Adenosine Monophosphate-activated protein kinase (AMP-activated protein kinase, AMPK), a serine/threonine protein kinase, regulates cellular energy homeostasis. Based on yeast two hybrid assay,63 proteins identified interacted with AMPK β1 subunit in the human universal cDNA library. Among these proteins, there are 30 enzymes including metabolic enzymes, kinases and SUMO protease, 9 transcription factors or their coregulators, 5 transport/cargo proteins, 4 GTP-binding proteins, 3 adaptors and some proteins involved in cell cycle, DNA repair and cell growth and/or maintainance. These results suggested that AMPK may play a key role not only in the metabolism but also in non-metabolism processes in the cellular functions.
Objective: To investigate the ability of multilineage differentiation of human umbilical cord-MSCs by subculturing to 23 passages.Methods: Mesenchymal stem cells were isolated from umbilical cord using collagenaseⅡdigestion and were digested by trypsin for continuous passage culture. A series of different generation cultured-cells were collected for following experiment. The cells surface antigens and cell cycle were measured by flow cytometry; The cell growth curve was analyzed by MTT assay; Adipogenic、osteogenic and chondrogenetic ability were evaluated by specially inducing culture; OCT-4、SOX-2 and Nanog mRNA expression level were assayed by real-time fluorescence quantitative PCR. Result: MSCs can be obtained by collagenase digestion method. They had uniform shape, and can be stably passaged over 23 generations. By subculturing to 23 passages in vitro, the expression rate of surface marker in cell was not significant changed; the shape of cell growth curves were similarity; there was also no obvious difference in cell cycle, (73.04±1.15)% of the cell were G0/G1 phase; cells can be induced into osteoblasts, chondrocytes and adipocytes; OCT-4、SOX-2 and Nanog mRNA expression level were no obvious difference. Conclusion: Subculturing to 23 generation in vitro, the ability of mutilineage differentiation of hUC-MSCs was not influenced with increase in numbers of subculture.
Interleukin 10 (IL-10) is a pleiortropic cytokine which plays a pivotal role in the inflammatory response, immunological reaction and diseases development. RGD peptide has been demonstrated to bind to integrin on neovasculature endothelial cells specifically. A novel hybrid protein combining RGD with IL-10 was designed. The DNA sequence encoding recombination fusion protein IL10-RGD was subcloned into the pET22b(+) vector for protein expression in E. coli strain BL21(DE3). SDS-PAGE analysis showed an induced expression product band, with a molecular weight of about 19.3kDa, which was consistent with the expected value. The recombinant protein was isolated and purified by Sephadex G25M, and then renatured. Cultured human dermal fibroblasts were treated with transforming growth factor β1 (TGF-β1) stimulation and IL10-RGD treatment. The protein levels of various fibrosis related molecules were assessed by Western blot. The change of α-SMA positive fibroblasts was analyzed by immunocytochemistry. The results showed that IL10-RGD can significantly down-regulate the protein expression levels of Col1, Col3 and α-SMA, and decrease α-SMA positive cells in cultured dermal fibroblasts stimulation with TGF-β1. These findings indicate that IL10-RGD has the antifibrotic effect on dermal fibroblasts, the potential usage in fibrosis diseases by improvement the abnormal deposition of ECM, and suggest that IL10-RGD may be beneficial for the treatment of skin fibrosis and pathological scars.
Objective: The celY gene encoding endo-1,4-β-D-glucanase from a strain of Erwinia chrysanthemi was amplified by PCR and cloned into plasmid pUC19. To improve the expression and secretion of CelY, different regulatory elements were compared in driving the expression of CelY in Escherichia coli. Methods: A series of celY expression vectors containing different regulatory elements, such as lpp promoter, T7 promoter, pelB signal peptide, which were compared with the regulatory elements of celY, were constructed. Results: The series of celY expression vectors driven by lpp promoter, T7 promoter, pelB signal peptide, in varying degrees, increased the secretion expression level of CelY. Conclusion: The lpp promoter, T7 promoter, pelB signal peptide could be considered as candidates for the construct of a high-effective secretion vector.
