Abstract:血管平滑肌细胞(vascular smooth muscle cells，VSMCs)的发育与血管壁的构建是目前相关领域中的重要学科前沿．国内外同行的工作多集中在血管发育初始阶段内皮细胞及其前体细胞在血管新生中的作用、调节因素及生物学机制．VSMCs参与血管壁早期构建，特别是VSMCs的募集与分化机制已经成为血管新生研究中的一个新领域． 本期发表的《 抑制Rac1蛋白活化阻碍胚胎发育早期血管新生 》(见696～701页)报道了韩雅玲教授及其合作者在这一领域取得的最新研究结果．Rac1是真核细胞内重要的一类信号传递分子，在细胞信号传递过程中发挥分子开关作用．他们采用胚胎干细胞(ESCs)为模型，建立稳定表达持续型Rac1和显性失活型Rac1编码序列的小鼠ESCs并制备胚胎小体，诱导分化后观察其对内皮细胞分化和迁移的影响，发现抑制Rac1可以干扰血管内皮细胞连接成血管网状结构，细胞骨架F-actin排列紊乱，细胞的迁移受到明显抑制，表明Rac1在胚胎早期血管发育过程中与内皮细胞的迁移有关[1]． 近年来，韩雅玲教授及其研究集体在VSMCs发育与血管构建、胚胎干细胞来源的拟胚体血管平滑肌发育与血管新生机制以及胚胎主动脉VSMCs起源等方面开展了研究，取得了一系列有价值的成果[2～11]，可能为闭塞性和增生性血管病的发生及防治提供理论依据和候选基因．详见“相关链接”．

Abstract:Selective temporal attention is a developing domain of attention. It refers to people can orient attention to a certain time point or a temporal window associated with an upcoming event. The behavioral and neuroimage researches of temporal attention were reviewed. Some arguments and future trends were pointed out based on the comparison with spatial attention.

Abstract:The shotgun strategy applying tandem mass spectrometry to identify proteins has been widely used in proteomics for its high reliability and efficiency. However, protein deduction is ambiguous due to uncorrected identified peptides and erased correlated information between peptides and their source proteins. Protein quality control methods can be divided into two categories: protein assembly and protein confidence evaluation. To date, parsimony principle, deriving the minimal proteins accounting for all identified peptides, has been widely used in protein assembly. Boolean and probability assembly are two kinds of methods in protein assembly. Protein confidence evaluation includes protein probability calculation of a single protein and false discovery rate estimation of all identified proteins. This review reveals the trend of developing a generalized probabilistic model with consideration of all influence factors, which can be applicable to a variety of peptide scoring system for protein identification.

Abstract:Photodynamic therapy (PDT) is an emerging therapeutic modality based on the interaction of light, photosensitizer and molecular oxygen. As PDT techniques continue to develop and find more potential clinical indications, accurate dosimetry becomes a critical factor for achieving satisfied individual treatments. The current status of strategies and corresponding technologies for PDT dosimetry are reviewed. Typical PDT dosimetry estimations include the direct measurement of three critical ingredients of PDT (i.e. fluence rate, photosensitizer concentration, and tissue oxygen pressure), indirect determination of the photosensitizer fluorescence photobleaching and photoproduct formation, monitoring biological responses, and detecting singlet oxygen production with its near-infrared luminescence around 1 270 nm. The advantages and limitations of each PDT dosimetric model will be presented and the main challenges in clinical PDT dosimetry will be discussed.

