Objective: The aim was to examine the potential of human adipose-derived mesenchymal stem cells (hAD-MSCs) transplantation to restore inner ear hair cells in mice. Methods: To test this approach, hAD-MSCs were delivered into the cochlea of deafened mice. The grafted cells were tested by immunostaining and RT-PCR. Results: Transplanted cells survived for at least 2 weeks postoperatively, evidenced by RT-PCR and immunofluorescence detection of hAD-MSCs. A few hAD-MSCs located in the cochlear sensory epithelium, with expression of myosin 7a, a specific marker for inner ear hair cells. There was no evidence of significant immunological rejection of the implanted hAD-MSCs. Conclusion: hAD-MSCs transplanted into the mouse inner ear after drug-induced injury can survive, migrate and differentiate towards hair cell-like cells.
The inverted microscope was used to observe the process that peripheral blood mononuclearcells (PBMCs) were cocultured with allogeneic human umbilical cord derived mesenchymal stem cells ( hUC- MSCs). The atomic force microscope was used to analyze the morphology and mechanical property after two kinds of cells were cocultured. The result showed that the morphology of adherence PBMCs changed greatly after cocultured, and was coated with different grain sizes of polymers. From the high spatial resolution of AFM force-curves, the adhesion force and stiffness values of PBMCs cocultured with hUC-MSCs were stronger than those of controlled PBMCs. CCK-8 indicated that PBMCs may appear to compete with hUC- MSCs in the co-culture system. The visualized data obtained from AFM can provide a better understanding the interactive mechanism of hUC-MSCs and PBMCs.
A new kind of p24 antigen positive control was developed to provide a better solution to the problems encountered in developing a novel HIV detecting kit, such as (a) inconveniency in storing p24 positive control, (b) difficulty in maintaining p24 antigen activity, (c) differences in protein structure and aggregation mode between recombinant p24 antigen and natural p24 antigen. Modified HIV1 skeleton plasmid pNL43 EGFP R-E-and envelope protein plasmid pGX-C were co-transfected into 293T cell, and then culture fluid containing single-round HIV pseudovirions were collected. This kind of HIV pseudovirions possess natural core shell protein p24, which could be used as the positive control for detection of HIV p24 antigen. Through comparison of activity and stability between pseudovirion p24 antigen and positive control in common detecting kit, it was proved that pseudovirion p24 is more convenient to store, more stable and more similar to natural p24 protein structure and aggregation mode than common recombinant p24 protein.
During research of the reported drug-resistance mutation T66I, it was found that this mutation can also improve the solubility of integrase. IN1–288/T66I and WT were expressed at the same time, 10μl of the supernatant was extracted for SDSPAGE analysis and His-tagged protein staining. Result shows that the solubility of IN1–288/T66I is 2~3 fold of WT. 4.72mg protein was obtained from 600ml of cells expressing IN1–288/T66I. The strand transfer activity of IN1–288/T66I and IN1-288/F185K /C280S were analyzed by advanced ELISA, which shows that two proteins are almost equal in catalytic activity.Another method, being different from F185K /C280S mutation, to improve the soluble expression of integrase has been provided. IN1–288/T66I can also be used to screen integrase inhibitor, which can avoid the drug-resistance brought by T66I mutation.
The Glyoxalase(GLO) plays a very important role in plant detoxification system.Glyoxalase gene was isolated from banana (Musa acuminata AAA subgoup)cDNA library, named as MaGLO14. To confirm the tolerance of MaGLO14 to abiotic stresses, a yeast expression vector PYES2-MaGLO14 has been constructed and transformed into Saccharomyces cerevisiae strain INVSC1 with uracil auxotrophic phenotype. Comparison of colony numbers of transgenic INVSC1 with non-transgenic INVSC1 grown in medium containing NaCl, under high temperature and low temperature, drought and ultraviolet radiation stress, the survival colony numbers of transgenic INVSC1 were more than that of non-transgenic INVSC1 respectively. The results confirm that MaGLO14 enhances the tolerance of Saccharomyces cerevisiae to abiotic stresses. It is suggested that MaGLO14 may play important roles in resistantance of plants to abiotic stresses.
