Objective: To express and purify AgrC, a receptor protein of Staphylococcus aureus and to construct its artificial supramolecular system. Methods: AgrC expression bacterial strain, E. coli TOP10-pBAD-AgrC was constructed. The induction conditions and the extracting methods of the protein were optimized. The recombinant protein was identified by Western blot and purified with HisTrap affinity column. The vesicle was prepared with peptide lipid N+C5Gly2C16, and the purified protein was subsequently inset into the vesicle. Results: The constructed engineering bacterium had an optimal expression at 18℃ and with 0.002% arabinose. The protein existing in membrane was maximally dissoluted with BDH. Conclusion: The recombinant AgrC was expressed and purified successfully, and an artificial supramolecular system was constructed.
To investigate the long term effect of glucose metabolism in gene therapy by using IGF-Ⅰ and MGF, the MGF and IGF-Ⅰ eukaryotic expression vector were constructed, and then imported into the left quadriceps of mice every 2 weeks. The sugar tolerance of these mice was tested at the 15th week after the first injection by Yicheng blood glucose meter (JPS-5). It was found that the balance of glucose tolerance was broken when IGF-Ⅰ gene were injected into mice, the blood glucose could reach 12.07 ± 1.35mmol / L, which is significantly higher than the control group (10.15±0.87mmol/L) and the MGF group (10.58 ± 0.61mmol/L). At the same time, the combine injection of both MGF and IGF-Ⅰcould significant reduce the ability of regulating blood sugar, the value (16.30 ± 2.69mmol / L) was rank highest in all of the tested group (P <0.001). Long-term presence of IGF-Ⅰ may antagonize insulin in vivo and then reduce the ability of glycometabolism, however, MGF does not show this function. Integrating the results of combination with IGF-Ⅰand MGF, the speculation that IGF-Ⅰantagonize to insulin mainly through its N-terminal, and its C-terminal which exposed do the formation and function of glucose metabolism could be made, or MGF promotes muscle cell to absorb and express exogenous DNA, which can be used as an adjuvant of gene vaccine.
Objective: To investigate and compare the effect of neuropilin-1(NRP-1) antisense oligodeoxynucleotides with that of vascular endothelial growth factor receptor-2 (VEGFR-2) antisense oligodeoxynucleotides on the proliferation and apoptosis of human gastric cancer SGC7901 cells. Methods: Transmit thiophosphorylate modified antisense oligodeoxynucleotides of NRP-1 and VEGFR-2 into human gastric cancer SGC7901 cells with liposome separately and simultaneously, detect NRP-1 mRNA levels and VEGFR-2 mRNA levels by reverse transcriptase-polymerase chain reaction(RT-PCR); Measure the changes of cell proliferation by methyl thiazolyl tetrazolium(MTT); Observe cell apoptosis by flow cytometry. Results: The transcription level of NRP-1 mRNA and VEGFR-2 mRNA in SGC7901 cells after transmission with ASODN decreased. NRP-1 ASODN and VEGFR-2 ASODN inhibit the proliferation of SGC7901 cells and meanwhile promotes the apoptosis of these cells. Such effect intensifies as the concentration of ASODN increases; When NRP-1 ASODN and VEGFR-2 ASODN are used separately, no significant difference is detected. Conclusion: NRP-1 ASODN and VEGFR-2 ASODN inhibited the transcription level as well as the proliferation while they promote the apoptosis level of SGC7901 cells. Compared with that of transmitting ASODNs separately,the effect manifests itself more significantly with the combination of NRP-1 ASODN and VEGFR-2 ASODN.