Abstract:PKHD1( polycystic kidney and hepatic disease 1), the causal gene of human autosomal recessive polycystic kidney disease(ARPKD), is located on chromosome 6p12.2 and covers a genomic region of ～500 kb. PKHD1 is among the largest human genes, with a minimum of 86 exons from which multiple transcripts may be generated by alternative splicing. The longest continuous open reading frame consists of 12 222 bp, encoding a 4 074 amino acid protein, designated as fibrocystin/polyductin(FPC). FPC is predicted to be a receptor like protein, with single transmembrane domain and a short cytoplasmic tail. The expression of mouse FPC can be detected in various duct-containing organs. In mouse embryogenesis, FPC appears in developing neural tube, bronchi, and the primordial gut as early as the day of E9.5. In fetal human kidneys, high level of FPC expression is present in ureteric bud and its expression continues throughout the process of renal tubuobranching. In adult human kidneys, FPC mainly expresses in the epithelia of renal collecting ducts. FPC is subcellularly localized to the primary cilia and concentrated on the basal bodies in renal epithelial cells. The detail function of FPC is still unrevealed, most recent studies demonstrate that FPC, as a receptor, may transduce cell signal by interacting with a trp superfamily TRPP2 (PKD2), which is a causal gene for ADPKD, and mediate intracellular calcium homeostasis to regulate differentiation, proliferation, migration and polarity of various duct/tubular epithelia, in turn, to modulate the formation of all physiologic ducts, tubules and tracts.

Abstract:Foot-and-mouth disease virus (FMDV) is a positive-sense RNA virus which has caused severe damage to world-wide livestock industry. The extensive genetic and antigenic diversity observed in the evolution of FMDV is generally the obstacle for controlling the disease. The homologous recombination, as a significant force driving the evolution of virus, has also effect on the epidemiological trait of FMDV. However，the role of homologous recombination in the diversification of FMDV in China has not investigated. So it is necessary to study the homologous recombination underlying the evolution of FMDV to control FMD. Based on a sound evolutionary framework, molecular evolutionary analysis was used to identify the putative recombinants. All complete FMDV genomes from China were respectively retrieved from GenBank. Homologous recombination was identified using Simplot program. Phylogenetic relations were analyzed to determine the recombination events among these FMDV isolates by using MEGA 4. The isolates O/NY00, O/China/1/99Tibet, O/Tibet/CHA/99, O/OMIII and O/ES/2001 among 16 FMDVs were identified as putative recombinants by analyzing the FMDV genomic sequences extracted from GenBank. The recombination events frequently happen between serological type Asia1 and O which are endemic FMDV circulating in China, suggesting frequent cross infection of FMDV in China. This situation further makes controlling FMDV in China more difficult. Moreover, serotypic conversion of FMDV between Asia1 and O was detected to be due to homologous recombination. These results provided clues for understanding the antigenic and genetic diversification in FMDV, and shed lights on the potential vaccination and treatment of FMD.

Abstract:Whether small G protein Rac1 plays a role in regulating blood vessel formation especially endothelial cells(ECs) differentiation during early embryonic vascular development remains unclear. Murine embryonic stem cells (ESCs) lines stably expressing the constitutively active Rac1 mutant (Rac1G12V) or dominant negative Rac1 mutant (Rac1T17N) and the resulting embryoid bodies (EBs) were established as models, to observe the effects of Rac1 on the differentiation and migration of ECs. Using contrast phase microscope to observe the development and differentiation features of EBs; pull-down assay to analyze the expression of Rac1 activity; immunofluorescence staining and Western blot to inspect the differentiation markers of ECs；matrigel model to observe the assembly of endothelial network. It showed that neither increase nor suppression of Rac1 activity significantly affect the differentiation of EBs to form the typical primitive germ layers. Expression of dominant negative Rac1 did not affect ECs differentiation, but it significantly inhibited cell migration, furthermore completely blocked the assembly of vessel network. Actin stress fibers were largely absent in Rac1T17N-expressing cells. Expression of Rac1T17N inhibited FAK activity while Rac1G12V did not affect it compared with control group. These results indicated that the effect factor of Rac1 on the vasculogenesis of EBs was to inhibit cell migration which probably mediated by F-actin mechanism.

