Abstract:Survivin is a recently identified member of the inhibitor of apoptosis protein (IAP)family, which inhibits processing of downstream effector caspase-3 and caspase-7 in cell receiving apoptotic stimulus. Unlike other IAP protein, found during embryonic and fetal development, survivin was completely down-regulated and undetectable in normal adult tissues, and became prominently reexpressed in all of the most common cancers. Survivin plays important pole in regulation of cell cycle. Its expression in tumors has been associated with increased aggressiveness, recurrence and decreased patient survival. Survivin may be a new target for cancer therapy.

Abstract:Insulin is one of the most important versatile hormone, and its functions include regulation of glycemia homeostasis, enhancement of anabolism, control of cell division and differentiation, modulation of cell growth and development. The molecular mechanisms of insulin release and its regulation can be considered as a paradigm of endocrine secretion. Insulin is stored in large dense core vesicles and released by exocytosis, a multistage process involving transport of vesicles to the membrane, their docking, priming and final fusion with the plasma membrane. SNARE proteins are molecular machinery of exocytosis. Pancreatic islet β cells integrate signals of nutrients and hormone/neurotransmitter to release proper quantity of insulin needed for various state. It is well established that glucose and other metabolizable nutrients depolarize the β cells membrane and ensure Ca²⁺ influx through the voltage calcium channel by change of ATP/ADP ratio and other metabolic coupling factors. Hormones and neurotransmitters exert their regulation effects on insulin secretion through the signal transduction of heterotrimeric and monomeric G-protein. There are two regulatory steps on exocytosis, proximal regulatory step via the change of second messengers and distal one at the level of exocytosis machinery itself.

Abstract:Stat3，one of the members of STATs (signal transduction and activators of transcription), activated by polypeptide ligand such as cytokin and growth factors, plays an important role in early embryonic development, epithelial cell apoptosis, skin remodeling and keratinocyte migration et al. The progress in study of Stat3 shows that Stat3 can be constantly activated in many kinds of tumor cell lines; constantly activated Stat3 can make cell malignant transformation happened and repress apoptosis. Study of Stat3 as an oncogene, sets an important basis for explanation of oncogenesis and screening the therapy medicine of many kinds of tumor.

Abstract:Concerns have been raised that the presence of selectable marker genes such as antibiotic or herbicide resistant genes might be unpredictable hazard to the ecosystem as well as to human health. The genes coding enzymes that catalyze special sugars have showed big practical potency in plant transformation as safe marker genes. This kind of sugar catabolic maker genes can give plant cells the ability to use selective agent sugars such as xylose or mannose, therefore the transformed cells could get sufficient energy and grow dominantly while the untransformed cells are starved and inhibited from growing but not be killed. It is called positive selection system. Now the safe marker genes such as xylA（xylose isomerase gene） and pmi（phosphomannose isomerase gene） have been successfully used in plant transformation.

Abstract:Tankyrase, a new telomeric poly (ADP-ribose) polymerase (PARP), acts as a positive regulator of telomere elongation. It can influence chromosomal stability and is closely related to cell senescence, death and carcinogenesis. Its complex pattern of subcellular localizatioon and different subtypes enable it to play varient roles in different cellular sites.

Abstract:Mechanical stresses play critical roles in normal cellular functions and pathophysiological processes.Integrins,which are transmembrane molecules that interact with both extracellular matrices and intercellular cytoskeleton and kinases in the focal adhesion,are important in mechanotransduction.There is increasing evidence that the dynamic and specific interaction between integrin and extracellular matrices is essential for mechanotransduction.

Abstract:With the development of the human genome project, the chicken genome research has made great progress and has significant impact on both animal breeding and basic biological research. The current development of the chicken genome research was reviewed in including the parameter of chicken genome, genetic linkage map, physical map, comparative genome map, expression sequence tag (EST) and identification of quantitative trait laci(QTL) in chicken and the future prospect of the chicken genome research was also discussed.

