channel shifted towards more negative potentials in irradiating 5 min and the action of laser irradiation reached stabilization in 7 min. There was no effect on peak currents of Na+ channel using laser irradiation. The peak current density of control group and irradiation group were (－383.51 ± 26.93) pA/pF and (－368.36 ± 33.14) pA/pF respectively (n=8, P > 0.05). －40 mV activated threshold potential and －30 mV peak potential for control group respectively dropped to －60 mV and －40 mV after irradiating 7 min. The half-activation voltage and the slope factor of the activation curves of Na+ channel were also changed by the laser's exposure. The former changed from (－42.091 ± 1.537) mV to (54.971 ± 1.846) mV (n＝8, P < 0.01) and the latter form (1.529 ± 0.667) mV to (2.634 ± 0.519) mV (n=8, P < 0.05). The results show that activation properties of Na+ channel are influenced by laser irradiating hippocampal neurons. Thus, depolarizing process of action potential is affected. And further physiological functions of neurons are altered as a result of low level laser irradiation.

Abstract:The DegP protein, functioning as both chaperone and protease, plays a critical role in degrading and removing denatured or damaged proteins in the cellular envelope during heat shock and other stresses. So far, several proteins have been identified as its natural targets. A carboxyle-terminal peptide derived from the PapG pilus, one of the in vivo substrates for DegP, has been shown to activate the protease. Nevertheless, neither the details nor the physiological implications of such activation have been studied. The evidence that DegP undergoes conformational changes upon binding the peptide derived from C-terminal sequence of pilus subunit PapG has been presented. It demonstrated that upon binding this peptide, detectable changes can be observed for both secondary and tertiary structures of DegP, as examined by CD spectroscopy. Gel filtration and dynamic light scattering analysis also revealed that the size of DegP becomes smaller to a minor extent. Moreover, both the hydrophobic surfaces and catalytic sites of DegP were found to expose slightly in the presence of the peptide. Upon peptide binding, a less flexible and more rigid conformation of DegP was obtained as analyzed by fluorescence anisotropy. The physiological implications of these observations for DegP are discussed.

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Abstract:The atherosclerotic animal model was established, whose pathological changes and the plasma lipid level are as same as the atherosclerotic injury in human, and then the stability of atherosclerotic plaque was studied. The miniswine resemble human in the physiology, biochemistry and anatomy. So it is a very appropriate candidate animal for atherosclerosis study. The miniswine atherosclerotic model by high lipid and high cholesterol diet was successfully established, whose plaque included a necrotic lipid core, a thin fibrin cap, a lot of foam cells and inflammatory cells. Calcify and intraplaque hemorrhage might be found in plaque. Based on the pathological character, the model might be as an appropriate tool for the study of plaque rupture and stability. Atherosclerotic plaque rupture was the major cause of the clinic cardiovascular diseases. To study the mechanics of plaque rupture had important significance. The establishment of the appropriate animal model of plaque rupture was the premiss to study the mechanism of plaque rupture. The established animal model existed deficiency on many aspects, such as uncontrolling and quantifying the pressure which led to plaque rupture, and whether the rupture was really existed. A swine model of plaque rupture was established in the experiment, which had much excellences, such as the pressure which led to plaque rupture could be controlled and qualified in vitro, and the way was very simple. So it provided a very good animal model for studying plaque rupture and the relation of rupture and related factors with rupture.