Objective To clone, express the Glycine-rich RNA-binding protein of tobacco. Methods Amplified the full length NtRGP-1a and NtRGP-3 gene of tobacco, then ligated the gene into pGEX4T-1.Expression vectors pGEX4T-1/NtRGP-1a, pGEX4T-1/ NtRGP-3 were constructed and transformed into E．coli rosetta for expression induced by IPTG. Recombinant proteins were purified through GSTrap 4B affinity chromatography. Results The recombinant gene can be overexpressed in E.coli. SDS-PAGE showed that the molecular weight of the expressed product were the same as expected. The purity of the protein is greater than 95%. Conclusion the NtRGP-1a and NtRGP-3 protein of tobacco has been successful cloned and expressed, which could be useful for developing monoclonal antibody and the research of stress resistance.
Taxus suspension cell culture has the potential to provide a sustainable source of anticancer drug paclitaxel (Taxol) and other taxoids. In the cell culture of Taxus chinensis, taxuyunnanine c (Tc) is the primary taxoid. To design a rational strategy for redirecting the precursor fluxes from other taxoids into paclitaxel production, this study employed quantitative real-time-PCR (RQ-PCR) to understand the dynamic profiling of key biosynthetic pathway genes of palcitaxel and taxoids during the culture process. Six genes —TASY, TDAT, T5αH, TαH, TDAT and T10βH were quantified under a process condition of double elicitation by 100μM 2,3-dihydroxylpropanyl jasmonate(DHPJA) on day 7 and day 12, 20g/L sucrose feeding and 100g/L XAD-7HP absorption on day 7 . This process treatment led to a high accumulation of Tc at 1517±37mg/L on day 30, 11.1 folds of that of control and 1.7 folds of that of double elicitation with sucrose feeding. The RQ-PCR results showed that with DHPJA elicitation, the expression of all the six genes was greatly increased, but sharply declined after 12 hours. This rapid decline suggested that the second DHPJA elicitation was required to sustain gene expression. Simultaneous absorbents addition would delay the improvement of gene expression but maintain the gene expression at a higher level, resulting in higher gene expression over treatments without absorbents. TαH expression was somewhat different which was markedly inhibited by XAD-7HP absorption while absorbants still maintained its expression.
Pyrolysis characteristics of Enteromorpha clathrata was investigated with thermogravimetric analyzer. Simultaneously, cornstalk and sawdust were references. The basic pyrolysis characteristics of the three kinds of biomass and the difference among them are analyzed using TG-DTG curves. The results showed that three stages appeared in this thermal degradation process and Enteromorpha clathrata has lower initial temperature and peak temperature of pyrolysis than two kinds of terrestrial biomass, which are sawdust and cornstalk. Moreover, with alumina catalyst in different concentration, the pyrolysis process of the three kinds of biomass, which are Enteromorpha clathrata, cornstalk and sawdust, were studied with thermogravimetric analysis method. The basic pyrolysis characteristics and the diffenence effect of the catalyst were analyzed by using TG-DTG curves . The results showed that the catalyst can reduce the conversion of the biomass and the maximum weight loss rate greatly. Specifically, alumina catalyst in 10% concentration has a notable impact on the conversion of the biomass and the maximum weight loss rate. Taking account of the surface of Al2O3 with tunable acidity and a variety of different crystal structure, Al2O3 catalyst has a great application value.
Urechis unicinctus living in the inter-tidal and hypotidal zone of the lower coastal mud-sand areas is a member of the group of worms classified as the Phylum Echiura since 1940, but increasingly thought to represent a clade within the polychaete annelids. A systematically analysis revealed that the highest thrombolytic activity was found in the haemocoelomic fluid and gut. Therefore the isolation of fibrinolytic enzyme (UFE)was undergone from the two tissues by centrifugation, filtration, ammonium sulfate precipitation, ultrafiltration, and chromatographic steps, including Q. Sepharose Fast Flow, Sephadex G-75, and Sephacry S-100. UFE was finally purified approximately 11 fold with a yield of 29.82%. UFE appeared to be homogenous by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with a low molecular weight of about 10.38 kDa.
Functional studies showed that using urokinase (UK) as a positive control and phosphate buffer saline (PBS) as a negative control, UFE mainly hydrolyzed fibrin directly, but it also exihibited some UK activity. UFE appeared to degrade fibrin by transformation of plasminogen into plasmin, which was similar to lumbrokinase from different earthworms, whereas UK did not directly hydrolyze fibrin without the presence of active plasminogen. These suggest that UFE is a potent thrombolytic agent with less immunogenicity and strong fibrinolysis for treatment of thrombosis.