S-mandelate dehydrogenase(SMDH) can catalyze S-mandelic acid to benzoylformic acid. The SMDH nucleotide gene(mdlA)was cloned from DNA of Pseudomonas putida NUST by PCR, and the amplicon was inserted into prokaryotic expression vector pET-30a (+). This recombinant was transformed into E. coli BL21(DE3) and then highly expressed by induction of IPTG. The result of SDS-PAGE showed that the molecular weight of cloned SMDH was about 43kDa. The recombinant strain could catalyze S-mandelate to benzoylformic acid.
Background: The etiology of Parkinson's disease(PD) involves genetic, environmental, endogenous neurotoxins and other factors. But the current weight of evidence strongly suggests that environmental neurotoxins may play an important role in the etiology of PD. Rotenone is a classical mitochondrial complex-I inhibitor and is the most potent member of the rotenoids, a family of isoflavonoids extracted from Leguminosae plants. More recently, chronic intraperitoneal injection of rotenone has been shown to cause dosedependent dopamine (DA) loss, reduced tyrosine hydroxylase (TH) immunoreactivity, and behavioral abnormalities in rats. PTEN-induced kinase 1 (PINK1) is linked to recessive PD. Pink1 deletion results in impaired DA release and decreased mitochondrial respiration in the striatum of mice. Objective: To test if overexpression of PINK1 in C57BL/6 mice striatum may alleviate the injury of dopaminergic neuron caused by Rotenone. Methods: Lentivirus of GFP-PINK1-LV, GFP-PINK1G309D-LV or GFP-vector-LV were injected into the C57BL/6 mice (male, 7 weeks old,18-20g weight) striatum. Two weeks later, Rotenone dissolved in DMSO was injected into the C57BL/6 mice lateral ventricles. After 2 weeks, TH protein level in C57BL/6 mice striatum was measured by Western blotting and Immunohistochemical staining. Meanwhile the behavioral performance was evaluated in Open Filed experment. Results: Western blotting and immunohistochemical experiments have proved that over-expression of wild-type PINK1 in C57BL/6 mice striatum may rescue the TH protein level reduction, but can not alleviate the behavioral injury caused by Rotenone.
To study the effects of carbamoyl phosphate synthetase and aspartate carbamoyl transferase on cytidine fermentation of E. coli, carAB and pyrBI were amplified by PCR from E. coli A39 (△cdd) chromosome and ligated into the pSTV28 vector. Then the recombinant plasmids pSTV28-carAB and pSTV28-pyrBI were transformed into E. coli A39 (△cdd). And the effects of pSTV28-carAB and pSTV28-pyrBI on cell growth, cytidine and uridine production and accumulation of acetic acid were studied by fermentation experiments. The results suggested that cytidine productions of E. coli A39-AB and A39-BI were 583.5 mg/L and 408.4 mg/L, which were increased by 85.3% and 29.7% compared with A39 (△cdd). It shows that Overexpressing carAB and pyrBI operon can promote the accumulation of cytidine.
The magnesium alloy (Mg-AI-Zn) covered with tricalcium phosphate (β-TCP) was electrosynthesized and evaluated its surface characteristics and biocompatibility. The β-TCP-Mg-AI-Zn has been prepared by electrochemistry, whereas the surface structure and spectrum analysis of material were measured. The osteoblasts from mice skull were cultured with magnesium alloys in vitro, and the cell proliferation and alkaline phosphatase(ALP)were detected with methyl thiazolyl tetrazolium and fluorescein isothiocyanate. The results show that the β-TCP-Mg-AI-Zn surface was porosity and high content of magnesium, calcium and phosphorus elements. The cell proliferation and the activity of osteoblasts were increased after cultured 24 h or 48 h with the β-TCP-Mg-AI-Zn (P＜0.05). Therefore, covering with β-TCP has improved surface properties and cell compatibility in vitro. The magnesium alloy covered with β-TCP would be a new biomedical material.
