Polypyrimidine tract binding protein-associated splicing factor (PSF), a multifunctional RNA/DNA-binding protein, can suppress the expression of proto-oncogene and function as tumor suppressor protein (TSP) in human and mouse. Using total RNA extracted from human fibroblasts, PSF-encoding cDNA was synthesized and inserted into vector pET-28a(+) to construct the recombinant plasmid pET-28-PSF. The PSF fused with His-tag was expressed in E.coli BL21 (DE3) by IPTG induction and then purified by affinity chromatography. SDS-PAGE and Western blotting results indicated the soluble His-tagged PSF could be produced with a prokaryotic expression system. Finally, the RNA-binding capacity of recombinant PSF to mVL30-1 RNA was determined by Gel-shift assay. The results indicate that recombinant PSF produced with a prokaryotic expression system can be biologically active and will therefore allow us to investigate the function of PSF in the future.
A new method to harvest stem cells was established by implanting biomaterials into mice. Gelatin Sponges (GS) were implanted into the spatium intermusculare of mice hind limbs. A large number of migrating cells were isolated from the transplanted biomaterials at 12 days after implanting. The characteristics of adherent cells were similar to that of bone marrow (BM) stem cells, including morphology, proliferation potential and multilineage differentiation capacity. The frequency of CFU-F in GS (82.2±10.6/105) was much higher than in BM (43.7±7.4/106) (P<0.05). In additional, BM transplantation demonstrated that stem cells in the GS originated from the PB. Moreover, it is possible for autologous cell therapy, so this method may be a promising alternative for the clinical application.
To reveal the effects of glutaraldehyde polymerization on the immunological properties of porcine hemoglobin, bovine hemoglobin and human hemoglobin, repeated ip. or sc. administration of an immunizing dose of pHb, bHb, hHb or their glutaraldehyde cross-linked derivatives were given to mice to stimulate IgG responses, which was measured by indirect ELISA. Western blotting analysis of the antibody-antigen cross-binding reations between pHb, bHb, hHb or its related glutaraldehyde polymerized hemoglobin derivatives and anti-hemoglobin or anti-glutaraldehyde polymerized hemoglobin antibodies raised in mice were used in order to investigate the effects of glutaraldehyde polymerization on the immunological properties of the proteins concerned.The results indicated that hHb, bHb and pHb are all weakly immunogenic, while polymerization significantly increases the immunogenicity of all their glutaraldehyde-cross-linked derivatives. There exist clear species-specific antigen-determinants in hHb, bHb and pHb. The effects of glutaraldehyde polymerization on immunological properties of porcine hemoglobin, bovine hemoglobin and human hemoglobin are significantly different, there were no neo-antigens formed in pPolyHb, some of the determinants of bHb were covered because of glutaraldehyde polymerization, however,neo-antigens produced in hPolyHb. Therefore,it is necessary to study the immunological characteristics of different HBOCs products separately and should not inferred the characteristics of the products from different kinds of Hb even when they were made by the same method.
Aim: To investigate the effects of Chitosan oligosaccharides (COS) on attenuating menadione-induced stress injury in macrophages. Method: MTT assay was used to examine the cell viability, and the reagent kits were used to investigate the activities of enzymes and the products of lipid peroxidation in redox system. Results: By using MTT assay and detecting the activities of LDH (lactate dehydrogenase) in the cultural supernatants, it was confirmed that chitosan oligosaccharides could attenuate menadione-induced stress injury in macrophages. Moreover, the results showed that COS could also restore activities of endogenous antioxidants including superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX), and suppressing the production of lipid peroxidation. Those results could explain the mechanisms of COS on attenuating menadione-induced stress injury in macrophages. Conclusion: Stress injury induced by menadione in macrophages could be blocked by COS.