Heart cell therapy provides an unconvention connective measure to compensate for myocyte loss in the infacted heart.Nevertheless poor survival of doner cells is one of the major concerns that hampers a better prognosis.Additionally,poor engraftment and lack of functional coupling of donor cells with the viable host tissue greatly impede cell-to-cell signaling and electric conmmunication. Connexins with its dual role as an antiapoptotic and as a gap-junctional protein,can effectively resolve both of these issues. CXCL12,a member of the chemokine CXC subfamily,may play a role in stem cell survival and proliferation.CXCL12 activates several signaling pathways in stem cells,particularly the survival kinase PI3K/Akt which is also an important mediator of Cx-43 expression.On the basis of these characteristics of CXCL12 and Akt, the potential of overexpressed CXCL12 to improve Cx43 expression via PI3K/Akt pathway was investigated.Mesenchymal stem cells were transfected with adenovirus for increasing CXCL12 secretion.Connexin40, Connexin43, Connexin45 and Akt enzyme were evaluted by Western blot analysis.Transfection CXCL12 resulted increased CXCL12 in situ.Increased CXCL12 induced elevated Connexin40, Connexin43 and Connexin45 expression,which was established by activated PI3K/Akt pathway.Increased CXCL12 leads to enhanced expression of Connexin40, Connexin43 and Connexin45 through PI3K/Akt pathway,which may augment mesenchymal stem cell survival and integration in the infracted heart.
Objective: Carbonic anhydrase I (CA1) not only enhances the hydration reaction but also promotes the formation of CaCO3, which is an essential step for new bone formation. However, there is no direct data to demonstrate the involvement of CA1 in bio-mineralization in cells and tissues. The aim is to determine the role of CA1 involvement in bio-mineralization and ossification with cultured cells. Methods: HeLa cells were incubated with dexamethasone (DEX), β-glycerophosphate (β-GP) and ascorbic acid (AA) that can induce bio-mineralization under physiological condition. The cellular calcification was determined by Alizarin Red-S staining. The expression of Runx2/cbfa1, a protein marker of ossification, was detected in the process of bone formation by real-time PCR. The CA1 expression was determined by Western blot. Acetazolamide, an anti-carbonic anhydrase drug was also added to the culture to determine the role of CA1 in bio-mineralization and new bone formation. Results: Following the stimulation, HeLa cells produced a great amount of calcium-rich deposits. Increased transcription of Runx2/cbfa1 was also detected in the HeLa cells, indicating that the DEX, β-GP and AA mixture launched the process of bio-mineralization and bone formation in the cells. CA1 has a significantly increased expression during this process. Following treatment of acetazolamide, the expression of CA1 considerably declined, and calcium nodule formation was significantly decreased. Conclusion: The above findings suggested that CA1 participates in bio-mineralization and ossification,and plays an important roles in the process.
The regenerated silk fibroin were obtained by separately prepared from the calcium nitrate tetrahydrate-methanol solution [molar ratio of Ca(NO3)2 4H2O∶methanol =1∶2], calcium nitrate tetrahydrate-ethanol solution (molar ratio of Ca(NO3)2 4H2O∶ethanol =1∶2), calcium chloride-methanol system(molar ratio of CaCl2∶methanol∶H2O= 1∶2∶8), and calcium chloride-ethanol system(molar ratio of CaCl2∶ethanol∶H2O =1∶2∶8). Then these regenerated silk fibroins were dialyzed against distilled water for 2 days. The regenerated silk fibroin was used as substrate to prepare silk fibroin powder by using freeze-drying method. The molecular mass of liquefied silk fibroin was measured by SDS-PAGE. Regenerated fibroin fibers were examined by the scanning electron microscope (SEM). To obtain fine images, the lyophilized samples were coated with gold. Their surface and fracture images were captured. The molecular conformation of regenerated silk fibroin was investigated by FTIR. SDS-PAGE results showed that regenerated silk fibroin treated with Ca(NO3)2 4H2O-methanol and Ca(NO3)2 4H2O-ethanol had lower molecular weight than CaCl2-ethanol-H2O and CaCl2-methanol-H2O. SEM images revealed that the Ca(NO3)2 4H2O-methanol and CaCl2-ethanol solvent system disolved the silk fibroin completely. FTIR results showed that the molecular conformation of regenerated silk fibroin was between with β-sheet and random coil. These provided a ground work in research of silk fibron which was suitable to serve as drug delivery material.