[Objective: AtrA (AtrA-c) was characterized as a transcriptional activator for actII-ORF4 gene which encoding a pathway-specific transcriptional activator of the actinorhodin biosynthetic gene cluster in Streptomyces coelicolor A3(2). Two atrA homologues in Streptomyces griseus NBRC13350 and Streptomyces avermitilis MA-4680 were identified to be regulators for the production of streptomycin and avermectin B1a respectively. This study aimed to identify and clone a new atrA homologue in Streptomyces globisporus C-1027 that can produce a chromoprotein antitumor antibiotic lidamycin. [Methods] Heterologous expression of AtrA-c, Southern blotting, PCR, sequencing and bioinformatic analysis were used in this study. [Results] The heterologous expression of AtrA-c in S. globisporus C-1027 reduced the production of lidamycin production. Southern blotting analysis revealed that the S. globisporus C-1027 genome contains at least one copy of atrA-c homologue (atrA-gl). atrA-gl and flanking gene fragments were successfully amplified with the primers designed by the alignment of amino acid sequences of AtrA from distinct Streptomyces (GenBank/EMBL/DDBJ accession number GU723707). The alignments of amino acid sequences showed AtrA-gl shares highly conserved sequences with other homologous AtrA proteins, with a percentage of identity ranging from 65% to 87% and similarity ranging from 70% to 89%. The alignments of amino acid sequences of the flanking gene fragments suggested four atrA genes have identical arrangement in their genomic context. Phylogenetic analysis showed that S. globisporus AtrA-gl and S. griseus AtrA-g have a relatively more closer relationship. [Conclusion] atrA-gl is the first cloned transcriptional regulator located outside the gene cluster in S. globisporus C-1027 for lidamycin production. The data presented here set the stage for subsequent studies to elucidate the regulation of C-1027 biosynthesis, as well as for designing strategies for the construction of strains with enhanced C-1027 production.
According to Genbank Kluyveromyces lactis Cu/Zn-SOD gene sequence primers were designed；the Cu / Zn-SOD gene were obtained by PCR amplification. Driven by the PGK1 promoter, the gene fused to the fluorescent reporter gene GFP were used to construct recombinant plasmid YEplac195-PSGA and YCplac33-PSGA, and transformed into yeast (Saccharomyces cerevisiae) W303α strain. By colony PCR and fluorescence microscopic observation we confirmed that the Cu / Zn-SOD gene of Kluyveromyces lactis was successfully expressed in W303α strain.20 mM paraquat were added to the positive transformants before fermentation. SOD activity and total activity were respectively 6.7 times and 4.7 times as high as those without paraquat in the biomass of the fermentation medium. To further investigate the impact of the Cu / Zn-SOD gene on the host sod1Δ strain EG118, heat shock treatment was applied. Results show that heat strikes capability of host tolerance in the following order of EG118 (YEplac195-PSGA)> EG118 (YCplac33-PSGA)> EG118. The results is not only necessary for the fermentation industry to prevent bacterial fermentation in the aging strains and furthermore, an enhanced capacity to provide some theoretical guidance, but also a foundamental of in vitro directed evolution of the Cu/Zn-SOD.
Pleuromutilin is one of the tricyclic structural diterpenoid antibiotics, and is currently being used as an intermediate for semisynthetic antibiotics such as veterinary antibiotics tiamulin, valnemulin, and human-used antibiotic retapamulin. These compounds inhibit bacterial protein synthesis by specifically targeting the large subunit (50S) of the bacterial ribosome and exhibit excellent in vitro and in vivo antibacterial activity against gram-positive pathogens . Recently, a series of novel water-soluble pleuromutilin derivatives bearing a purine ring have been developed. These developing pleuromutilin derivatives stimulate the need of pleuromutilin from Clitopilus prunulus. But the strain improvement about Clitopilus prunulus has not been reported. A large number of pleuromutilin high-producing strains were obtained from the original basidiomycetes strain Clitopilus prunulus after nitrosoguanidine mutagenesis and following continuous subculture in the broth with nystatin, terbinafine, sodium dodecyl benzene sulfonate, and lauryl trimethyl ammonium chloride respectively. Mutants with antibiotic resistance which antibiotics targeted on ergosterol biosynthesis pathway, got a higher forward mutation rate. The pleuromutilin yield of mutant strain pn163, a nystatin resistant mutant with genetic stability, was increased by 38.50% while ergosterol was decreased to 29.29%, which was supposed to correlate with cell membrane permeability.