Abstract:Nuclear factor κB(NF-κB) is an important transcriptional factor and maintains in a cytoplasmically localized inactive state by the inhibitory protein κB(IκB). Following stimulation, NF-κB and IκB import into nucleus through nuclear pore complexes(NPC),mediated by nuclear localization signal(NLS), respectively. In nucleus, IκB binds to NF-κB again. Dependent on the CRM1 pathway, the complexes export from nucleus to cytoplasm, mediated by nuclear export signal(NES). Nucleocytoplasmic transport of complexes is an energy-dependent process which involves small Ran protein and several soluble factors.

Abstract:In order to study the effect of NAG7 gene on NAG7 down-regulated nasopharyngeal carcinoma (NPC) cell line HNE1, the pcDNA3.1(+)/NAG7 mammalian expression recombination was constructed and transfected into HNE1 cells. G418 was used to obtain the neomycin-resistant transformants of which indicated the vector was present in the HNE1 cells. The expression of NAG7 gene was detected by RT-PCR and Northern blot. The cytobiologic characteriztions of the transfected HNE1 cells were probed by population double time (PDT), xenograft of nude mice and cell cycle analysis. The results showed that the PDT of G418-resistant HNE1 cells with expression of NAG7 was longer than that of vector transfected HNE1 cells and untranstected HNE1 cells, and the more NAG7 transfected cells went into phase G0-G1 compared with the two other cells. It also presented inhibited the tumor formation in mude mice. Thus, these data suggested that NAG7 gene play an important role in the cell growth of HNE1, and might be a good candidate of tumor suppressor gene correlated with NPC.

Abstract:Overexpression of the genes induced by nitric oxide (NO) in EC109 esophageal carcinoma cell line was studied by using suppression subtractive hybridization (SSH), reverse mRNA dot blot and Northern blot. The nucleotide of their expressed sequence tag (EST) was sequenced and analyzed by NCBI database. The six mitochondrial DNA coding genes, ND-4L, ND-4, COX-2, Lys-tRNA, ATP-8 and ATP-6 were identified from 69 SSH positive clones. The results indicated that NO can distinctly induce overexpression of mitochondria DNA encoded genes in the esophageal carcinoma cells. In addition, it was discovered that there were single nucleotide substitution in three sites of the fragment of ND-4L and ND-4 genes (10 736～11 449, 10 872 T→C, 11 001 A→G, 11 346 A→G), and one single nucleotide deletion (8 380，A) which will lead to occur the frame-shift mutation in the peptide in the fragment of COX-2/Lys-tRNA /ATP-8/ATP-6 genes (8 011～8 589). The analysis of amino acid sequences showed that an incorrect structural ATP-8 subunit existed possibly in the EC109 cell induced by NO. These results provided a new clue for further exploring the mechanism of NO effecting on carcinoma cells.

Abstract:In order to investigate the relation between Cyclin D₁ protein expression in multistage tissue of pathological changes during the nasopharyngeal carcinogenesis and nasopharyngeal carcinoma(NPC), the simple hyperplasia/ metaplasia, atypical hyperplasia/metaplasia in the nasopharyngeal paracarcinoma mucous epithelium and NPC were studied for Cyclin D₁ expression with immunohistochemical streptavidin-peroxidase (SP) method by tissue array techenique. The positive rates were 30.0%（3/10）,90.0%（18/20）and 62.9%（39/62）respectively,among which hightening “instantaneously” in the atypical hyperplasia/metaplasia appeared. It is shown high Cyclin D₁ expression may be an early incident in the course of nasopharyngeal carcinogenesis, and that atypical hyperplasia/metaplasia probably be an important “toll-gate”.

Abstract:The cDNA of gloshedobin was synthesized and amplified by RT-PCR from the total RNA of snake (Gloydius shedaoensis) venom gland. The 711 bp nucleotide sequence, which encodes the mature gloshedobin, was cloned into expression vector pPIC 9K and transferred into yeast Pichia pastoris, strain GS115. Transfermants with phenotype His⁺Mut⁺ were selected to study their expression. Recombinant protein was conveniently separated and purified from the supernatant by two chromatographic steps: ion exchange chromatography on Q-Sepharose FF and affinity chromatography on Benzamidine-Sepharose 4BCL. Like intact gloshedobin, the recombinant enzyme exhibited strong esterase activity using tripeptide p-nitroanilide derivatives as substrate, but hydrolyzed N-p-tosyl-L-arginine methyl ester (TAME) very weakly. The recombinant protein displayed extreme instability at 37℃ in neutral buffer but higher stability at 0℃, and also, pH is not a key factor to affect its stability while the optimal pH for its enzymatic activity is pH 8.0.

