Abstract:Mx proteins are one of the key components of the antivirals induced by interferons in many species. They are a class of dynamin-like GTPases and share structural and functional properties with dynamin, such as self-assembly and association with intracellular membranes. A unique property of some Mx proteins is their antiviral activity against a wide range of viruses. These viruses are inhibited at an early stage in their life cycle, soon after the entry into the host cell and before their genome amplification. It is known now that some Mx proteins recognize the viral nucleocapsid components and interfere with their role in viral genome replication. Crystal structures of some members of dynamin-like GTPase have been solved. Solution of the crystal structures of Mx proteins is of major significance for not only understanding their antiviral function but also prophylaxis and control of the emerging and re-emerging viruses.

Abstract:PML NBs (promyelocytic leukaemia nuclear bodies) is subnuclear structure in mammalian cells, which widely takes part in kinds of biological events such as transcriptional regulation, genome stability, response to viral infection, apoptosis and tumor suppression. As protein post-translational modification, SUMO located in nucleus plays important roles in PML NBs formation and degradation. Recent research suggests that the human E3 ubiquitin ligase-RNF4(RING finger protein 4), mediates PML NBs degradation depending on SUMO-2/3 modification of PML, ATO(arsenic trioxide) could facilitate in this process. FRET(fluorescence resonance energy transfer)technology could be used in studying the protein-protein interaction of PML NBs and SUMO protein in living cells. Thus, it is far-reaching and significant to research deeply in the mechanism of the formation and degradation of PML NBs and the interaction between the important proteins during this pathway.

Abstract:Orexins (hypocretins) are novel neuropeptides released by some neurons identified in lateral hypothalamus. They are involved in the regulation of energy metabolism, neuroendocrine, sleep and waking. Recently, more and more evidence have shown that orexins play a key role in the stress and reward including drug addiction. The evidence was sumarized, showing that orexin neurons are well positioned to regulate stress-related neural systems, and orexin systems are involved in the physiological, neuroencocrine, psychological and behavioral process of stress. More recently, the role of orexins in stress-induced relapse of drug abuse has been established. All these evidence implicated that orexin system play a critical role in the process of stress, and their role in stress is dependent on the type of stress, their distribution and connection to other stress-related neurotransmitter systems.

Abstract:Cardiovasculature forms during early stages of embryonic development and enables other organs to develop, maintain and regenerate. Imbalanced growth of blood vessels can give rise to numerous pathological disorders. However, the genes involved in blood vessel development remain largely elusive. Zebrafish (Danio rerio) is an ideal vertebrate model organism for the study of developmental processes, especially for that of cardiovascular formation. 26 transgenic fish lines with blood vessel-specific EGFP expression were identified via a large scale enhancer trap screen mediated by Tol2 transposon in zebrafish. The EGFP expression in some of these lines shows different and unique patterns in different part of blood vessels. The genomic sequences flanking the Tol2 insertion sites have been successfully cloned from 22 lines via linker-mediated PCR, among which 17 sequences could be mapped to a unique location within current zebrafish genome assembly. Expression of 8 flanking genes from 9 transgenic lines was confirmed to recapitulate the expression of EGFP reporter gene in their corresponding lines via RNA whole mount in situ hybridization (ISH). Three of these genes, hhex, ets1a and dusp5 are known to be important for vasculargenesis. Since hhex and ets1a are also expressed in hematopoietic precursors, these transgenic zebrafish should be very useful for the study of both hematopoiesis and vasculargenesis. The rest of these genes, namely zvsg1, micall2a, arl8b (1 of 2), zgc:73355 and hecw2 (1 of 2), are either novel or functionally unknown in zebrafish. Further investigation of these fish lines and corresponding genes will give important insights of blood vessel developmental mechanisms including hemangioblast formation and differentiation, as well as genes and enhancer elements important for cardiovascular system. In addition, these transgenic fish lines could also make invaluable contributions to small molecule screen for drug discovery.

