BlotMan is a protein blotting device that allows generating multiple membranes from a single polyacrylamide gel. To transfer all proteins uniformly with the same efficiency regardless of protein size, BlotMan employs ...BlotMan is a protein blotting device that allows generating multiple membranes from a single polyacrylamide gel. To transfer all proteins uniformly with the same efficiency regardless of protein size, BlotMan employs pulse-width-modulated (PWM) voltage that applies a higher average voltage to a larger protein species. BlotMan can be controlled not only by its custom-made interface but also by a smart phone via Bluetooth technology. In this study, we examined effects of PWM signals (50%, 60%, and 80% duty cycle) on transfer efficiency and signal intensity in comparison to a constant voltage signal (100% duty cycle). The result revealed that in response to the same average voltage of 150 V, a lower duty cycle with a higher maximum voltage increased transfer efficiency as well as sharpness of transferred proteins. We validated BlotMan’s capability using a chondrosarcoma cell line (SW1353 cells) and a breast cancer cell line (MDA-MB231 cells) in response to antitumor chemical agents. BlotMan successfully generated 5 membranes from a single gel and detected 5 protein species such as c-Src, eukaryotic translation initiation factor 2 alpha (eIF2), phosphorylated eIF2, lamin B, and actin. Collectively, we demonstrated herein that BlotMan reduces an amount of protein samples by generating multiple membranes from a single gel and improving signal intensity with PWM voltage signals.展开更多
Cuscuta species(dodders)are holoparasites that totally rely on host plants to survive.Although various mobile proteins have been identified to travel within a plant,whether and to what extent protein transfer between ...Cuscuta species(dodders)are holoparasites that totally rely on host plants to survive.Although various mobile proteins have been identified to travel within a plant,whether and to what extent protein transfer between Cuscuta and host plants remain unclear.We found that hundreds to more than 1500 proteins were transferred between Cuscuta and the host plants Arabidopsis and soybean,and hundreds of interplant mobile proteins were even detected in the seeds of Cuscuta and the host soybean.Different hosts bridge-connected by dodder were also found to exchange hundreds of proteins.Quantitatively,the mobile proteins represent a few to more than 10%of the proteomes of foreign plants.Using Arabidopsis plants expressing different reporter proteins,we further showed that these reporter proteins could travel between plants and,importantly,retained their activity in the foreign plants.Comparative analysis between the interplant mobile proteins and mRNAs indicated that the majority of mobile proteins were not de novo synthesized from the translocated mRNAs,but bona fide mobile proteins.We propose that large-scale inter-plant protein translocation may play an important role in the interactions between host plants and dodder and even among the dodder bridge-connected hosts.展开更多
AIM: To identify the two polymorphisms of microsomal triglyceride transfer protein (MTP) gene in the Chinese population and to explore their correlation with both hepatitis B virus (HBV) self-limited infection and per...AIM: To identify the two polymorphisms of microsomal triglyceride transfer protein (MTP) gene in the Chinese population and to explore their correlation with both hepatitis B virus (HBV) self-limited infection and persistent infection. METHODS: A total of 316 subjects with self-limited HBV infection and 316 patients with persistent HBV infection (195 subjects without familial history), matched with age and sex, from the Chinese Han population were enrolled in this study. Polymorphisms of MTP at the promoter region -493 and at H297Q were determined by the allele specific polymerase chain reaction (PCR). RESULTS: The ratio of males to females was 2.13:1 for each group and the average age in the self-limited and chronic infection groups was 38.36 and 38.28 years, respectively. None of the allelic distributions deviated significantly from that predicted by the Hardy-Weinberg equilibrium. There was a linkagedisequilibrium between H297Q and -493G/T (D’ = 0.77). As the χ2 test was used, the genotype distribution of MTP -493G/T demonstrated a significant difference between the self-limited infection group and the entire chronic group or the chronic patients with no family history (χ2 = 8.543, P = 0.015 and χ2 = 7.199, P = 0.019). The allele distribution at the MTP-493 position also demonstrated a significant difference between the study groups without family history (χ2 = 6.212, P = 0.013). The T allele emerged as a possible protective factor which may influence the outcomes of HBV infection (OR: 0.59; 95% CI: 0.389-0.897). CONCLUSION: The polymorphism of the MTP gene, T allele at -493, may be involved in determining the HBV infection outcomes, of which the mechanism needs to be further investigated.展开更多