PAC1 is a specific receptor for pituitary adenylate cyclase-activating polypeptide (PACAP).The N terminal first extracellular region of the PAC1 (PAC1-EC1) is involved in the regulation for the activation of PAC1. To study the effects of the EC1 domain of the PAC1 normal (N) isoform on the cell lines expressing different PAC1 isoforms, the recombinant protein PAC1-EC1 (N) with 6-His-tag at the C-terminus was expressed in an engineered Escherichia coli strain and purified by Ni-NTA affinity chromatography. Mass spectrum, SDS-PAGE and Western blot were used to characterize the recombinant PAC1-EC1(N). Western blot were used to identify the expression pattern of PAC1 isoforms in three cell lines including rabbit corneal stromal cells, human neuroblastoma cells SH-SY5Y and human prostate cancer cells 22RV1. MTT assays were used to detect different biological effects of the recombinant PAC1-EC1 (N) on the cell lines with different PAC1 isoforms. The results showed that PAC1-EC1(N) had significantly different biological effects on the cell lines with different PAC1 isoforms. PAC1-EC1(N) stimulated the viability of rabbit corneal stromal cells expressing only one PAC1 isoform with a smaller molecular weight, but inhibited the viability of human neuroblastoma cells SH-SY5Y expressing one type of PAC1 isoform with a larger molecular weight. Meanwhile, PAC1-EC1(N) caused complex biological effects on human prostate cancer 22RV1 cells with three types of PAC1 isoforms. These results showed that PAC1-EC1(N) exerts different effects on the viabilities of the cells with different expression patterns of PAC1 isoforms, indicating that the PAC1 owns a complex self-regulation mechanism of activation.
The G1/S transition is one of the key regulatory points during the plant cell cycle. D-type cyclins (CYCDs) play critical roles in controlling the progression through the G1 into the S phase. Since CYCDs are responsive to stimulatory signals, they are functional in integrating mitogenic signals into cell division. However, the expression patterns and functional characteristics of different CYCD families in woody species remain to be elucidated. Six putative CYCD genes designated as PdCYCD1-7 from a hybrid poplar clone (P.deltoides × P.nigra, NE19) by complementing a yeast mutant lacking G1 cyclins were identified. But their effects on promoting yeast cell division after ectopic expression are divergent. Sucrose and phytohormone treatments on in vitro-grown poplar seedlings led to the alteration of morphological traits in roots. The effects on the transcript levels of PdCYCDs were monitored. Differential expression characteristics of the CYCDs in response to the mitogen supply were observed. Though CYCDs were induced by sucrose, their extents of induction among CYCD subgroups varied. PdCYCD6;4 and PdCYCD3;1 may have wider roles in responding to external signals than others, for they were modulated with sucrose and most hormones treatments. The induction of PdCYCD2;1 and PdCYCD1;1 by hormones depended on the presence of sucrose. PdCYCD5;1 was greatly stimulated by ethylene and the strengthening effect on induction was observed when sugar and hormones were added together. PdCYCD7;1 was not so sensitive to sucrose but was upregulated by gibberellin and ethylene. Together, these results suggest that the six populus CYCD genes identified here are functional in rescuing yeast cyclin mutant but may still have group-specific functions during the development of plants.
The expressed sequence tags (ESTs), fragments of mRNA sequences, have been widely used for effective gene discovery and complementation of the genome annotation. It is also beginning to be applied in the fields of phylogenetics, transcript profiling and proteomics recently in combination with quantitative real-time RT-PCR (qRT-PCR). The analysis of genes expression level from reproductive organs of the oilseed-bearing shrub called Jatropha curcas (J. curcas) may reveal some interesting genes related to regulation of lipid biosynthesis. The outcome may someday be used to improve the oil productivity of oil seed-bearing woody plants. Two cDNA libraries were constructed separately. A total of 9289 EST sequences were obtained with an average length of 603 bp (base pair), where as 4502 unique sequences (UniSeqs) were obtained with assembly of these EST sequences, including 1427 contigs (containing more than one reads) and 3075 singletons (containing a single read), respectively. These assembled sequences were annotated with gene names of Gene Ontology (GO) terms. In these annotated UniSeqs, the relative expression level of 50 ESTs was quantified with qRT-PCR in tissues of leaf, flower and seed. It was found that the most abundant 6 ESTs (Contig1452, Contig1482, Contig1510, Contig1514, Contig1534 and Contig1535), which are frequently detected, in two cDNA libraries were also expressed highly in one or two different tissues. These highly expressed genes encode lipid biosynthesis-related proteins or transcript factors. Numerous high quality ESTs for J. curcas genome annotation and increase in the number of sequences deposited in public databases were provided. The qRT-PCR assay has validated the abundance of six interesting ESTs, which were frequently found in cDNA library. Especially, those genes, which are related to both lipid biosynthesis and transcription, have been shown to be actively expressed according to qRT-PCR analysis. These genes may play an important role in regulation of lipid biosynthesis in J. curcas.