Agrobacterium tumefaciens-mediated wheat genetic transformation system was set up by using immature embryos of spring wheat cultivar Longchun 22 as explant. 4-day-preculture immature embryos were transformed with pta gene using Agrobacterium tumefaciens C58c1. Three transgenic plants were gained after G418 resistant screening and PCR analysis. Real-time PCR analysis showed that pta gene copy number in transgenic wheat plant is 2,1,3, respectively. The exogenous pta gene have integrated into three plants out of six T1 progeny plants confirmed by PCR analysis, and the transgenic wheat showed good resistance to aphids in greenhouse.
Apoptosis plays an important role in limiting viable cell density and production performance in large-scale animal cell culture. Overexpression of anti-apoptotic genes is one of the most common strategies used to improve apoptosis resistance of industrial cells. HEK293 cells with adenoviral anti-apoptotic E1B-19K gene were transfected and picked out several representative clones. These clones were further investigated in the apoptotic percentages under various inducing culture conditions and cell growth and metabolism under normal culture condition. The results showed that the overexpression of E1B-19K in HEK293 cells, endowed cells anti-apoptotic ability by decreasing cell death by 60%~80% under inducing culture conditions with reduced glucose, low serum or without glutamine, delayed cells' entry into decay phase by 2 d under normal culture conditions; and had no significant affect on cell metabolisms of glucose, lactate and glutamine. The above results indicate that the overexpression of E1B-19K is a promising strategy to reduce cell death in mammalian cell culture.
Objective: To obtain the hybridoma cell line stably secreting monoclonal antibody (mAb) specific for F glycoprotein (F) of human respiratory syncytial virus (RSV) for the further study of early detection and passive immunotherapy against RSV infection. Methods: The mAbs against F were prepared by hybridoma technology and then the associated characteristics were identified in vitro. Results: The hybridoma cell line of F8 capable of stably secreting mAb was established. It grew well after continuous culture for two months, the resultant supernatant titer of the mAb is 1∶1000. The mAb is IgG1 and its relative affinity (Ka) is 6.8×108 L/mol. The potential epitope recognized by F8 is located around AA 205-222 of F1 subunit of RSV F. Through the immunoenzyme method, the neutralizing activity and fusion inhibition capacity of mAb F8 in vitro were also confirmed. Conclusion: The mAb F8 against RSV F with neutralizing activity was obtained. It lays a foundation for early diagnosis and passive immunotherapy of RSV infection.
Using a small cassette DNA encoding library construction strategies, DNA template encoding 10 random amino acids was synthesized according to degenerate codon of a 15 kinds amino acids set. After three consecutive small cassette library assembly process including PCR amplification, digestion and connection, a small cassette DNA encoding library which a random coding region with length up to 97 amino acids was successfully constructed, and library capacity was 1.31 × 1012/ml.
HIV-1 integrase (IN) mediates the integration of viral cDNA into the host genome, which is a key process for viral replication. The 3'-processing of viral DNA extremities is the first step in the integration process, and kinetic studies on the 3'-processing reaction will be helpful in mechanism studies of IN and anti-IN drug design. Wild type IN recombinant plasmid was constructed and IN was expressed in E. coli BL21 cell. Subsequently, IN was purified through denaturation and refolding procedure from inclusion bodies. Based on molecular beacons, fluorophore and quencher-labeled DNA substrate was designed and applied in real-time monitoring of 3'-processing reaction as well as subsequent kinetic study of the reaction. As a result, the purified IN was active, and IN prefered Mg2+ as the cationic cofactor in the reaction. The studies on its enzymatic kinetics (Km = 131.79 nmol/L and Kcat = 0.0042 min -1) elucidate that both the molecular beacons based real-time assay and the designed DNA substrate are suited for use in 3'-processing kinetic study as well as other reaction characters studies.