Objective: A comparative study of mice surviving under 8Gy high-dose γ ray irradiation by gavaged Acai berry based herbal clean supplementary blend and Noni fruit powder was reported. It was evaluated for eliminating free radicals, reducing the hematopoietic cell apoptosis and immuneregulation. Methods: C57BL/6 mice were randomly divided into groups of half male and half female, Acai or Noni powder used at the same dose, respectively or combinedly gavage use for 10 days before or/and after 60Co-γ irradiation at 8Gy. Firstly in the following 40 days after irradiation the surviving rate was observed; a duplicated test under the same treatments were employed for peripheral white blood cell count, CD4+ and CD8+ lymphocyte type, reactive oxygen species, as well as bone marrow cell apoptosis analysis. Results: The saline group C57BL/6 mice irradiated by 8Gy all died in the first 16 days (n = 20, the same in other groups), the mortality rate 100%, while among the pre-gavaged groups, the survive rate were Acai plus Noni 10/20, Noni fruit powder group 9/20, Acai group 8/20 in the following 40 days after irradiation. And the survive rate in the groups of gavaged after irradiation were: Acai alone 2/20, Noni alone group 7/20, Acai with Noni group 4/20 in the following 40 days after irradiation(30 days after gavage). Gavaged the fruit powder before and/or after irradiation were found all increased leukocytes in peripheral blood, and decreased bone marrow cells apoptosis, and in all the test groups the Th/Tc ratio was maintained in the normal range compared with the control group. But red blood cells and platelets without significant changes in the data, yet showed no correlation change in reactive oxygen levels. Conclusion: Acai berry and the Noni fruit powder were helpful for mouse anti-radiation and had a preventive effect, and they maintained hematopoietic cellular proliferation, decreased apoptosis under radiation, and maintained Th/Tc immune balance that were contributions to the anti-radiation. It was demonstrated that Acai berry and Noni fruit powder can improve hematopoietic ability, decrease the hematopoietic cell apoptosis, and keep immune resistance to radiation damage. It also showed that taking Herbal Clean Energy Acai berry powder and NoniGIA Noni fruit powder obviously had positive effect for preventing the radiation damage to human health.
In order to study the expression and regulation of LEAFY homolog in Populus tomentosa, a 1575 bp 5' flanking sequence of PtLFY gene was cloned by PCR from genomic DNA of Populus tomentosa. The results analyzed by PLACE and PlantCARE showed that the sequence contains TATA-BOX,CAAT-BOX and other regulatory elements, such as MYB binding site involved in drought-inducibility, cis-acting regulatory element involved in the ABA responsiveness, light-responsive elements and other transcription factor-binding sites. It was supposed that the expression of PtLFY was regulated by drought, ABA and light etc. Compared the PtLFY promoter with that of other 5 species by FootPrinter, Both conservation and diversity were found in these promoter sequences. It indicated LEAFY homologues genes perform a similar function in Plants. Furthermore, a plant expression vector carrying PtLFYp∷GUS, called PtLFYp1304, was constructed. The results of Agrobacterium-mediated transient expression showed that the PtLFY promoter was competent to drive GUS gene expression in roots, stems, leaves and floral organs of Nicotiana tobaccum. Compared with CaMV 35S promoter, only slight expression of GUS gene was detected in roots, stems and leaves, whereas strong GUS expression driven by PtLFY promoter in the sepal and stamen of Nicotiana tobaccum.
Objective: Based on Escherichia coli MG1655-ΔtktA, histidine-operon leader region was replaced by strong promoter T5, 6-phosphoglucose dehydrogenase(zwf), 6-phosphogluconate dehydrogenase(gnd) and phosphoribosylpyrophosphate synthetase(prs) were overexpressed. Effects of the above modifications on L-histidine accumulation were investigated. Methods: In strain with tktA interrupted, leader region in histidine operon was replaced by T5 promoter by Red recombination system from bacteriophage λ. By cloning technology, zwf and gnd were coexpressed on pSTV28 and prs was expressed on pQE30. Effects of the above modifications on L-histidine accumulation were compared by fermentation in flasks. Results: Quantified by HPLC, trace L-histidine was accumulated in fermentation mediums for those strains with leader region replaced by promoter T5——MG1655-ΔtktA-PT5, MG1655-ΔtktA-PT5(prs-pQE30), MG1655-ΔtktA-PT5(zwf-gnd-pSTV28), and MG1655-ΔtktA-PT5 (prs-pQE30, zwf-gnd-pSTV28) with 60.12 mg/L, 66.47 mg/L, 89.69 mg/L and 111.56 mg/L, respectively. Conclusion: Modification of leader region in operon strengthened the ability to synthesize L-histidine. Afterward, elevation in oxidative pentose phosphate pathway level and PRPP synthetase activity resulted in increased accumulation.