In order to investigate floral inhibition through over-expression of AGM3 in heterologous plants, a dominant negative mutation construct gene, 35S-AGM3-E9 was transformed into Nicotiana tabacum via the Agrobacterium-mediated method. The results of PCR and Southern analysis showed that the gene of AGM3 was integrated into tobacco genome. Furthermore, the data of real-time qRT-PCR analysis showed that AGM3 was expressed in all the transgenic lines and the transcripts of AGM3 were significantly different among these transgenic lines. The results of the investigation indicated that 46.7% of the transgenic lines did not flower during their life cycles, and flowering time of the 33.3% lines was delayed 29.7 days averagely, while the rest 20% of transgenic lines flowered at the same time with the wild type control. In addition, over-expression of AGM3 in transgenic tobacco plants caused decreased number of sepals, deep clefts on corolla, morphological changes of petals and stamens, and numerical change of stamens. The facts suggested that over-expression of AGM3 effectively suppress floral development, which laid a foundation for genetically engineered sterility.
Objective: Detect bioactivities of fermentation crude extracts originated from previously constructed deep sea metagenomic libraries. Methods: Functional screening of antitumor(cytotoxic),antibacterial and anti HBV activity from fermentation crude extracts was performed by CCK-8 method, agar diffusion method, double agar method and real time PCR method. Results: No antibacterial and anti HBV clones were identified and three mixed clones showed good cytotoxic activity. HPLC analysis of these three mixed clones fingerprints indicated they possess obviously different fingerprints with E. coli host which could be useful to next isolation of cytotoxic compounds from deep sea metagenomic library.Conclusion: These results indicated that chemical screening could be potential way for screening bioactivity substance from deep sea metagenomic libraries besides functional screening and homology screening.
Cyanobacterial NADPH dehydrogenase (NDH-1) is an important photosynthetic membrane protein complex, and is essential to CO2 uptake, cyclic electron transport around photosystem I and cellular respiration. This enzyme accepts electrons from NADPH and consists of at least 17 subunits, i.e., NdhA to NdhQ. Recently, an ndhO gene inactivation mutant, ΔndhO, has also successfully been obtained. However, little is known regarding the functional roles of NdhO subunit in cyanobacteria. Therefore, the encoding gene, ndhO, was PCR amplified from the unicellular cyanobacterium Synechocystis sp. strain PCC 6803, the expression plasmid pET32a(+)-ndhO was constructed and transformed into BL21(DE3)pLysS, and the expression of NdhO protein was induced by IPTG. After purification, the fusion protein pET-NdhO was used to immunize Japanese white rabbit to obtain the polyclonal antibody. The titer of the polyclonal antibody was detected by ELISA and its specificity was analyzed by immunoblotting. The titer of polyclonal antibody was found to be up to 1 ∶ 1 025 000, and thus possessed a high specificity. Further, immunoblotting results using the polyclonal antibody showed the presence of NdhO in active NDH-1 mediumcomplex, and not active NDH-1 supercomplex. Therefore, the antibody of NdhO obtained will further help us to reveal the functional roles of cyanobacterial NdhO subunit.
The rapeseed straw contains large quantities of lignocelluloses and its structure is stable and hardly degraded. By comparing the enzyme produced ability and characteristic in ten strains microbe, including bacteria, yeast and white rot fungi, and co-culture testing, the five strains symbiotic microbe, including BS09、BL、PC、TS and KS,were screened out. Investigating the rapeseed straw degrading ability of the five strains, the results showed that PC had a strong degrading ability for lignocelluloses, that degradation rate of lignin, cellulose and hemicellulose was 16.5%, 22.7% and 20.3% respectively. The BS09 and BL had a significant effect in degrading cellulose and hemicellulose, and degradation rate of cellulose and hemicellulose reached 19.5%,15.0%, 16.0% and 22.1% respectively.
Glycerol dehydratase was the critical enzyme in the metabolic pathway of converting glycerol to 3-hydroxypropionic acid. It was subjected to suicide inactivation by the natural substrate glycerol during catalysis. The inactivated glycerol dehydratase will influence the production of 3-hydroxypropionic acid. Therefore, the dhaB gene encoding glycerol dehydratase and gdrA, gdrB gene encoding glycerol dehydratase-reactivating factor from Klebsiella pneumoniae, the aldH gene encoding aldehyde dehydrogenase from Saccharomyces cerevisiae W303 were amplified by PCR. dhaB, gdrA, gdrB and aldH gene was employed to construct the recombinant strain Escherichia coli JM109/pEtac-dhaB-tac-aldH and E. coli JM109/pEtac-dhaB-gdrAB-tac-aldH. The effects of gdrA, gdrB expression on 3-hydroxypropionic acid production from glycerol were investigated. When cultivated aerobically on medium, the recombinant E. coli JM109/pEtac-dhaB-gdrAB-tac-aldH and E. coli JM109/pEtac-dhaB-tac-aldH produced 3-HP at a maximum of 4.0 g/L and 0.54 g/L. The results showed that the gdrA, gdrB expression could reactivated the activity of the inactivated glycerol dehydratase. Compare with E. coli JM109/pEtac-dhaB-tac-aldH, E. coli cells coexpressing both gdrA and gdrB with the glycerol dehydratase genes showed that the production of 3-HP was increased by 6.4fold.