Objective: To effectively express Geotrichum candidum lipase (GCL) in Pichia pastoris, optimize the culture conditions of the recombinant strain in shaking flask and investigate its high-level expression in a 3L bioreactor. Methods: The inducible secretion vector pPIC9K-gcl was transformed into P. pastoris GS115 by electroporation. Clones with high lipase activity were screened by olive oil-rhodamine B plates and shaking flask cultivation. Real-time PCR based TaqMan probe was applied to determine the copy number of G. candidum lipase gene in the target clones. Then six key factors of shaking flask cultivation were optimized, including induction time, methanol concentration per 24h, culture medium initial pH, inoculation concentration, culture medium volume in shake flask and induction temperature. Based on the results of shaking flask cultivation, fed-batch fermentation was implemented in a 3L bioreactor. Results: A clone GS115/pPIC9K-gcl 78# which possessed three copies of G. candidum lipase gene was obtained. Its initial lipase activity was up to 220 U/ml. The results also showed that when the cultivation conditions were 96 h after methanol induction, 1% of methanol addition per 24 h, 2% of inoculation concentration, initial pH 7.0 of culture medium, 50 ml of culture medium volume, and induction temperature at 25 ℃, the lipase activity reached 735 U/ml. In the 3L bioreactor, lipase activity achieved 3360 U/ml and the total protein concentration reached 4.30 g/L after 176.5 h cultivation. Moreover, the cell viability was above 96% during the high cell-density fermentation. Conclusion: Pichia pastoris GS115 is a suitable host for high-level expression of G. candidum lipase. Multiple gene copies had a positive effect on expression level of GCL. Single factor optimization can effectively enhance lipase expression. This high cell-density fermentation would offer a solid basis for large-scale production of GCL.
Monoclonal antibody (mAb) with increasing importance to modern medicine is currently one of the major biopharmaceutical products in development. Mammalian cells are the preferred host for the manufacture of mAb, but production costs are still high with a low productivity. A range of rational engineering strategies have been pursued in order to increase volumetric yield of production from mammalian cells, such as delaying apoptosis, manipulating the cell cycle, and improving metabolism and protein processing. Many engineering strategies developed to date are reviewed and the possible areas of research in the future are discussed.
Methicillin-resistant Staphylococcus aureus (MRSA),is a typical member of superbugs and a serious threat to people's health. However current antibiotics and the active vaccination can not control immunization of MRSA infection effectively. In recent years, with the rapid development of antibody-drug industry, many drug RD companies and researchers working on therapeutic antibodies begin to focus on the development of new antibody drug to control of MRSA infection. The progress and current status of therapeutic antibodies against MRSA were discussed.
Tissue engineering is a new idea of the modern reconstruction medicine. Biological scaffold and seed cells are two important elements for this new technology. Self-assembled peptides nanofiber scaffolds(SAPNS) are amphiphilic peptides,These peptides self-assemble into a new type of scaffold which has three-dimensional network structure under certain conditions. Its Properties such as structure, biological function, mechanics are similar to native extracellular matrix(ECM), and its internal functionalized modifications of epitope present in high concentrations on surface of nano-fiber and high- efficient selectively regulate the biological behavior of seed cells. Seed cells is the necessary condition for successful organization regeneration. Bone mesenchymal stem cells (BMSCs),due to its strong self-renewal and multiplex differentation potential,is the best candidate cell for tissue engineering. In vitro experiments showed that these specific functionalized modified of SAPNS can promote adhesion, proliferation, migration and directional differentiation of BMSCs with/without auxiliary factor.In vivo animal models found that the combination of SAPNS and BMSCs build up tissue engineering grafts can repair organic structure and function of the defect sites,so it have a good prospect in the restoration medicine. A review of SAPNS,self-assembled,BMSCs and directional differentiation was given.