Abstract:rAdinbitor was cloned from Gloydius blomhoffi brevicaudus in the laboratory. Previous researches had proved that rAdinbitor could inhibit proliferation of C6 glioma cells as well as promote their apoptosis. The molecular mechanism of rAdinbitor’s effects on C6 cells need to be further studied. rAdinbitor was expressed in E. coli BL21/pET23b-adinbitor and purified with Ni Sepharose 6 Fast Flow. The purified protein was confirmed by Western blotting. C6 cells were induced with fibronectin (FN). The effects of rAdinbitor with different concentrations on the expression of FAK, MEK1/2 and Caspase-3 as well as on activity of FAK and ERK1/2 in FN-induced C6 cells were studied by immunoblotting and immunoprecipitation. Results showed that rAdinbitor with different concentrations could obviously reduce the expression of FAK and MEK1/2, increase the expression of Caspase-3, as well as decrease ERK1/2 phosphorylation; besides 10 mg/L rAdinbitor, other concentrations’ rAdinbitor could inhibit FAK phosphorylation obviously. All those effects were dose-dependent. Results indicate that the effects of rAdinbitor on decreasing expression and activity of FAK and inhibiting Ras-MAPK signaling pathway play an important role in suppressing the proliferation of C6. Furthermore, the increase in Caspase-3 expression implies that the increase in apoptosis of C6 cells might be due to the suppression of rAdinbitor on the activity of ILK and PI-3K/Akt pathway.

Abstract:NOR1 is a novel nasopharyngeal carcinoma(NPC) associated gene. It is significantly down-regulated in NPC cell line HNE1 and spontaneous NPC biopsies. Over-expression of NOR1 in HNE1 cell line is effective to inhibit HNE1 cell growth and proliferation. To establish a clearer picture of what the functions of NOR1 might be and to identify cellular proteins that might bind to NOR1, a yeast two-hybrid screen was performed to search for proteins that interact with NOR1. 10 positive clones which encoded for 7 polypeptides were successfully isolated. Among these 7 candidate interaction proteins, one candidate was mitochondria ATP synthase subunit OSCP. NPC cell line 5-8F cells were transfected with pCMV-myc-NOR1 plasmids, then the mitochondrial proteins and cytoplasmic protein were isolated by cellular subftration and subjected to Western blot assay. The data showed that myc-NOR1 protein distributes in mitochondrion and cytoplasm. Immunofluoresence assay also showed the endogenous NOR1 protein co-localized with mitochondrion in human normal nasopharyngeal epithelial cells NP69, which indicate that NOR1 is a novel mitochondrial protein. The interaction between NOR1 and OSCP was confirmed by specific yeast two-hybrid assay, immunofluoresence co-localization and co-immunoprecipitation assay. These primary work suggests NOR1 may be involved in energe metabolism.

Abstract:To investigate the biosynthetic genes and the biological functions of carotenoids on the radioresistant mechanisms of the extremely radioresistant bacterium, Deinococcus radiodurans, the carotenoids of this bacterium were extracted with cold methanol and acetone, then were isolated and analyzed by LC-MS technique. Two colorless mutants knocking out of the phytoene synthase (crtB) and phytoene desaturase (crtI) genes were constructed by PCR and in vivo homologous recombination. The successful construction was confirmed by phenotype observation and carotenoids analysis by high performance liquid chromatography (HPLC). Comparative analysis of the sensitivities among the two colorless mutants and wild R1 showed that the two colorless mutants were more sensitive to ionizing radiation (IR) and ultraviolet (UV), indicating that the deletion of crtB and crtI genes made the mutants unable to biosynthesize the crucial intermediate lycopene and series of downstreaming carotenoids. The scavenging abilities of the two carotenoids were measured using the ultraviolet induction of bacteriophage λ (UIB) system and electron spin resonance (ESR) spin trapping technique in vivo and in vitro, respectively. The results showed that the main two carotenoids exhibited significant antioxidant capacities to superoxide anion (O₂^·) and hydroxyl radicals (·OH) which were two main types of free radicals produced during IR. This study has provided for the first time a new and direct evidence to explore the biosynthesis genes of carotenoids and their biological functions for the radioresistant mechanisms of D. radiodurans.