The gene for NDM-1 (New Delhi metallo-beta-lactamase 1) is one member of a large gene family that encodes beta-lactamase enzymes. Bacteria that produce NDM-1 often referred to in the news media as "superbugs" because these bacterial are usually resistant to most antibiotics and infections caused by them are difficult to treat. NDM-1 gene was used for design of PCR primers and Taqman probe. Then a real-time quantitative PCR array for rapid detection of bacteria that produce NDM-1 was developed and its specifcity, sensitvity and repeatability were tested. Simulated urine specimens were used to assess the assay. A linear relationship was consistently obtained for input loads of 5×100~5×108 copies per assay. And the sensitivity of constructed assay method was 5 copies per reaction. No products were observed for strains of Enterobacteriaceae and only positive controls showed positive amplifications, indicating that the PCR assay is specific. The detection limit for simulated urine specimens was 10 CFU. These results indicated that the TaqMan real-time PCR assay described here is specific, sensitive and rapid for the detection of bacteria that produce NDM-1, and this assay could be used to detect the clinical specimens.
Objective: A simple way to construct an adenovirus vector containing a peptide-RGD in the HI loop of the fiber knob was developed and then its infection efficiency on RD and T24 cells in vitro and the humoral responses to transgenes in vivo were investigated. Methods: The pAdEasy-RGD-1 was modified by inserting RGD motif into pAdEasy-1 based on simple PCR method. Enhanced green fluorescent protein (EGFP) and codon optimized fusion protein (Fsyn) of human respiratory syncytial virus (RSV) were cloned and constucted into pAdEasy-RGD-1 to generate adenovirus vectors of FGAd-RGD-EGFP and FGAd-RGD-Fsyn, respectively. FGAd-RGD-EGFP infected RD and T24 cells to investigate its infection efficiency using fluorescent microscope and flow cytometry. FGAd-RGD-Fsyn was exploited to vaccinate BALB/c mice via intramuscular and imtranasal routes, respectively, to evaluate humoral responses to transgenes in vivo. Results: The infection efficiency on RD and T24 cells was higher with FGAd-RGD-EGFP than FGAd-EGFP, and immunization with FGAd-RGD-Fsyn generated similar serum IgG specific for RSV-F. Conclusions: PCR is a simple and practical method to construct adenovirus vector containing a peptide-RGD in the HI loop of the fiber knob. The resultant modified adenovirus vector displays improved infection efficiency on RD and T24 cells and similar humoral immune responses to transgene as compared to original adenovirus vector. Therefore, it will be an useful platform for oncotherapy.
The PCR technique has been widely used in various fields of molecular biological studies. However, multiplex PCR for determination of plasmid copy number has not been reported. To further explore the research in this field, based on the multiple PCR primer design and evaluation computer programs, Two-plex PCR primers were disigned to amplify the bacterial genome and plasmid DNA; then using bacterial liquid of E. coli with different plasmid vectors as the template for multiple PCR reactions. Finally, the integrated optical density of the gel image was analyzed to determine relative copy number of the plasmids. The data showed that the result of relative copy number of the plasmids determined by multiplex PCR was consistent with which determined by Real-time PCR and Southern blot, indicating that multiplex PCR was an alternative and valuable method compared with traditional techniques in routine experiments. So a rapid and efficient method in identification of the plasmid copy number was established, which provides a suitable way to host cell selection by easily and rapidly determining the relative copy number of clone vector and expression vector.
Objectives: pRNA, the packaging-RNA molecule of Bacillus subtilis is a new nanometer-molecular carrier. The recombination of pRNA and the hammerhead Ribozyme can be used to construct stable, self-deliverable pRNA-Ribozymes for entering cells and actively binding and cutting RNA. However, it is technically difficult to synthesize RNA molecules longer than 100 nt by chemistry. Therefore, 170 nt pRNA-Ribozyme was prepared by genetic recombination and in vitro transcription. Methods: The DNA of pRNA and Ribozyme were amplified by PCR before recombination according to their molecule sequences. The pRNA-Ribozyme was reconstructed in two forms: the chimeric ribozyme linked to pRNA vector as a cis-connected open structure, and the chimeric ribozyme harbored in pRNA vector as a closing inner structure. The pRNA-Ribozymes were obtained by in vitro transcription. Results: The pRNA-Ribozyme of cis-connected open structure and the embedded inner structure were prepared by in vitro transcription. Both molecules could form the polymer structure in vitro through correct folding. The pRNA-Ribozyme had the activity for binding and cutting its target mRNAs, which provides the foundation for further application of pRNA-Ribozyme in intracellular.
