Objective: To sieve molecular biomarkers associated with heart failure derived from arrhythmogenic right ventricular cardiomyopathy (ARVC). Methods: We performed comparative gene microarray analysis using Individual left ventricular myocardial samples from 5 patients with heart failure resulting from ARVC undergoing transplantation and 5 matched samples from normal adult heart. The accuracy rate of the differentially expressed genes obtained by gene microarray was further verified by quantitative real time RT-PCR. Results: We identified 83 genes (from a total of 35,000) that were differentially expressed in diseased hearts versus normal hearts. Among them thirty-seven genes were up-regulated and forty-eight genes were down-regulated in ARVC hearts compared with the normal hearts. Changes of gene expressions were most prominently observed in those belonging to the "metabolism" category. Eighty percent of the selected 30 differentially expressed genes from microarray analysis were confirmed by quantitative real time RT-PCR. We reported the highly expressed level of atrial natriuratic peptide (ANP) in ARVC hearts that was confirmed by quantitative real time RT-PCR and immunohistochemistry. Conclusion: We identified, for the first time to our knowledge, the altered expressed genes in ARVC hearts compared with the matched normal hearts. Our results are the base to further study the molecular mechanism and identify diseased-specific molecular biomarkers in heart failure derived from ARVC.
The ORF3 gene from a full-length swine HEV genomic clone was amplified by polymerase chain reaction(PCR) and inserted into vector pMD18-T, then subcloned to prokaryotic expression vector pET28a(+). The recombinant plasmid pET28a-ORF3 was transformed to E.coli BL21 (DE3) for expression under induction of IPTG. The expressed product was identified by SDS-PAGE, Western blot, then purified by Ni-NTA affinity chromatography and used as an antigen for the determination of HEV antibody in swine and human serum by ELISA. It indicated that the target gene was amplified by PCR. The results of restriction and DNA sequencing confirmed that the prokaryotic expression vector pET28a-ORF3 was constructed correctly. The expressed product, with a relative molecular weight 6 500~16 500, was consistent with the designed. Western blot showed specific reaction of the expressed fusion protein with mouse anti-His Tag serum. ELISA indicated that the expressed fusion protein can reacted with the HEV positive swine and human serum. The expressed product showed good reactionogenicity, which laid a foundation of developing a swine HEV diagnostic reagent.
This study employed surface amino-modified magnetic nanosphere prepared by our research group. First prepared Staphylococcal protein A (SPA) immobilized magnetic nanosphere carrier (SPA-MP) by covalent coupling method, and discussed the optimization condition for carrier preparation. Then according to the specific affinity between biological molecules, application feasibility of the SPA-MP carrier for antibody purification field was investigated through the oriented controlling of additional magnetic field, affinity adsorption, washing and elution operations. The optimization experiments appeared that we can prepare SPA-MP carriers with different surface density by changing the protein concentration, cross-linker concentration and activation time of cross-linker. Under the conditions of 300 μg SPA,2.5% (V/V) glutaraldehyde concentration and 3h activation time, the SPA-MP carrier with surface density as high as 35 mg SPA per gram magnetic nanosphere can be prepared. In addition, application results indicated that 1g SPA-MP carrier can bind CD25 monoclonal antibody up to 14 mg, and is effective for IgG antibody purification from human antiserum samples.
Eukaryotic translation initiation factor 5A (elF5A ) is involved in celI proliferation, senescence, development and environmental response. cDNA of Chinese rose was amplified by PCR with degeneracy primer. The special fragment with 480 bp nucleic acid encoded a deduced amino acid sequence of 159 residues was obtained and named as RceIF-5A(GenBank No. EF177192). The gene was cloned into pET32a-vector and the recombination vector was named as pET32a-eIF5A. E.coli line BL21(pET32a-eIF5A) had more obvious tolerance than E. coli BL21 (pET32a) under the high(50℃) and low（4℃）temperature. By all accounts, thermotolerance enhancing was attributed to the heterogenous expression of eIF-5A gene in host cell line.
A Staphylococcus aureus JH strain which can produce lipase was obtained from environment．According to polysequence comparision of prokaryote’s lipase gene published on NCBI ，we found that its sequence is very conservative． Lipase gene was obtained by PCR from genome DNA of Staphylococcus aureus JH，and then it was incorporated into plasmid pC194 and transformed into B.subtilis ．We selected the recombinant strain which is antipenicillin．The recombinant lipase was purified by ammonium sulfate fractionation and ion exchange ．SDS-PAGE was used to identify the recombinant lipase ．It was revealed that the molecular mass of the recombinant lipase is 24kDa；the recombinant lipase show maximum activity at 41℃ and pH8.0；the values of Km and Vm were found to be 0.34mM and 308 μmol•mg-1•min-1；and the metal ions Ca2+,K+and Mg2+ can activate the lipase, wheras Fe2+,Cu2+,and Co2+inhibit it.