Cholesteryl ester transfer protein （CETP） is a plasma glycoprotein that plays an important role in decreasing high-density lipoprotein cholesterol （HDL-C） levels and increasing low-density lipoprotein cholesterol ...Cholesteryl ester transfer protein （CETP） is a plasma glycoprotein that plays an important role in decreasing high-density lipoprotein cholesterol （HDL-C） levels and increasing low-density lipoprotein cholesterol （LDL-C） levels. Inhibition of CETP may be a new therapy for treating atherosclerosis. Herein, we report the development of a ligand-based pharmacophore model and pharmacophore-based virtual screening of the ZINC/big-n-greasy database, leading to the identification of compound H-10 as a potential CETP inhibitor in vitro. Based on H-10, a series of 3-（（3,4-dichlorophenyl）（4-substituted benzyl）amino） propanamides were designed, synthesized, and evaluated against CETP. Compound 41 was found to have the best activity, resulting in 85.0% inhibition of CETP at l0 μmol/L.展开更多
Objective: To assess whether the TaqIB polymorphism of cholesteryl ester transfer protein (CETP) is associated with coronary artery disease (CAD) in Chinese population, we performed a meta-analysis in this paper. Meth...Objective: To assess whether the TaqIB polymorphism of cholesteryl ester transfer protein (CETP) is associated with coronary artery disease (CAD) in Chinese population, we performed a meta-analysis in this paper. Methods: We searched PubMed, Embase, the Science Citation Index (SCI), the China Biological Medicine database (CBM), the China National Knowledge Infrastructure (CNKI), and the Wanfang database for relevant articles. Data were extracted, and pooled odds ratios (ORs) and 95% confidence intervals (CIs) were calculated. Results: The lit- erature search yielded 448 studies, in which 10 case-control studies including 1 694 cases and 1 456 controls matched the selection criteria. The combined B1 and B2 allele frequencies were 0.587 and 0.413, respectively. The pooled OR was 1.10 (95% CI, 0.89-1.34) for comparing the B1B1 or B1B2 carriers with B2B2 carriers, and was 1.27 (95% CI, 1.09-1.49) in the B1B1 carriers versus B2B2 or B1B2 carriers. Conclusions: In the present study, the TaqIB poly- morphism of CETP was found to be associated with CAD in the Chinese population.展开更多
Non-specific lipid transfer proteins(nsLTPs) are small, basic proteins that are characterized by an eight-cysteine motif. The biological functions of these proteins have been reported to involve plant reproduction and...Non-specific lipid transfer proteins(nsLTPs) are small, basic proteins that are characterized by an eight-cysteine motif. The biological functions of these proteins have been reported to involve plant reproduction and biotic or abiotic stress response. With the completion of the barley genome sequence, a genome-wide analysis of nsLTPs in barley(Hordeum vulgare L.)(HvLTPs) will be helpful for understanding the function of nsLTPs in plants. We performed a genome-wide analysis of the nsLTP gene family in barley and identified 70 nsLTP genes,which can be divided into five types(1, 2, C, D, and G). Each type of nsLTPs shares similar exon and intron gene structures. Expression analysis showed that barley nsLTPs have diverse expression patterns, revealing their various roles. Our results shed light on the phylogenetic relationships and potential functions of barley nsLTPs and will be useful for future studies of barley development and molecular breeding.展开更多
Objective: To investigate the relationship between polymorphisms of rs1532624 and rs289741 loci in cholesteryl ester transfer protein(CETP) genes and atherosclerotic cerebral infarction(ACI). Methods: The CETP gene rs...Objective: To investigate the relationship between polymorphisms of rs1532624 and rs289741 loci in cholesteryl ester transfer protein(CETP) genes and atherosclerotic cerebral infarction(ACI). Methods: The CETP gene rs1532624 and rs289741 in 95 patients with ACI and 177 healthy subjects were genotyped by Mass ARRAY mass spectrometry. Each locus genotype and allele frequency distributions were compared. Results: The difference of allele frequency distribution between the rs1532624(χ~2=1.723, P=0.189) and rs289741(χ~2=2.466, P=0.116) were not statistically significant. The frequency distribution of rs1532624 genotype between the cerebral infarction group and healthy control group was statistically significant(χ~2=7.096, P=0.029), while rs289741 genotype frequency distribution between the two groups was not statistically significant(χ~2=2.906, P=0.234). Conclusion: ACI have a positive correlation with rs1532624 polymorphism, and AA genotype may be susceptible factors of ACI.展开更多