Abstract:Liver progenitor cells are co-precursor cells of hepatic cells and bile duct epithelial cells, a reporter gene was used to research the differentiation of liver progenitor cells. First of all, the cytokeratin 19 promoter segment was cloned from hepatocellular carcinoma cell line HepG2 and then following the promoter a renilla luciferase and a red fluorescence protein's fusion gene were inserted to finish the double report lentiviral vector. Second, the liver progenitor cells were transducted with lentivirus, and then GFP positive cells were enriched by flow cytometry sorting. Third, the GFP positive liver progenitor cells were co-cultured with PT67 cells which could express the molecule-epimorphin for 5 days. Then, it was found that the stable transducted liver progenitor cells' shape were not only transformed and arranged as cord like structure, but also renilla luciferase and red fluorescence protein which enhanced by CK19 promoter were detected. So, these results proved that the liver progenitor cells had been induced to bile duct epithelial cells. The vector enhanced by CK19 promoter can monitor the differentiation of liver progenitor cells in different environment. In a word, this lentivirus vector can help us study the differentiation mechanism of liver progenitor cells, and scan the molecules which can do a help in differentiation.

Abstract:Molluscan engrailed proteins and bone morphogenetic proteins have attracted lots of interests in their potential important roles during embryonic shell compartment boundary formation. Engrailed is also proposed to be one of the important regulators of matrix proteins regional expressions in oyster mantle tissue, therefore determination of the regulation mechanisms on the particular expression patterns of engrailed in mollusks would be of great scientific value. However the study has been bogged down because the whole genomes of the oysters have not been sequenced completely and no oyster cell lines are available at present so that many genes of the regulators and mediators need to be cloned and classic signal pathways assays are hard to be applied. In previous study, the bone morphogenetic protein 2 (BMP2) and the oyster mothers against decapentaplegic 3 homolog (Smad3) have been identified from the pearl oyster Pinctada fucata. It is interesting to investigate the relationships among the oyster engrailed, BMP2 and Smad3 expressions in the oyster. Thereby a partial fragment of an engrailed homolog has been amplified from the genome template of the pearl oyster Pinctada fucata. Alignments with the deduced amino acids sequence show that the DNA-binding homeobox (EH4) domain of the oyster engrailed shares extremely high similarities with other engrailed proteins. Shell notching experiment and semi quantitative polymerase chain reaction results suggest that the change pattern of the oyster engrailed mRNA level is similar to that of the oyster mothers against decapentaplegic 3 homolog (Smad3) in shell repair process. Then the Smad3, the engrailed and the BMP2 mRNA expression levels in primary cultured oyster mantle tissue cells that are treated with dexamethasone (DXM) and hydrogen peroxide (H₂O₂) respectively have been examined. Real time quantitative polymerase chain reaction results show that the engrailed, the Smad3 and the BMP2 mRNA levels in the oyster mantle cells exhibit significant correlations with each other. All the results provide important clues and basis for further study on developmental and signal transduction mechanisms on oyster biomineralisation.

Abstract:Previously it was reported that the 27-nucleotide(nt) repeats in intron 4 of endothelial nitric syanthase (eNOS) were the source of 27-nt microRNA, which play an important role in regulation of eNOS expression. In order to further study the molecular mechanisms in regulation of eNOS gene expression and endothelial cell proliferation by the 27-nt microRNA, it was constructed the 27-nt microRNA highly expression plasmid, which was transferred into HUVEC with lipofectamine. The level of eNOS protein and mRNA, as well as Sp1 and Ap-1,were measured by Western blot and RT-PCR, respectively. The proliferation of the HUVEC was analyzed by MTT. The results showed that the 27-nt microRNA can significantly decrease eNOS mRNA level by 94.6% (0.015 ± 0.006 vs 0.277 ± 0.012, P < 0.01) and inhibit its protein expression by 94.9%(0.012 ± 0.005 vs 0.237 ± 0.010, P < 0.01); Sp1 and Ap1 protein were significantly decreased by 14.0% (0.860 ± 0.013 vs 1.000 ±0.015, P < 0.05) and by 22.0% (0.780 ± 0.033 vs 1.000 ± 0.052, P < 0.05), compared with control, respectively. The growth rate of HUVEC treated with the 27-nt microRNA high expression was significantly decreased, particularly the inhibition in the cell lines transfected with double-length and mutant of the 27-nt microRNA plasmid, by which the doubling-time of growth was increased by 49.4% (29.22 ± 0.25 vs 19.55 ± 0.19, P < 0.05), compared with control. The data suggested that intronic 27-nt microRNA significantly inhibit the eNOS expression and the proliferation of HUVEC, by the time the expression of transcription factors Sp-1 and Ap-1 were altered, strongly suggesting that 27-nt microRNA and transcription factors cooperatively regulate the expression of related genes, consequently the proliferation of HUVEC. Data from the present study may serve as one model of the critical mechanisms through which the intronic microRNA and transcription factor synergisticly were involved in the auto-regulation of disease-related gene expression.