Abstract:A new type of functional material: aminated silica gel was prepared through the hydrolysis and polycondensation of precursors tetramethoxy-silicane (TMOS) and γ-aminopropylmethyldimethoxysilane by sol-gel process.Glucose oxidase (GOD) was cross-linkly immobilized on the carrier aminated silica gel by cross-linking agent glutaraldehyde.The effects of immobilization conditions such as TMOS content，glutaraldehyde concentration，enzyme content given，temperature and pH on the activity of immobilized GOD were discussed in detail.The heat performance and storage stability of immobilized GOD were also investigated. The feasibility of GOD immobilized on the carrier was confirmed by the correlative infrared spectra.The optimum conditions were acquired as follows：TMOS content 10%，glutaraldehyde concentration 2.0%，enzyme content given 1 600 U，temperature 32℃ and pH 5.2.The immobilized GOD had high bioactivity.

Abstract:Under the simulated microgravity, the microtubulin content and the alkaline phosphatase activity of cultured chicken embryonic chondrocytes reduced remarkably, which indicated that the simulated microgravity induced the changes of microtublar system and the calcification of chondrocytes. The antagonistic effects of Mg²⁺ and SeO₃^2- against the changes were studied. The results showed that these changes can be partly antagonized by 1 mg/L Na₂SeO₃ and completely antagonized by 5 mmol/L Mg²⁺.

Abstract:The gene expression profiles of 1 176 genes were analyzed using Atlas^TM Mouse cDNA array 1.2 in Babl/c mice lung tissues during endotoxic shock. Results showed that the expression of 128 genes was up-regulated and 3 genes down-regulated at 2 hours after exposure to endotoxin; the expression of 51 genes was up-regulated and 21 genes down-regulated at 20 hours after exposure to endotoxin. The changes in gene expression were further confirmed by RT-PCR. The significance of above changes in gene expression was analyzed. The results would be helpful to elucidating the molecular mechanism of endotoxic shock.

Abstract:In order to investigate the role of Toll-like recepter 4(TLR4) in lipopolysaccharide(LPS)-induced NF-κB activation in human endothelial cells,LPS-stimulated ECV-304 cells were used as experimental model and the expression of TLR4 and effect of LPS on the expression were analysed with RT-PCR and Western blot assay. Moreover, the role of TLR4 in LPS-induced NF-κB activation in endothelial cells was explored with gene transfection of non-signaling mutant forms of TLR4 and anti-TLR4 monoclonal antibody. The results showed that LPS could upregulat the expression of TLR4 in time and dose-depentent manner and that transfection of non-signaling mutant forms of TLR4 and anti-TLR4 monoclonal antibody inhibited LPS-induced NF-κB activation in human endothelial cells obviously.These data indicated that TLR4 mediates LPS-induced NF-κB activation in human endothelial cells and it may play important role in endothelial cell activation and injury induced by LPS.

Abstract:728 human genes were divided to four groups according to the GC contents of their coding sequences (from GC＜0.43 to GC＞0.58). Examination of synonymous-codon bias in the 4 groups show that NTG (N represents any base of T, A, C, G) is most favored and NCG is most avoided in all four groups. Statistical correlation analysis of GC content in genetic environment with C3/G3 content (the C or G in the 3rd position of codons) and the codon bias in the four groups suggest that C-ending codons are special preferred. This is in favor of accurate translation. Frequency of each amino acid in the four groups was also examined.