Abstract:To observe the effect of Helicobacter pylori(H. pylori) on cell gap junction ultrastructure of gastric epithelial cells, and to explore carcinogenic mechanism of H. pylori from the changes of cell gap junction, BGC-823 cells were co-cultured with different H. pylori strains for 24 h and 48 h. The cell gap junction ultrastructure was observed under transmission electron microscope with sample preparation of fixation and embedding in situ. In 70 patients with gastric cancer(GC), H. pylori was detected by rapid urease test, basic fuchsin stain and ¹⁴C-urea breath test. The CagA gene of H. pylori was determined by PCR and the cell gap junction ultrastructure was observed under transmission electron microscope. More cell gap junctions and junction complexes of BGC-823 cells were found in control group without H. pylori. Groups with H. pylori had less number of cell gap junctions, less number of junctions/ unit perimeter, shorter length of junctions /unit perimeter, and bigger width of the intercellular space, comparing to control groups without H. pylori(P < 0.001 or P < 0.005). The number of cell junctions and the number of junctions/unit perimeter in the groups co-cultured with NCTC J99, GC 01 and NCTC 11639(CagA+) were less than that in the groups co-cultured with NCTC 12908(CagA^-) (P < 0.001 or P < 0.05), and the length of junctions/unit perimeter in the groups co-cultured with NCTC J99 and GC 01 was shorter than that in the groups co-cultured with NCTC 12908 (P < 0.001). In patients with GC, the number of cell junctions, the number of junctions/unit perimeter and the length of junctions/unit perimeter in group H. pylori infection were all less than those in group without H. pylori infection(P < 0.001), and that in CagA⁺ H. pylori group were less than that in CagA^- H. pylori group, but its smallest width of the intercellular space was longer than that in CagA^- H. pylori group. The above results showed that the changes of cell gap junction of gastric epithelial cells were associated with H. pylori infection especially CagA⁺ H. pylori infection.

Abstract:A preliminary 3D structural analysis of caveolae from porcine aorta endothelial (PAE) cell has been done by electron tomography. Caveolae of PAE cell were distributed irregularly around the cell surface and aggregated locally to form cluster. The striated structure with a width of 14～16 nm around the caveolae can be seen at the inner or external side of the membrane, and more concentrated striations were found at the narrow neck. A three-dimensional structural model based on tomographic reconstruction shows that caveolae interact with potent microtubule network, suggesting a possible caveolae traffic path in the cell during endocytosis.

Abstract:Shiga toxin 2 (Stx2) toxoid produced by formaldehyde treatment of purified toxin was used to immunize BALB/c mice for monoclonal antibody (MAb) production. The neutralizing activities of positive clones against Stx2 were screened by in vitro cytotoxicity assay. The isotype and specificity of resultant clone was determined, and its efficacy to neutralize the activity of purified Stx2 was evaluated by in vitro and in vivo toxicity model. Lastly, its spectrum of activity against Stx2 variants was also accessed by mouse toxicity model. It was demonstrated that one of the 12 positive MAb clones against Stx2, designating S2C4 had neutralizing activity. S2C4 belongs to the immunoglobulin G1 subclass and has a κ light chain, and it reacts with the A subunit of Stx2 and does not bind to Stx2 B subunit or to Stx1. S2C4 could efficiently neutralize the cytotoxicity of Stx2 to Vero cells and mice. It also protected mice against lethal doses of Stx2 variants challenge including Stx2c and Stx2vha. S2C4 is a promising candidate molecule in preventing the progression of hemolytic-uremic syndrome (HUS) mediated mainly by Stx2 in Stx-producing Escherichia coli (STEC) infection.