Protein glycosylation is the important step involved in post-translational modifications within eukaryotes, and it plays a significant role in protein folding and transportational processes. The oligosaccharide moieties of glycoconjugates have a major impact on the structure and functions of protein, and influence biological phenomena such as lectin binding, viral infection and cellular recognition. In the field of glycobiology, it is necessary to investigate the homogeneous glycopeptides and glycoprotein for detailed structural and functional studies of glycoproteins. As a result, how to synthesize this kind of glycoprotein and glycopeptides efficiently has become an important subject in glycotechnology. At the present time, three major strategies have been developed to synthesize homogeneous glycopeptides. One is using pre-formed glycosyl amino acids as building blocks in solid-phase or solution-phase peptide synthesis. Another strategy is combining an oligosaccharide glycosylamine with a pre-assembled polypeptide which contains a free or selectively activated aspartyl side chain to synthesize glycoprotein. The main disadvantages of these approaches are that the strong acidic conditions may cause partial hydrolysis of the attached oligosaccharide moiety. To deal with these problems mentioned in the above strategies, a new chemoenzymatic approach combined with endoglycosidase which from microorganisms to synthesize homogeneous glycopeptides is developed. This approach requires only monosaccharide-tagged polypeptides, and the reaction condition is very moderate. The sugar chain extension is combined with free polypeptides in aqueous solutions, without the using of protecting groups. Both glycosyltransferases and endoglycosidases have been explored for this chemoenzymatic strategy. Compared with glycosyltransferase-based methods, the endoglycosidase-based approach is more attractive because it can connect a large intact oligosaccharide to a GlcNAc polypeptide in one-step. Although this endoglycosidase-based chemoenzymatic approach is better than other methods, it still has shortcomings, such as low transglycosylation efficiency and product hydrolysis issue. The synthesis of these homogeneous glycopeptides is still a challenging work because of every strategy has its own advantages and limitations. But compared with other strategies, endoglycosidase-based chemoenzymatic synthetic approach is more attractive owing to its faster developing and more practical significance. Following aspects are focused on: the research progress of endoglycosidase-based chemoenzymatic synthetic strategy, including 4 aspects: (1) Studies on the catalytic mechanism of endoglycosidase. Such as substrate-assisted mechanism. (2) Researches of glycosyl donors of endoglycosidase. Based on the substrate-assisted mechanism of ENGase-catalyzed reaction proceed, sugar oxazolines was used as activated substrates for ENGase-catalyzed transglycosylation, and this substrate surprisingly induced substantial improvement of the synthetic efficiency and high-yield of large glycopeptides. (3) Investigations of mutants of endoglycosidase. Which lack hydrolytic activity but can still combine an activated glycosyl donor to peptide acceptor for catalysis to form a new glycosidic bond. (4) The applications of glycoprotein and glycopeptides which have homogeneous sugar chains and synthesized by endoglycosidase-based chemoenzymatic approaches. These glycoproteins could be used in the study of structure-function relationship of protein, and the study of modifications of protein glycosylation.
Microbial biofilms are composed of a hydrated matrix of biopolymers including polypeptides, polysaccharides and nucleic acids and act as a protective barrier and microenvironment for the inhabiting microbes. The resistantce of biofilms to antimicrobial agents leads to a range of problems, including medical treatment, which highlights the significance of biofilm dispersal. The mechanisms that result in active dispersal of bacteria from biofilm, which include the synthesis of enzymes, the return of motility, surfactant production and cell lysis were reviewed.
The yeast two-hybrid system was one of conventional methods to probe of the protein-protein interactions. This model which was first developed by Fields and Song in 1998, explored the interactions between cellular proteins in vivo. Many researchers favoured this method in the study of protein-protein and protein-DNA interactions because of its high sensitivity. The development of the construction of the bait protein vector in yeast two-hybrid system, its self-activation and toxic to the host yeast were summarized.