Abstract:The envelope glycoprotein D gene of pseudorabies virus Ea strain was cloned by PCR technique. Sequence analysis displayed 98% nucleotide sequence homology and 97% deduced amino acid sequence homology between the cloned gD gene and PRV Rice strain gD gene. Then gD gene was expressed highly in the baculovirus GST fusion vector system. Both SDS-PAGE and Western-blot verified that the expression product was GST-gD fusion protein with the molecular mass of 71 ku. Expressed GST-gD fusion protein accounted for about 20% of total cellular protein. The protective immune assay was performed in mice by using GST-gD fusion protein as antigen. The result showed that all mice immunized by GST-gD could produce a certain level of antibody against PRV gD(antibody titer: 1∶128), and the mice in immunized group could be partly protected against challenge infection of highly virulent PRV Su strain at 2×10⁵ PFU per mice. A good foundation has been laid down for developing PRV genetically-engineered subunit vaccine.

Abstract:Being cultured in high cell density, E.coliBL21(DE3) harboring plasmid PET22b-mETIa were induced by IPTG and then recombinant ETIa were highly expressed.Expressed rETIa were above 40% of total bacterial protein.After primary purification through breaking E.coli, dissolving inclusion bodies, refolding,and further purification by two-step chromatographies, rETIa of electrophoretic purtity has been obtained.Inhibitory activity of rETIa against tPA deletion variant (NTA) has been detected and inhibitory constant (K_i) was 8.72×10^－8mol/L.So affinity chromatography column of rETIa-Sepharose 4B was prepared for purification of NTA.After only one-step purification with this column from refolded NTA,13.2-fold puritied NTA with the specific activity of (565.7±71.3) U/μg and above 90% of purity,have been obtained with the recovery rate of 96.2%.

Abstract:A cDNA library of the venom of Inimicus japonicus was constructed. The cDNA was cloned into eukaryotical expression plasmid pcDNA3.0. SMART^TM protocol was used for cDNA library construction and bioinformatics analysis was carried out. 94 novel EST clones were obtained from 150 sequences in the library, of which there were 35 full-length clones, including cytolysin genes, short neurotoxin gene, C-type lectin gene, macrophage migration inhibitory factor gene and so on. Most of those genes were reported for the first time in Inimicus japonicus. Further studies on those genes and large scales of sequencing in the library are going on, which may be helpful to make clear the components of Inimicus japonicus venom and understand the function of those proteins at molecular level.

Abstract:The cDNA encoding angiogenin was isolated by RT-PCR from human peripheral white blood cell. The bait protein plasmid of pAS-2-1-Ang was constructed, and it's transcription activity was analyzed. Two positive clones were obtained from human fetal liver cDNA library screened by yeast two-hybrid system. The result of sequence analysis and homology comparison showed the candidate protein was lambda-crystallin and human granulin, respectively. The tag plasmid of angiogenin and candidate protein were contructed and cotransfected into COS-7 cell line. The interaction between angiogenin and candidate protein was identified by the assay of co-immunoprecipitation and Western blotting.

Abstract:Rab protein family belongs to the superfamily of ras-like GTP-binding protein. Osrab5B，designated for rab5B gene from rice, encodes a putative Rab protein. In order to investigate the physiological and biochemical functions of this protein OsRab5B, the complete coding sequence (CDS) of Osrab5B was amplified and inserted into a prokaryotic GluGST expression vector, pGEX-4T1, thus constructing the prokaryotic expression vector. The in-frame recombinant plasmid was confirmed by sequencing and was named pG-5BE. Then the vector pG-5BE was transformed into prokaryotic cell E.coli strain BL21 (DE3). The GST fusion protein expressed by this positive strain was purified with GSTrap^TM column. The result from GTP-binding assay proved that the fusion protein has GTP-binding ability.

Abstract:Gene expression profiles of hippocampi and cerebral cortex of genetical epilepsy-prone P77PMC rats and normal Wistar rats were established by using Atlas^TM Rat cDNA Expression Array. The difference of gene expression profiles was analyzed using image analysis instrument. And 15 differential expression genes were discovered each in hippocampi and cerebral cortex. In the hippocampi, 12 genes were high expression in the P77PMC rats but low expression in the normal Wistar rats, 3 genes were high expression in the Wistar rats but low expression in the P77PMC rats.In the cerebarl cortex,13 genes were high expression in the P77PMC rats but low expression in the normal Wistar rats, 2 genes were high expression in the Wistar rats but low expression in the P77PMC rats. Thus, there are several differential expression genes between P77PMC rat and Wistar rat. All these differentially expressed genes may play important roles in the pathogenesis of epilepsy.