Abstract:In order to explore the role of 14-3-3σ gene methylation in nasopharyngeal carcinoma pathogenesis, biopsy specimens of 75 nasopharyngeal carcinoma tissues and 25 normal nasopharyngeal mucosal tissues were harvested for research. Then, methylation specific PCR (MSP) experiment was introduced to reveal 14-3-3σ gene methylation status of tissues, and reverse transcriptional PCR (RT-PCR) assay was employed to quantify the expression of 14-3-3σ mRNA, further more, immunohistochemical staining was undertaken to assess the 14-3-3σ protein expression level. The MSP experiment results indicate 14-3-3σ gene methylation status in nasopharyngeal carcinoma tissues was 4 exhaustive methylation, 59 partial methylation and 12 unmethylation, while in normal nasopharyngeal mucosal tissues was 7 partial methylation and 18 unmethylation. The frequency of 14-3-3σ methylation in nasopharyngeal carcinoma tissues was significantly higher than that in normal nasopharyngeal mucosal tissues (84% vs 28%, χ2=28; P < 0.05). The results of RT-PCR and immunohistochemical staining discovered that 14-3-3σ gene exhaustive methylation leads to absent 14-3-3σ mRNA as well as protein expression in nasopharyngeal carcinoma tissues and normal nasopharyngeal mucosal tissues. Tissues characterized with partial methylation 14-3-3σ gene represented remarkable down-regulated 14-3-3σ mRNA and protein expression level when compared with tissues characterized with unmethylation 14-3-3σ gene. Graded correlation analysis discovered that 14-3-3σ gene hypermethylation status is positively correlated with NPC lymphoid node metastasis and NPC clinical staging. The research demonstrated that 14-3-3σ gene was frequently hypermethylated in nasopharyngeal carcinoma tissues and leads to decreased or depleted gene expression, 14-3-3σ expression level was correlated to nasopharyngeal carcinoma lymphoid node metastasis and NPC clinical staging.

Abstract:GAP-43, netrin-1, collapsin-1, and neuropilin-1 have been regarded to play crucial roles in the formation of patterned neural connections. The cerebellum consists of five distinct concentric layers: white matter, internal granule layer (IGL), Purkinje cell layer (PCL), molecular layer (ML), and external granule layer (EGL) in young rodents. Cells in EGL are generated after birth. In contrast Purkinje neurons are born before birth, which receive main innervations of climbing fibers from the inferior olivary nucleus and parallel fibers from the internal granule cells. These innervations are mostly established in the first three postnatal weeks, accompanying the sprouting and maturation of Purkinje cells. The potential roles of GAP-43, netrin-1, collapsin-1 and neuropilin-1 in the postnatal development of cerebellum remain unclear. To get insights into the above issue, the expression of GAP-43, netrin-1, collapsin-1, and neuropilin-1 mRNAs and proteins were examined in the cerebellum of mice at postnatal days (P) 5, P10, P20 and adulthood. The results showed that these four molecules were expressed in different temporal and spatial patterns in the postnatal cerebellum of mice, which was in match with axonal synaptogenesis, elongation and synapse formation during postnatal development and adulthood. By using double immunohistochemistry, it was found that the Purkinje cells stained for GAP-43 were also positive for either netrin-1 or collapsin-1 at P10, and cells stained for collapsin-1 were also positive for netrin-1 or neuropilin-1. It was suggested that the four molecules are involved in the postnatal development of cerebellum.

Abstract:Growth hormone (GHRH) and pitutary adenylate cyclase activating peptide (PACAP) are the members of the PACAP/Glucagon superfamily, who are related in both sequence and function. Their stimulation of GH secretion and animal growth is concerned. A series of expression plasmid, pIRES1-GHRH-PACAP (P-G-P), pIRES1-GHRH (P-G) and pIRES1-PACAP(P-P), were constructed, extracted and purified, then transfected into CHO cell line with Lipofectamine. The expression was examined by RT-PCR, dot-ELISA and Western blotting. The biological activity of expression products was detected in rats. At 8 h after injection of transfection supernatant, serum IGF-Ⅰ concentrations in P-G-P group were significantly higher than that in other groups(P < 0.05). PLGA encapsulating plasmid microspheres were prepared and injected intramuscularly into rabbit legs. Growth behavior and IGF-1 level were measured at day 0, 15, 30 and 45 after injection. Greater body weights gain and higher serum IGF-Ⅰlevels were observed in three plasmid microsphere injection groups, compared with control group. At day 30, the body weight gain in P-G-P group was greater than saline group (81%, P < 0.01), P-G microsphere group (15%, P < 0.05) and P-P microsphere group (7%, P > 0.05), serum IGF-Ⅰ concentration in P-G-P microsphere group showed a 16.68% increase to P-G microsphere (P > 0.05), a 17.14% increase to P-P microsphere(P > 0.05) and a 50.46% increase to control (P < 0.05). These results suggest that co-expression of GHRH and PACAP in one expression plasmid might exert an additive stimulation of GH secretion and growth when delivered into rabbit skeletal muscle with PLGA microsphere. The results may provide a new approach to regulate animal growth.