The transesterification reaction conditions of lard with methyl acetate with combined use of immobilized lipases as catalysts were conducted. Initially, according to single factorial experiments, the studies on Lipozyme TL IM and Novozym 435 respectively catalyzed transesterification of lard showed that the optimal parameters of transesterification reaction were: the molar ratio of methyl acetate to oil of 14:1, 40% enzyme added based on oil weight, temperature 50℃. Combined use of Lipozyme TL IM and Novozym 435 was proposed further to improve the catalytic performance by the response surface method (RSM). Herein, a 5-level-3-factor central composite rotated design was employed to evaluate the effects of lipase loading, the proportion of the two lipases and amount of methyl acetate. The optimum conditions were as followings: 40% lipase loading based on oil weight, 50%/50% the proportion of lipases (Novozym 435/Lipozyme TL IM), and the molar ratio of methyl acetate to oil of 14:1. And under the optimal conditions, the highest biodiesel yield of 97.6% could be attained, which was higher than the biodiesel yield with each single one of the two lipases. The results suggested that the technics of combined use of certain immobilized lipases catalyzed transesterification reaction of lard for biodiesel production with methyl acetate as the acyl acceptor could raise the FAME yield and save the production cost.
Photoautotrophic gametophyte cells of the brown macroalgae Laminaria japonica were cultivated in 500 mL stirred tank photobioreactors under seven pulse feeding modes and one batch mode. It is the first time for the study of effects of the feeding time points and feeding quantity on macroalgal cell growth and nutrient consumption. Results showed that, with inoculum density of 50 mg DCW L-1, in modified APSW artificial seawater medium at 13 ℃, light intensity of 60 ?E m-2 s-1, light cycle of 16/8 h L/D, aeration rate of 50 mL min-1, and agitation speed of 100 rpm, feeding the culture with small nutrient quantity was beneficial for the synchronization between nitrate and phosphate absorption, and further for biomass production. Feeding when ambient nutrient was abundant or depleted was quite weak for large amount of biomass accumulation, which might be due to the slowing nutrient absorption, nutrient storage, or the divergence absorption between nitrate and phosphate. Feeding nutrient frequently with small quantity from mid-exponential growth of macroalgal cells, that is maintaining medium nutrient concentration between 1/3 and 1/2 of its initial concentration in this paper, was the most effective way for biomass production, with biomass increased by 12.270 times of for 51 days' cultivation.
Lipopeptide biosurfactant from Bacillus natto TK-1 was purified via acidic precipitation, methanol extract and thin-layer chromatography (TLC) system. The surface-active substance migrated at the position of Rf 0.58-0.65 as a single line on the TLC. The critical micelle concentration (CMC) was estimated to be around 115 mg/L and the minimum surface tension of lipopeptide was 30.1 mN/m at a concentration of 512 mg/L. Meanwhile, the anti-adhesion activity of lipopeptide was investigated in vitro. The results indicated that lipopeptide was found to significantly inhibit the adhesion of Salmonella typhimurium, Escherichia coli and Staphylococcus aureaus to 96- well microtiter plates at various concentrations. The highest anti-adhesion activity was exhibited against Salmonella typhimurium with inhibition percentage till 51.9% at 12.8 g/L. The antimicrobial activity of lipopeptide was evaluated against ten microorganisms using disc diffusion method and the lipopeptide showed a broad spectrum of antimicrobial activity. The strongest inhibitory effect was found against Botrytis cinerea and Fusarium moniliforme.
A novel reverse emulsification-phase transition process was designed to prepare alginate-based nanocapsules in the present study. Ca alginate nanocapsules with an average hydrodynamic diameter below 300 nm were obtained by adjusting surfactant and alginate concentration. Instrumental analysis observations revealed a morphologically spherical shape with a relatively uniform size distribution without aggregation. ?-potential measurements indicated an topically electrostatically negative nature of capsules in neutral aqueous solution. These nanocapsules were proven to have an equivalent potency to stimulate the maturation of dendritic cells originated from CD14 positive cells isolated from human peripheral blood. Protein loading via covalent conjugation was demonstrated feasible with EDC/NHS facilitated reaction between carboxyl and amino groups. These nanocapsules are considered to have important applications as a new adjuvant in cancer cell therapy, vaccine design and targeted drug delivery for active components of protein nature.