Abstract:The human VEGF₁₂₁ cDNA was amplified by RT-PCR, and was inserted into the Pichia pastoris expression vector pPIC9K to form the expression plasmid p9KVEGF₁₂₁. This recombinant plasmid was transformed into GS115. Transformants were screened by G418-YPD plates and were induced by methanol. The expression product of r-hVEGF₁₂₁ amounted to 900 mg/L by a 5-liter fermentor, over 70% of the total secreted protein. The purified r-hVEGF₁₂₁ can stimulate the proliferation of bovine capillary endothelial cells and shows high vascular permeability.

Abstract:To elucidate the phenomenon of protein blowthrough in electrophoretic transfer and its effects on Western blotting. Using survivin antibody (a novel inhibitor of apoptosis protein), several kinds of cancer cell lysate were analyzed by Western blotting. Comparing with the traditional transfer method, two pieces of nitrocellular membranes were applied in the transfer “sandwich”. Then these two membranes were analyzed by Western blotting under same conditions. In some electrophoretic transfer circumstances, the specific Western blotting band corresponding to survivin were detected in both of them. This revealed the phenomenon of protein blowthrough in electrophoretic transfer. The length of transfer time, electric current and the molecular mass of targeted protein all affected the effects of protein blowthrough. Protein blowthrough will lead to “blotting distortion” and affect the result of quantitative analysis and qualitative analysis in Western blotting. The result revealed an important problem which required to pay more attention to in Western blotting. And it will make some help for scientifically mastering and using this technique.

Abstract:A piezoelectric DNA sensor based on self-assembled monolayer technology was developed.In the experiment, 3,3′-dithiopropionic acid was applied to immobilize DNA, 1-ethyl-3-［3-(dimethylamino)propyl］ carbodiimide (EDC) and N-hydroxysuccinimide (NHS) can coat avidin on gold electrode, and then avidin can combine to different segment (25-mer biotinylated oligonuleotide and 169 bp biotinylated DNA probe by PCR) nucleotide on gold electrode surface. The consistency and speciality of the piezoelectric DNA sensor adopted above immobilization method was well. The sensor can be reused by electrode regeneration.

Abstract:The FⅩⅢB short tandem repeat(STR) alleles, four-base-pair difference in length, could be separated and identified by capillary gel electrophoresis(CGE).The gene types of these alleles were determined by runing with them a known-length DNA fragment. This study paved the way for gene typing of STR locus or individual identification by capillary electrophoresis.

Abstract:A miniSAGE (minimal serial analysis of gene expression) technique was established for determining the gene expression pattern from minimal amount of starting material(1 μg RNA). The gene profiles of two fibroblast cell lines were analyzed. One was from a coronary artery disease (CAD) patient, another one was from a normal person. Two high quality SAGE libraries were synthesized. Comparing the SAGE tags in the two libraries, abounding data about the differential expression genes was provided for the research on the mechanism of CAD. The new gene related with CAD could be isolated based on these data. The miniSAGE technique is a powerful gene expression analysis tool for study on complex disease, and can be applied on only 1 μg RNA.

Abstract:The low-affinity neurotrophin receptor p75 plays an important role during retina development and regeneration. But its localization in human retina has not been explored till now. The expression of p75 in human retinae from embryonic 5 to 7-month old was studied using immunohistochemistry method under light microscopic level. The most intensive p75 immunoreactivity appeared in ganglion cell layer, and weak p75 reactivity also presented in other part of the retina. In the retina of 6 and 7-month old embryos, relative strong p75 immunoreactivity appeared in inner limiting membrane, which is formed by the end-feet of Müller cells. The result indicates that, like in rat retina, p75 mainly localized on Müller cell processes though the possibility of p75 on retinal ganglion cells could not be completely excluded.