Objective To express Mycobacterium tuberculosis DnaA protein in Mycobacterium smegmatis and identify the expression product. Methods The dnaA gene of M. tuberculosis was amplified by PCR and cloned into expression vector pMF406 to generate the shuttle vector of E.coil and Mycobacterium pMF-dnaA. It was comfirmed by restriction endonuclease digestion and sequence analysis. The recombinant plasmid was transformed into M. Smegmatis mc2155 by electroporation. The recombinant M. smegmatis was induced by 0.02% acetamide and the expression product was analyzed with SDS-PAGE and Western blotting. Results The recombinant M. smegmatis was successfully constructed and the M. tuberculosis DnaA protein was expressed in the recombinant M. Smegmatis at a relatively high level identified by SDS-PAGE and Western blotting. Conclusion The successful homologous expression of the M. tuberculosis DnaA protein will be very helpful for the further study on the mechanism of M. tuberculosis DNA replication .
One hundred and ninety eight bacteria were isolated and tested for their nematicidal activity from the soil in Nanyang, Henan Province, China. Six strains which had nematicidal activity to Bursaphelenchus xylophilus were found. A bacterium, named Bacillus sp. strain NS-3, showed the highest nematicidal activity to B xylophilus. The bacterium was classified by phenotypic and genotypic characteristics, which belonged to Bacillus genus. The culture supernatant and the crude extracellular protein extract from the strain NS-3 separately killed 50 % and 100 % of the tested nematodes (B. xylophilus) within 48 h, which suggested their high toxic activity toward the nematodes. Most of the dead nematodes were degraded and only minor fragments were left. However, the crude protein extract lost nematicidal activity after boiled, which showed the namaticidal materials produced in the cultural filtrate by the bacterium was not stable to the heat.
C65-2 strain with high chitinase producing ability was isolated from soil and identified it as Aspergillus fumigatus by observing its morphology character and measuring its 18S rDNA sequence. The chitinase producing conditions was optimized. The maximum activity could be up to 6.9 U/ml and the enzyme activity increased 210% than before. The study of enzymatic properties of the chitinase showed that the molecular weight was 20 Kda. Thermostable stability of the chitinase was not good and the enzyme activity reduced to 0 at 60 ℃ for 50 min. The optimal enzyme reaction temperature was 55 ℃ and the optimal pH of enzyme reaction was 7.0. The chitinase activity can be enhanced by Mg2+，Cu2+ and inhibited by Fe3+.
Objective:Set a new chromatography method to remove impurity proteins from meningococcal polysaccharide type A sample instead of the traditional phenol abstraction method. Method:This new method includes two purification steps by using Capto adhere and Sephardex G-25. Result:The contaminative protein concentration is 0.2% in final meningococcal polysaccharise type A sample, which is below the WHO regulation standard (1%),the content of nucleic acid and endotoxin are also blow the current regulation standard,the recovery is over 65%. Conclusion:Compare with the tradition phenol abstraction method, the chromatography method can remove impurity proteins effectively with high recovery and reducing environment pollution and easy to scale up.
1,5-Pentanediamine is one of the most important industrial chemicals for its highly desired properties and its wide applications as a key component of an emerging polymer business. Biological production of 1,5-Pentanediamine has been a novel and competitive way. An about 2.2 kb fragment of Hafnia alvei lysine decarboxylase gene encoding lysine decarboxylase (LDC) was proliferated by polymerase chain reaction by using chromosomal DNA of Hafnia alvei as the template. The obtained ldc fragment was inserted into E. coli/C. glutamicum shuttle vector pXMJ19 to construct an expression plasmid pXMJ19-ldc. It was then introduced into C. glutamicum TK260512 via electrotransformation, and a recombinant C. glutamicum TK260512/pXMJl9-ldc was obtained. The 1,5-Pentanediamine in the broth was detected by HPLC when the culture in the shake flask was indueed with 0.5 mmol/L IPTG. The results showed that after 36 h cultivation,the recombinant stain could produce 0.96 g/L 1,5-Pentanediamine.