The Bacillus thuringiensis C-2 strain was isolated from activated sludge and it can tolerant 250mg/L Cr6+ and have better reducing capacity.The results showed that xylose, fructose, corn cake powder, malic acid, succinic acid, citric acid and ions Cu2+、Fe2+、Ca2+ had an active role for reduction and the inoculum size had influence for reduction. The conditions of 37℃ and pH9.0 were good for C-2 strain to reduce the Cr6+.The optimum pH and temperature of chromium reductase was pH7.0 and 37℃. The Co2+, Cu2+, Fe2+, DTT, NADH effectively promoted the reduction.
Objective: The nucleocapsid (N) protein of Canine distemper virus(CDV) was expressed base on Bac-to-Bac baculovirus expression system. CDV-N protein was used as a diagnostic antigen for detecting positive serum of CDV. Methods: N gene fragment was amplified by RT-PCR. Recombinant plasmid Bacmid CDV-N transfected into Sf9 cells was builded.CDV-N protein was expressed and shown by Western blot analysis and indirect immunofluorescence. Results:The N protein was correctly expressed. Thirty six serum were tested at anti-dilution 1∶80, respectivly diluted 1∶40(6.3μg/100μl) with CDV-N protein as the best reaction concentration by indirect ELISA, compared to virus neutralizing sensitivity 96.0%, specificity 81.8% and compliance rate 91.7%. Conclusion:The recombinant CDV-N protein was used as antigen in iELISA and shown to react with CDV antisera as a serological tool for the mass screening of CDV antibodies.
Aim: Construct rat skin flap model for the research of ischemia/reperfusion injury. The flap samples were harvested from the proximal, middle, and distal part of the viable flap and cytokines expression were detected separately in order to assess whether there were significant differences among the three parts and confirm an equitable way of sampling on ischemia/reperfusion injury of rat skin flap model. Method: Samples were harvested on postoperative day 5. The level of IL-1β, IL-6, TNF-α and EPO were detected to assess the sampling way. Statistical analyses were performed using SPSS. Results:Significant difference in cytokines levels was noted between the two groups. But no statistical difference in cytokines levels in either inter-group. Conclusion:Samples can be harvested at any arbitrary point on a skin flap to stand for the inflammatory level of the whole flap. And it should be sampled from the same position on different flaps in the same experiment. However harvest from the proximal point were encouraged for taking parallel samples.
Objective: To establish a NADH depending enzyme activity detection method. Methods: FDH and LeuDH were co-expressed in E.coli and appropriate substrate was added; the FDH activity was measured by NH3 production and the LeuDH activity measured by TLC analysis, which were also detected by measuring the NADH absorbance. The results from both methods were compared. Results: Both NH3 production and the NADH absorbance detection proved FDH activity; at the same time, tertiary leucine production and the NADH absorbance detection proved LeuDH activity. Conclusion: The detection of NADH absorbance changes could be employed in measuring the absorbance of NADH.
Studies on protein expression, modification and interaction have become important in proteomics in the post-genomics era. Phosphorylation/dephosphorylation of proteins is the main mechanism of signal transduction and regulation of enzymes in sperm cells, what’s more, it plays a key role during the sperm-egg recognition and fertilization process. Researches about function of phosphorylated proteins contribute to the understanding of the molecular mechanism of sperm capacitation, hyperactive motility and acrosome reaction process. The progress in the researches of mammalian sperm phosphoproteomics was reviewed that include the phosphoproteomics technology, phosphorylated proteins identification and quantitation, function analysis benefits in finding new important fertilization related biological markers and revealing the sperm development, reproductive potential changes and molecular mechanism of fertilization.