Abstract:Microencapsulated osteoblasts were cocultured with hematopoietic stem/progenitor cells (HSPCs) under hematopoietic niche oxygen concentration to investigate the promoting effort of hematopoietic microenvironment on the expansion of umbilical cord blood HSPCs. The osteobalsts were isolated from human iliac bone and cultured, the third passage of osteoblasts at a density of 8×105 cells/ml were encapsulated in gelatin-alginiate-chitosan (GAC) beads with a diameter of 0.5 mm by the polyelectrolyte-complexation method. Three groups of cells were cultured in 5% oxygen incubator, A′ group with microencapsulated osteoblasts and hematopoietic cells, B′ with only hematopoietic cells and C′ with only microencapsulated osteoblasts. Meanwhile, the similarly grouped cells were cultured under 20% oxygen condition, named as A, B and C groups, respectively. The expansion of HSPCs was evaluated by flow cytometry analysis and colony-forming assays. And the concentrations of two kinds of cytokines, LIF and IL-6, were tested to investigate the mechanism of osteoblast's action. The results showed that human osteoblasts dispersed uniformly and grew well in microbeads. There were amount of micro holes in the beads for nutrients transmission. Lots of hematopoietic cells adhered weakly on the surface of microbeads. After 7 days of culture, the hematopoietic cell expansion folds were (49.0 ± 4.6), (3.3 ± 0.5), (17.7 ± 1.2) and (1.9 ± 0.2) respectively for group A′, B′, A and B. And CD34+ cells in groups A′, B′ and A were expanded (87.6 ± 8.3)-fold, (2.2 ± 0.3)-fold and (14.9 ± 1.0)-fold, but CD34+ cells in group B descended. CFU-Cs expansion folds in group A′, B′, A and B were (9.8 ± 0.8), (3.5 ± 0.4), (6.9 ± 0.7) and (2.6 ± 0.2) respectively. It was indicated that Hypoxic co-culture system could promote HSPCs expansion much more than normoxic co-culture system and somatic cell-free culture system. IL-6 and LIF concentrations in A′, B′ and C′ were significantly higher than those in groups A, B and C, which were consistent with their expansion results. Moreover, microencapsulated osteoblasts could support the expansion of umbilical cord blood HSPCs, especially in 5% oxygen condition. Osteoblasts lived in low oxygen condition could secrete more cytokines and thus regulate HSPCs expansion.

Abstract:The theoretical and experimental studies have showed that the topology of the native structure of protein plays an important role in determining its folding process. The complex network approach was used to analyze the topological characters of the native structure of protein and then explore the relationship between these characters and the experimental folding rates. Several types of network were constructed, including all-amino-acids network, hydrophobic amino acid network, hydrophilic amino acid, hydrophobic-hydrophilic amino acid network and their corresponding long-range interaction network. The statistic characters of the assortativity coefficient and the clustering coefficient were studied. The results indicate that all types of network except for the hydrophobic-hydrophilic one are of the positive assortativity coefficient. Furthermore, there is an obvious linear positive correlation between the assortativity coefficient and the folding rate for the all amino acids network and the hydrophobic amino acid network, which implies that the cooperative interactions of the hydrophobic amino acids are important for proteins rapidly folding into their native states. Moreover, it is found that there is a clear linear negative correlation between the clustering coefficient of the hydrophobic amino acid networks and the experimental folding rates of the corresponding proteins, which indicates that the formation of the triangle construction among the amino acids reduces the folding rates. It is also found that in the long-range interaction networks, the formation of contact pairs linking two distant residues in sequence would slow down the process of protein folding.