Abstract:Using recombinant human calmodulin(rhCaM), cyclophilin(rhCyP), cardiolipin and double strained DNA (dsDNA) as coating antigens, an indirect enzyme immunoassay (EIA) for autoantibodies against 4 antigens was developed. and investgated the pre- and post UV-irradiation of polystyrene (PS) microtitre plateas solid phase. It was found that, compared with non-treated PS, the irradiated PS plate could significantly improve the performance of assay result, and its detective sensitivity as well as reproducibility of 4 autoantibodies were significantly elevated. Surface characterizations were done by AFM, and AFM photo-pictures furnished the powerful direct evidence of improving EIA. Antigens were well-distributed on the surface of UV-irradiated PS, whereas non-treated samples shown not uniform, the percent of moleculars which adsorbed was very low and accumulative. XPS analysis revealed the bottom of the wells which irradiated were oxidated, and induced peroxide reactive groups. The ratio of oxygen and carbon (O/C) was 6.9-fold higher than that of non-treated PS, and increased the surface hydrophylicity as well as chemical reactivity. This is the main reason for improved performance of UV-irradiated EIA plates.

Abstract:In order to determine the function of, in vivo, human rennin gene and to establish an animal model for drug inhibitory experiment of human rennin, the transgenic mice were produced via microinjection method by which the mice fertilized ova were injected with the purified human rennin gene. Assay of transgene integration was determined by DIG DNA blotting hybridization and PCR analysis.One mouse among 13 survived mice was the positive transgenic mouse. Integration efficiency was 7.7%. Overall efficiency was 0.3%. Transgene was steadily transfered from generation to generation. The human rennin transgene was found, by RT-PCR, to be expressed in the heart, kidney, and lung of transgenic mice, but not in the liver or skeletal muscle. The mean levels of rennin activity in the plasma of the transgenic mice was also found to be significantly higher than that of the control mice. However, no significant differences were seen in the mean levels of rennin activities in the kidney and heart between transgenic and control mice. These transgenic mice will provide the opportunity to investigate the rennin gene function in circulation (or tissue) rennin angiotensin system, and may, provide an experimental model for testing human rennin inhibitors as drugs.

Abstract:The changes of bacteriorhodopsin's structure and function were monitored by the UV/VIS absorption spectrum technique and the flash photolysis technique over the wide pH range (2.1～12.3). The results of the UV/VIS absorption spectra showed that: 1) the wavelength of the characteristic absorption peak was about 568 nm (pH=5.0～10.0), 2) the red shift of the absorption spectra was observed (pH＜5.0) and 3) the blue shift of the absorption spectra was observed (pH＞10.0). The results of the flash-induced kinetics spectra indicated that: a)M₀ (relative concentration of M₄₁₂) kept around 0.038 constantly (pH =7.3～9.5); b) M₀ decreased gradually as pH was below 7.3; c) when pH was above 9.5, M₀ increased obviously and reached the maximum 0.1355 (pH =11.8) then dropped down sharply; d) t^s_1/2 (halftime of the slow component of M₄₁₂) was (4.1±1.1)ms approximately (pH=2.1～7.3) and e) t^s_1/2 was prolonged to 40 677.4ms sharply (pH＞7.3). It can be concluded that under the condition of high pH, the novel pathway and mechanism of bacteriorhodopsin's photocyle may exist.

Abstract:With advances in areas such as genome sequencing，robotic，proteomics，microeletronics and bioinformaticd，the field of protein and antibody microarrays has recently had an explosion of interest. Current strategies used to generate protein and antibody arrays, their analytical principle and related detection procedure, as well as their current applications in biological research, medicine and diagnostics are reviesdy. The shortcomings of these approaches, future developments required, and the potential applications of this technique will be discussed.

Abstract:Activity of electron transfer in cytochrome c oxidase remains in its thin solid film. The characteristic metal active sites and their structural cycle during catalytic reaction, characteristic spectra, physical electron donor or donor-physically controlled, and switch property, which make the oxidase a candidate for study of molecular electronics and design of nanodevices, for example, erasable byte nanostorage.