Abstract An accurate and high-throughput fluorescence based method was developed for quantification of double strand DNA (dsDNA) using SYBR Green I dsDNA-binding dye. A series of diluted genomic DNA and ?DNA were mixed with SYBR Green I (4×), and the fluorescence signal was measured with real-time PCR instrument and calibrated with ROX (1×) as reference dye. The performance characteristics of the method were compared with spectrophotometric quantification based on ultraviolet absorption. The new dsDNA quantification using SYBR Green I exhibited linearity over a range of ?DNA concentrations from 0.015 to 2 ng/?l and was about 100-times more sensitive and accurate than spectrophotometric quantification. The new dsDNA quantification allowed DNA sample may contain impurities and only required a small quantity of tissue sample. It could be widely used in molecular biological and diagnostic applications as an accurate and high-throughput assay tool.
A method to determine dihydroxyacetone (DHA) in fermentation broth was developed by high performance liquid chromatography (HPLC). DHA was separated on a Alltima C18（5μm，250×4.6mm）. The mobile phase was 0.5% methanol solution (pH adjusted to 3.0 with H3PO4), the flow-rate was 1.0 mL/min and the detective wavelength was 200 nm. The detection limits of DHA was 0.1 g/L～10.0 g/L. 6.2 g/L DHA in the fermentation broth was detected by HPLC method, which was in agreement with the result by spectrophotometric method. The method in this paper was simple, rapid and applicable for DHA determination in the fermentation broth. The method was applicable for DHA determination in the fermentation process.
A separation technology of catalpol from Rehmannia with macroporpus adsorbent resins was investigated in this paper. The content of catalpol in the extract was determined by high performance liquid chromatography (HPLC). Nine different kinds of macroporous adsorbent resins were studied on the static capacity of adsorption and desorption, and D101 resin was best for the separation of the extraction solution of Rehmannia. The results showed that D101 resin had the highest static adsorption capacity of 69.2mg/g dry resin and its isotherm curve can be well described by Langmuir and Freudlich equation. The 5% ethanol elution on removal of the solvent under reduced pressure provided a brown powder, which was subjected to an open column chromatography on silica gel eluted with a CHCl3–MeOH gradient. The fraction eluted with CHCl3–MeOH (8：2) was identified as catalpol and the purity of the compound was more than 90% purity by HPLC analysis. The yield of this separation technology was 6%.
Objective: Preparation of monoclonal antibody to detect the wild-type HBsAg as well as variant forms. Methods: BALB/c female Mice were immunized with HBsAg isolated from serum of infected patients. mAbs were prepared by hybridoma technology. Identify the purity and characteristics of the selected mAb purified with saturated ammonium sulfate and give preliminary evaluation of the quality and effectiveness of its application, in which polyacrylamide gel electrophoresis and agarose gel electrophoresis and emzyme-linked immunosorbent assay (ELISA) were used. Results: A monoclonal antibody can detect the wild-type HBsAg as well as variant forms was obtained designated as D12. The purity of D12 was high. D12 was highly sensitive and specific to HBsAg and was tested for subtype as IgGl . The recognition sites existed in the nature antigen. The ability to detect the wild-type HBsAg using D12 was better than that of three commercial HBsAg diagnostic kits and the ability to detect the variant HBsAg using D12 was better than that of five commercial HBsAg diagnostic kits. Conclusion: The preparation of mAb D12 is very important to further improve the detection rate of variant hepatitis B virus and enhance HBV prevention.
Honggumycin, which showed a significant antibacterial activity, was a bioactive compound obtained by the fermentation broth of Streptomyces 702. Under the shaking-flask culture, unsterile fermentation process of honggumycin produced by Streptomyces 702 were studied by single-factor experiment design. The optimum unsterile shaking-flask cultivation condition consisted of: 7.5% of inoculation size (v/v), 1.5mL of antimicrobial agent addition. Under the optimal conditions, honggumycin yield of unsterile fermentation was up to 1.46g/L, which was increased by 25.7% than that of sterile fermentation. The result showed that fermentation for Streptomyces 702 under non-sterile conditions was an potentially efficient fermentation mode, which not only saved fermentation cost, but also increased fermentation productivity.
HIV-1 integrase enzyme is a 32 KDa protein encoded by HIV pol gene. It is responsible for integration of viral cDNA into host chromosomal DNA, which is indispensable for HIV replication.Since there was no functional equivalent for this enzyme in human cells, inhibition of integrase will bring little side effect to human body. Thus HIV integrase has become an attractive and rational target for therapy of AIDS after reverse transcriptase and protease. This review focuses on the recent research on HIV integrase structure ,inhibitors and also new therapeutic method target at HIV-1 integrase.