In recent years, carbohydrate-protein interaction has been taken attention by more and more researchers. it plays an important role in the life process and is the bases of signal transmission, immune response, cell adhesion, bacterial infection, fertilization, proliferation and differentiation in living organisms According to carbohydrate-protein interaction and the types of carbohydrates, main three different applications are as fellows: it is used in the immobilization of enzymes, basically there are chitosan, starch and agarose; it is used in recognition of proteins, in this aspect some sugar chips are mainly mentioned; it is used in separation of proteins, basically there are agarose, heparin, glucan. Finally, the applications of carbohydrate-protein interaction in biological chemistry are prospected.
The microalgae photoelectrode prepared by immobilizing microalgae on the porous carbon paper displayed light-dependent electrogenic activity with electron mediators in a three-electrode system. The effect of different immobilization methods, diverse genera of microalgae and different electron mediators on photocurrent responses were investigated. The results showed that microalgae immobilized on the anode via silica sol-gel encapsulation exhibited the best photocurrent response. The diverse genera of microalgae including eukaryotic alga (Tetraselmis subcordiformis, Chlorella pyrenoidosa, Chlamydomonas reinhardtii CC 124, Isochrysis zhanjiangensis) and prokaryon alga (Synechococcus sp. PCC 7942) had similar response, demonstrating that the electrons from photosynthetic electron transfer chain of diverse genera of microalgae could be transferred to the electrode via exogenous artificial electron mediator. Benzoquinone and its derivatives as the electron mediator had anodic photocurrent response due to high redox potential, while methyl viologen exhibited low cathodic photocurrent response because of its low redox potential.
The bioactivity of many natural products including antibiotics and anticancer therapeutics depends on their sugar moieties. Changes in the structures of these sugars can deeply influence the biological activity, specificity and pharmacological properties of the parent compounds. A unique characteristic of sugar moieties is their structural diversity which is greater than that of many other biological compounds. Modification of these parts has become an important strategy to achieve many clinical drug candidates. Research groups have focused upon the development of chemical and enzymatic tools to diversity natural product glycosylation.The glycosylation process of natural products and four strategy to diversity glycosylation were discussed in detail.
tRNase Z is an endonuclease that catalyzes tRNA 3'-end processing in many bacteria, most eukaryotes and all archaea. tRNase Z generates a mature tRNA ending with the 3'-OH of the discriminator nucleotide and a trailer sequence with a 5'-phosphate from CCA-less tRNA precursors, which is absolutely essential for the addition of the CCA sequence, tRNA aminoacylation and protein synthesis. tRNase Z enzymes belong to the superfamily of metallo-β-lactamases. tRNase Z exists the short (tRNase ZS) and long (tRNase ZL) forms. tRNase Z has many functions, such as RNA 3'-end processing, guiding the positioning of protein, rRNA-processing, complementation with the REX2 gene, regulation of cell proliferation and differentiation etc. Further analysis of the function and properties of tRNase Z will play a potential and actual role in the treatment of AIDS and prostate cancer in the coming years.
Multi-gene expression in the same host by genetic engineering is an effective approach in studying the regulation of cell development regulation and in manipulating the cellular metabolism of E. coli. There are mainly three types of vectors to coexpress genes in E. coli, including vectors expressing multiple genes from a single transcription unit, vectors expressing multiple genes from multiple transcription units and vectors expressing a single gene.These types of vectors in their construction principles, characteristics, advantages and transformation strategies had been compared, with a major focus on the principle and methodology of using LIC linker in gene cloning with a multi-gene vector.
A brief analysis about National High Technology Research and Development Program(863)drug design and development aiming at serious diseases topics in "Eleventh Five-Year Plan" was given. The details about topics plan, institutions carrying out the topics and representative research results were presented, these informations maybe be useful to scientific and technical workers.