Diethylstilbestrol was activated by ethyl 4-bromobutyrate to introduce carboxyl group and then conjugated to BSA via EDC/NHS as immunogen, New Zealand rabbits were immunized and antisera were prepared. The results showed that complete antigen was successfully synthesized and specific antibody was generated, with titer of 1.28×105 the antisera cross-reactivity with hexestrol (9.34%), dienestrol (1.1%) and no cross-reactivity with 17β-estradiol. An indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) was developed for determination of DES. In the linear range between 1 and 30 ng/ml, IC50 of 7 ng/ml and a detection limit of 0.3 ng/ml for DES were reached. Therefore the assay as screening method can meet the demand of DES residue detection for animal food products.
Adenovirus is a kind of virus vector which is used widely in gene therapy, especially in treating cancer and heredopathia. However, its non-specificity and high immunogenicity have inhibited its application in clinical. Virus which modified with macromolecular to the capsid of virus through covalently conjugate or electrostatic interaction have lower immunogenicity and high tropism, and the modifications have little effect in their gene transduction efficacy. It is becoming an important modification method in adenovirus. The progress of modified adenovirus with polymers were reviewed.
Most of cellular proteins work as the subunits of protein complexes which are the basic functional unit in cell. The structure research and functional analysis of macromolecule complexes became popular in the post-genomic era. One major challenge is to produce these complexes in sufficient amounts for biochemical and structural research. One efficient way which named mutigene expression system is used widely to obtain the protein complexes by co-expressing subunits simultaneously in host cells. Multigene expression system is becoming more and more important on both basic and application researches. The studies on multigene expression system have been developed quickly, especially in the recent five years. The studies on multigene expression system, which provided the knowledge on it's characters and usages were summarized. The detailed construction methods of different kinds of multigene expression vectors, especially the newest vectors, which will be helpful to choose the most efficient system for protein complexes studies were introduced. The primary applications of the multigene expression system are also introduced.
The detection and quantification of apoptosis with molecular probes would be desirable to provide clinicians with intuitive, noninvasive and real-time dynamic information, assisting in diagnosing early diseases, monitoring the course of disease, assessing the efficacy of disease treatment and developing new therapeutic strategies. According to related literatures in the past decade, The progresses in the fields of probes design, mechanism of molecular probes and application in the related research areas were summarized. The molecular probes are classified based on the mechanism of affinity components.
Articular cartilage has limited ability to repair and regeneration by itself. At present, the clinical treatment and drugs for articular cartilage injury are difficult to achieve satisfactory results. Mesenchymal stem cells present themselves as a promising cell source for cartilage tissue engineering, because they have great potential for multilineage differentiation, expandable in vitro without losing their differentiation type, low immunogenicity and easily obtainable in high numbers. The Mesenchymal stem cells differentiate chondrocytes was focused on and many effect factors of Mesenchymal stem cells differentiate chondrocytes, such as growth factor, oxygen concentration, scaffold and so on were systematically introduced. In addition, some opinions on MSCs as seed cells present disease and development for cartilage tissue engineering were given.
L-tryptophan (L-Trp) is widely used in food, animal feed and pharmaceutical industries as an essential amino acid for humans. Chemical synthesis, enzymatic/microbial conversion and microbial fermentation are the methods for industrial production of L-Trp. Recently, with successful application of metabolic engineering in strain improvement, microbial fermentation gradually becomes the major method of L-Trp production. The strategy of metabolic engineering for improving L-Trp production is reviewed, involving the regulatory mechanism and genetic modification of L-Trp biosynthesis. Furthermore, the prospect of L-Trp production is also discussed.
Modern tissue engineering that can only repair simple tissue defects is still in the primary stage of development, in which many basic problems remain currently unknown. With these scientific problems gradually resolved, the definition of the term tissue engineering is also expanding, which can speed up the industrialization process of tissue engineering and promote the application of tissue engineering in clinic. The main problems and research progresses and future prospects involved in modern tissue engineering are reviewed.