It is important that the regulation of the expression level and expression time of a target gene in gene therapy and study the gene function. Constitutive or inappropriate expression of the genes with traditional expression system may interfere with the effect of the gene therapy，even may lead to lethal side effect. Inducible expression systems can be used to regulate the expression level of genes of interest in appropriate time according to the aim of the experiments. Up to date, several inducible expression systems have been established and become the potent tools for gene therapy and studying the function of genes of interest. One of the most promising regulatory systems is the mifepristone-regulated system, which has much lower basal transcriptional activity and high inducibility. Recent developments in system design and application was reviewed in this article.
Abstract: RNA interference (RNAi) is a phenomenon of special post-transcriptional gene silencing mediated by induced dsRNA with endogenous or exogenous homogenous sequences, a lot of researches have been applied to demonstrate it as a reserved mechanism for gene regulation in animals, plants and fungi. In the past decade, RNAi aimed at composition of crop for nutrition improvement had been developed and tested, which included improving the quality of fat and oils, reforming starch quality, improving crop quality through altering composition of crop, elevating anti-browning nature, prolonging fruit-store, procuring secondary metabolite by metabolism regulation. This technology, as a knockdown technology, has been applied to a wide range of species to research plant gene function,silence the expression of specific genes for crop genetics breeding and offers alternative approaches for improving crop quality. The progress and potential of these approaches and studies mentioned above were reviewed and the problem about RNAi technology also was preliminarily discussed in this paper.
The metabolic pathway of anthocyanin including synthesis, modification, transportation and sequestration were described. The molecular regulating mechanism of the anthocyanin pathway was discussed on both transcriptional and post-transcriptional levels. Particular attention was paid to the functions of various transcription factors. The effects of external factors, such as illumination, UV-A, UV-B, low-temperature, water depletion, sugar and other biotic stresses, on the accumulation of anthocyanin were summarized. A model was proposed to explain the regulating mechanism of anthocyanin pathway, and a prospect of anthocyanin research was depicted.
TFL1 homologs play important roles in maintaining vegetative growth and inflorescence meristem identity. The plants without the function of this gene usually are flowering earlier. Their normal inflorescence development is inhibited, the inflorescence meristem eventually acquired floral identity, which producing a terminal flower. Up to now, the TFL1 homologs have been isolated from 28 species of plants, including Arabidopsis, Snapdrogen and Tomato. The phylogenic tree of TFL1 homologs is almost accordance with the relative of those plants. The inflorescence identity gene TFL1 interacted with floral meristem identity genes LFY and AP1, so as to retard the transformation from inflorescence identity into floral identity. These meristem identity genes such as TFL1 and LFY can be applied in breeding of earl-flowering cultivars, there also have plenty of potentials in breeding fruit-free plantanus, popular or willows.
The methods for aptamer selection have been modified and developed in recent years. Selection efficiency has been increased due to the improvement in separation of bound and unbound nucleic acids; applicability of aptamers has been improved due to the development of primer-free SELEX and the utilization of short oligonucleotide library; more research requirements have been satisfied due to the functional modifications of aptamer selection methods. All the methodological advances enhance the potential value of aptamers in basic researches and clinical applications.
The progress in separation and purification technology of nattokinase is reviewed. Based on the analyze of normal purification technology, the novel technologies about the integration of bioseparation processes are introduced, such as aqueous two-phase partitioning integrated with affinity ligand, expanded bed adsorption, and mixed-mode adsorption technology. In general, integration of bioseparation processes provides a good way to improve and optimize the whole separation process of nattokinase and other proteins.
Microbial volatile organic compounds (MVOCs) are important components of metabolites produced by microorganisms, and serve as chemical windows through which the fundamental information about the molecular basis of microbial activities is released. Understanding of the MVOCs, as well as the insights of the molecular basis associated with the MVOCs, provides a real-world capability to control and utilize microorganisms better than ever. MVOCs can be classified into species such as alcohol, aldehyde, organic acid, ester and ketone etc. Traditionally, successful detection of MVOCs requires sample pre-treatment such as separation and enrichment, because of the diversity of MVOCs in terms of physical/chemical property, concentration and matrices present in practical samples. In this review, the process and mechanism of formation of MVOCs are briefly summarized; recent progress made in applications of mass spectrometry for measurement of MVOCs are systematically reviewed, with emphases on the GC-MS and the comparison of different ionization techniques such as APCI, DESI, EESI, SDAPCI, SIFT and PTR. Clearly, novel ionization techniques such as EESI, SDAPCI and DESI requires no sample pretreatment, and provide rapid online analysis of MVOCs in real time. Prospectively, more advanced applications of these novel techniques based on mass spectrometry are anticipated.