Abstract:Previous pharmacological studies found that a water-soluble extract (C-MED-100^TM) of Uncaria tomentosa has antioxidant activity and beneficial effects on DNA repair and immune function. Batch-2 is a novel aqueous extract from cat's claw, however, the free radical scavenging ability and neuroprotective effect of batch-2 have not been reported. Firstly, the neuroprotective effect of batch-2 on the 6-OHDA-induced SH-SY5Y cell damage was detected. Secondly, the component analysis of batch-2 was tested by infrared spectroscopy, HPLC and spectrophotometry.The results showed that batch-2 has scavenging ability to several kinds of free radicals, especial the hydroxyl radical (25 mg/L of batch-2 can scavenge 60% of the hydroxyl radical). The dose dependently attenuated 6-OHDA-induced cell death, lipid peroxidation, mitochondrial potential loss and increase of intracellular reactive oxygen species (ROS) and nitric oxide (NO) in SH-SY5Y cells were also observed. Meanwhile, batch-2 can suppress the 6-OHDA induced increase of expression of iNOS and activity of NF-κB. The results show that the neuroprotective effect of batch-2 on 6-OHDA-induced damage of SH-SY5Y cells by scavenging ROS and NO, inhibiting activation of iNOS and NF-κB. Component analysis shown that the content of polyphenol and quininic acid from Batch-2 was 6.43% and 0.0958% respectively. These results provided evidence to support that the mechanism of batch-2 for antioxidative activity and neuroprotective effect may be similar with (-)-epigallocatechin-3-gallate(EGCG) partly. It is obvious that batch-2 is a potential neuroprotective antioxidant for the precaution candidate of Parkinson's disease.

Abstract:Oxidative stress is proved to play an important role in the pathogenesis of Alzheimer's disease (AD). The protective effect of anthocyanin against endogenous Aβ was investigated in N2a/Swe.△9 cells, which are widely used as AD model cells. Anthocyanin belongs to the family of flavonoids extracted from plants. It was demonstrated that anthocyanin at 100 μmol/L significantly inhibited the oxidative stress by decreasing the vulnerability, intracellular ROS and [NO]_i in N2a/Swe.△9 cells. Oxidative stress induces increased activation of C-JunN-terminal kinase (JNK). It was demonstrated that anthocyanin can decrease the activation of JNK in N2a/Swe.△9 cells, suggesting that anthocyanin exerts its protective effect by inhibiting the activation of JNK. Therefore, anthocyanin could act as an oxidative stress suppressor in protecting the N2a/Swe.△9 cells against Aβ induced cell injury and it is a promising candidate for AD treatment in the future.