The fermentation conditions of lipase production by Bacillus subtilis CICC 20034 were optimized. Initially, the most suitable intruder tributyrin, nitrogen source urea, carbon glucose, and MgSO4 were selected according to single factorial experiments respectively. Based on the result, screening methodology Plackett-Burman design was used to evaluate the effects of twelve factors related to lipase production and four statistically significant factors tributyrin, urea, KH2PO4 and pH were selected. The path of steepest ascent was used to approach the optimal region of lipase production subsequently. Then, the optimal combined concentration for maximum enzyme activity were further optimized by response surface methodology and determined as follows: tributyrin 2. 62%, urea 8.57g/L, KH2PO4 2.59g/L and pH 9.47. The optimization of culture conditions of B.subtilis CICC 20034 led to a 6.7-fold increase in lipase production relative to initial result 0.072 /ml, which indicate that single factor in combination with response surface methodology, is an effective method for optimization of lipase production conditions by B.subtilis CICC 20034.
Response surface methodology was used to optimize the autotrophic cultivation conditions for lipids production by Chlorella vulgaris in a 2-L airlift photobioreactor. In the first optimization step, a Plackett-Burman design was used to evaluate the influence of ten related factors and it was found out that KNO3 concentration, temperature and CO2 concentration influenced lipids production significantly. Subsequently, the path of steepest ascent was used to approach the optimal region of the cultivation conditions. In the third step, KNO3 concentration, temperature and CO2 concentration were further optimized using central composite designs and response surface analysis and the optimum conditions were that KN03 concentration was 0.31 g/L, temperature was 26.5 ℃ and CO2 concentration was 6.80%. Under optimum conditions, the lipid yield was 0.42 g/L and it was increased by two times than that under the conditions before optimized. Chlorella vulgaris was cultured in a 10-L airlift photobioreactor under optimum conditions.
Plackett-burman and response surface methodology were applied to optimize the Bacillus sp.HB20 fermentation medium components for synthesizing（R）-mandelic acid. The important factors influencing the yield of（R）-mandelic acid, which identified by initial screening method of Plackett–Burman，were Peptone, Beef extract and Maltose. The path of steepest ascent was undertaken to approach the optimal region of the three significant factors. Box–Behnken design and response surface methodology were adopted to further investigate the mutual interaction between the variables and identify optimal values that bring maximum（R）-mandelic acid yeild. Experimental results showed that Peptone, Beef extract and Maltose concentration all had significant influence on the specific（R）-mandelic acid yeild potential (Ps). The optimal conditions for the maximal Ps were: Peptone 11.507 g/L, Beef extract 6.708 g/L and Maltose 10.907 g/L, the maximum predicted value of（R）-mandelic acid yeild was 66.87%. After the checking experiment , the maximum predicted value was consistent with mean value of verification test and the （R）-mandelic acid yeild increased 25.87% at optimized condition.
RNA interference (RNAi) is an efficient tool for gene silencing. Due to enormous advance of molecular biology and biotechnology achieved in recent years, it has evolved into another excellent technology for gene silencing with higher specificity called artificial microRNA interference (amiRNAi). amiRNA, approximately 21 nucleotides long, which could be generated by endogenous miRNA precursors, can mediate the silencing of single or multiple genes targeted without affecting expressions of unrelated genes. Compared with the conventional RNAi, this novel technology has advantages of high specificity, satisfactory stability as well as precise predictability of silencing effect. Thus the amiRNAi definitely proves to be one of the most powerful tools for the functional analysis of heterogeneous genes. Also it promises a bright future for both study and application of genes'negative regulation.The very principle of amiRNAi was reviewed and its superiority and potential applications were discussed.
Plant cuticle plays an important role in the plant life cycle. The cuticle is highly hydrophobic layer of cutin intermeshed and coated with waxes that covers essentially all aerial organs and mainly composed of fatty acids and their derivatives. Plant cuticle can be divided into the inner cutin and outer wax layer and forms a protective layer against temperature extremes, drought, high salinity and other abiotic stresses. The cuticle also protects inner tissues from bacterial and fungal pathogens, herbivore attacks. The recent research progresses in the relationship between plant cuticle and stress resistance, especially drought tolerance were reviewed.
Japan is the world leader in the biotechnology area, bioindustry is its priority of all industries, the government accelerated bioindustry through optimizing development strategies, perfecting related institutions, rising financial input and promoting the combination of government, business and research and so on. The current status of bioindustry in Japan were reviewed, and its development directions and focuses in the future on the basis of Japanese situations and advantages were analyzed, the aim is providing references for our country's bioindustry development.
South Korean' s Bioindustry has been developed greatly since 1980' s. Its policies, funds, organizations and the current status were reviewed and its future development trends were analyzed to provide useful references for our country' s bioindustry development.