Abstract:Peripheral blood mononuclear cell (PBMC) was induced apoptosis when infected by influenza A virus. To observe the interaction between lymphocyte and macrophage, the lymphocyte and macrophage were induced by H1N1 subtype influenza virus. The result showed that apoptosis of PBMC including lymphocyte and macrophage was promoted by virus infection in the first 48 h, but the apoptosis of macrophage was inhibited after 48 h cultivation. According to fluorescent staining and flow cytometry there was no difference in apoptosis of lymphocyte after virus adsorption at different time. The result showed that there was no relationship between apoptosis and adsorption time. The apoptosis of lymphocyte and macrophage was detected when PFT-α was added, the result show that the apoptosis of lymphocyte and macrophage was inhibited. It hinted that p53 may be impact the apoptosis of lymphocyte and mononuclear macrophage that treated with influenza virus.

Abstract:In order to investigate the mechanisms of pregnancy associated with diabetes harm to gravida and fetal, The Kunming mice models for pregnancy associated with diabetes were simulated, The effects of glucose in different concentrations was detected on the growth of embryo cells in vitro culture. The effects of IGF-1 in different concentrations was determined on the development of blastocyst in hyperglycemic conditions (30.0 mol/L glucose) in vitro, as well as the apoptosis of embyro cells by using nuclear DNA double-dyed assays. Moreover, the expression of igf-1 and other genes associated with apoptosis in vitro embryo was detcted by Real-time quantitative PCR. The results showed that the total cell number of blastocyst fell off along with the increasing of glucose concentration. High concentrations of glucose (≥30 mmol/L) could significantly inhibit the growth of embryo cells (P < 0.01). The results of real-time quantitative PCR showed that the expression of igf-1 was down-regulated in mouse blastocyst with pregnancy associated with diabetes, and positively correlated with the concentration of glucose. The expressions of apoptosis-related genes bcl-2 and bcl-xl were up-regulated along with the increasing of IGF-1 concentration, while the p53 gene and the apoptosis-related gene Bax were down-regulated. The embryo cells apoptosis had been gradually reduced with the increasing of IGF-1 concentration in vitro, and in the IGF-1 concentration of 100 μg/L, there was almost no apoptosis in embryo cells. These experiments demonstrated that hyperglycemic conditions in vitro can inhibit the growth and development of the embryo cells resulting in the down-regulated expression of igf-1, and IGF-1 could inhibit the apoptosis of blastocyst and be benefit for the growth of blastocyst.

Abstract:The identification of genes responsible for human diseases based on functional consistency and network topological features is of great importance for both understanding human disease pathogenesis and improving clinical practice. A novel method based on the functional consistency and network topological features was introduced to establish an association between genes and diseases. Using this method, candidate disease genes were predicted from disease risk loci. Then, the candidate genes sharing the same or similar functions with known disease genes in the functional enrichment analysis of GeneOntology and KEGG databases as final disease genes were determined. 51 genes were predicted to be the disease genes for coronary artery disease and most of them participate in the development of disease by literature retrieval. The method provided additional insights for the finding disease genes, which will be helpful for the studies on the pathogenesis of human complex diseases.

Abstract:To investigate the attachment and neurite growth of patterned striatal neurons on the surface of polyethyleneimine(PEI) in vitro, by a soft lithography technique. Three different substrates, including Laminin(LN), poly·L-lysine(PLL) and PEI characterized with strong positive surface charges were micro-patterned using soft lithography techniques. Striatal neurons from postnatal rats were cultured in vitro. The condition of cell adhesion, living and the neurite growth of the cultrued neurons on different substrates were observed, and the differences of neural network patterns fabricated on the three substrates were evaluated. The quantity of neuronal growth on the surface of PEI and PLL is apparently larger than that on LN. Moreover, the coated pattern of neurons on the PEI is more integrated than that on the PLL and LN. The PEI characterized with strong positive surface charges is capable of well fabricating more continuous and integrated neural patterns, which is an ideal interfacial surface to realize the artificial neural network in vitro．