Abstract:Converging evidences show that face categorization and recognition are represented in specific brain areas. It is also known from behavioral studies that spatial frequency of pictures takes different roles in different face categorizations, e.g. identity information is carried more by low frequency features, and gender is represented by both high and low frequencies while high frequency is more important for face expression. However, there is no solid experimental data about the neural mechanism of the different contributions of spatial frequencies on face categorization up to now. ECoG was collected from epilepsy patients while they underwent a monitoring session to locate their epilepsy foci with implanted subdural surface electrodes. Pictures with different gender, facial expression and identities were presented to the patients, while they were required to perform a simple detection task to maintain their focus on the pictures. The changes of traditional face related components, N170, were analyzed by event related potentials and the significant changes was also verified on each electrode by permutation test. The N170 latency is found to be delayed for high spatial frequency (HSF) pictures. For faces of unknown person, low spatial frequency (LSF) presentation results in a longer N170 latency, but not for faces of well-known movie stars. Female faces show longer N170 latency in HSF conditions compared to LSF, but male faces have no such effect. No significant effect was found on N170 component for expression. However, analysis based on individual electrodes showed that more frontal electrodes are involved in expression representation; the identity-specific sites are more likely to respond to LSF stimuli, and the gender-specific sites have equal responses to both LSF and HSF stimuli, while the expression-specific sites are also more likely to respond to the LSF stimuli, which is inconsistent with existing behavioral studies, and show significant differences as early as 114 milliseconds. It fits with the cognitive model that expression related information has been analyzed briefly in occipital-temporal areas in the early stage before other brain areas are involved for further processing. In summary, spatial frequency's contributions to identity and gender could be represented by N170 in traditional face related brain areas while expression information, instead of carrying by N170, is distributed in a wider area in occipital temporal region for fast analysis and relayed to other areas, e.g. frontal cortex. This is the first study to study the neural mechanism of spatial frequency's contributions in face categorization by ECoG, and provides a new perspective to understand the brain dynamics of feature processes for face perception.

Abstract:K-ras gene mutation is generally regarded as a significant indicator for colorectal cancer screening and early diagnosis and in favor of filtering out colorectal cancer patients benefiting from epidermal growth factor receptor (EGFR)-targeted therapy. The chip-based temperature gradient capillary electrophoresis (TGCE) was successfully used to detect K-ras gene mutation in 98 cases of paraffin-embedded colorectal cancer tissues with high sensitivity. Mutation-positive detection rate from chip-based TGCE (47.96%) was significantly higher than that from the PCR product direct sequencing (23.47%). Clone sequencing showed that this detection system could detect at least 2.08% of the mutant K-ras gene. Analysis of the relationship between K-ras gene mutation and clinical pathological parameters revealed that K-ras gene mutation rate in rectum cancer was significantly higher than that of colon cancer (P < 0.05), but no remarkable correlation between K-ras gene mutation rate with age, gender, histological type and tumor stage was observed. Therefore chip-based TGCE is very sensitive and rapid method for mutation detection, and it can be applied for large-scale screening, early diagnosis and guiding clinical treatment.

Abstract:The characteristics of recovery cycles in inferior collicular (IC) neurons of leaf-nosed bat (Hipposideros armiger) and effect of the recovery cycle on the following pulse repetition rate were studied using mimic CF-FM sound stimuli emitted by free flying bat. Recovery cycle of 93 IC neurons were obtained from IC of five bats with normal hearing. These neurons were classified into three types, i.e. long recovery (LR, 47.4%), moderate recovery (MR, 35.1%), and short recovery (SR, 17.5%), according to their inter pulse interval (IPI) (ms) of 50% recovery under two CF-FM sound stimulation condition. Each type of the neurons could also be categorized into different sub-types according to changes induced by IPI increasing such as single-IPI response area neurons, multi-IPI response area neurons, and monotonic-IPI response neurons. Mean IPIs of 50% recovery of LR, MR, and SR neurons were (64.0 ± 24.8), (19.6 ± 5.8), and (7.1 ± 2.4) ms, respectively (P < 0.001). The calculated theoretically following pulse repetition rate (pulse per second, Hz) of LR, MR, and SR neurons by mean IPI of 50% recovery for each type were (18.2 ± 7.0), (55.4 ± 15.7), and (171.3 ± 102.9) Hz, respectively (P < 0.001). These three types of IC neurons were well corresponding to their three hunting phases, search, approach, and catch phases. The sub-types of single-IPI response area neurons and multi-IPI response area neurons had hunting phase selectivity, and sub-type of monotonic-IPI response neurons had well sensitivity to IPI change, but their hunting phase selectivity was of a sort. These results demonstrated that recovery cycle of IC neurons determined the ability to follow pulse repetition rate and matched this bat's echolocation behavior.