Cholesteryl ester transfer protein （CETP）, an important regulatory factor in reverse cholesterol transport, has been investigated in relation to plasma HDL cholesterol, which is associated with restenosis after perc...Cholesteryl ester transfer protein （CETP）, an important regulatory factor in reverse cholesterol transport, has been investigated in relation to plasma HDL cholesterol, which is associated with restenosis after percutaneous transluminal coronary angioplasty （PTCA） and stent implantation. We examined the effect of plasma CETP activity and the Taq1B polymorphism on restenosis in Chinese HaM population. Methods and Results Our study included 228 patients with symptomatic coronary artery disease （CAD）. The genotype of the subjects for Taq1B polymorphism of CETP was analyzed by using polymerase chain reaction-restriction fragment length poly- morphism （PCR-RFLP）. In-stent restenosis （ISR） was observed in 93 patients （40.8 %）. Baseline clinical, angiographic,and procedural characteristics data were not significantly different in patients with and without restenosis,except previous history of myocardial infarction （MI）, left ventricular ejection fraction （LVEF）, statin medication, and stent type. Taq1B polymorphism and plasma activity of CETP were not associated with the incidence of ISR （P = 0.68, P = 0.30, respectively）. Linear regression analysis revealed that the risk of ISR was statistically significant correlation with stent type, LVEF, statin medication, and lesion length （P 〈 0.05）. Conclusions Useful markers for risk of ISR were stent type, LVEF, statin medication and lesion length but not the Taq1B polymorphism and plasma activity of CETP.展开更多
Plant nonspecific lipid transfer proteins (nsLTPs) are widely distributed through plant kingdom and are characterized by the presence of a central hydrophobic cavity, suitable for binding various hydrophobic molecules...Plant nonspecific lipid transfer proteins (nsLTPs) are widely distributed through plant kingdom and are characterized by the presence of a central hydrophobic cavity, suitable for binding various hydrophobic molecules. Despite extensive research on nsLTP in different plant species, mostly angiosperm, and the great diversity of physiological processes in which they seem to be involved, their exact functions still remain unclear. Also, very limited experimental data are available on nsLTP in gymnosperm. In this study, we report for the first time on the molecular cloning of nsLTP, from Pinus sylvestris L.(PsLTP1, GenBank accession JN980402.1) and the expression pattern of PsLTP1 during ontogenesis and in response to environmental stress conditions. Total RNA from roots of 7-day old pine seedlings was used to isolate the cDNA clone, corresponding to Scots pine lipid transfer protein. The open reading frame of PsLTP1 consists of 372 bp encoding a protein of 123 amino acids. Amino acid sequence alignment revealed that mature PsLTP1 shares high level of similarity with nsLTP from other conifers and with well-studied nsLTPs from angiosperms. The PsLTP1 contains a 27-amino-acid N-terminal signal sequence and presents all the features of a plant nsLTP. Amino acid comparison analysis and 3D structure prediction showed that PsLTP1 is a type 1 nsLTP. The results of the expression analysis of Scots pine PsLTP1 gene revealed that its transcripts accumulate in actively growing tissues. Furthermore, transcription of PsLTP1 was upregulated in response to cold and salt treatments, and downregulated during acidic, osmotic and water stresses.展开更多
AIM: To determine the significance of cholesteryl ester transfer protein(CETP) in lipoprotein abnormalities in chronic hepatitis C virus(HCV) infection.METHODS: We evaluated the significance of the serum concentration...AIM: To determine the significance of cholesteryl ester transfer protein(CETP) in lipoprotein abnormalities in chronic hepatitis C virus(HCV) infection.METHODS: We evaluated the significance of the serum concentration of CETP in 110 Japanese patients with chronic HCV infection. Fifty-five patients had active HCV infection, and HCV eradication had been achieved in 55. The role of CETP in serum lipoprotein abnormalities, specifically, in triglyceride(TG) concentrations in the four major classes of lipoproteins, was investigated using Pearson correlations in conjunction with multiple regression analysis and compared them between those with active HCV infection and those in whom eradication had been achieved. RESULTS: The serum CETP levels of patients with active HCV infection were significantly higher than those of patients in whom HCV eradication was achieved(mean ± SD, 2.84 ± 0.69 μg/m L vs 2.40 ± 1.00 μg/m L, P = 0.008). In multiple regression analysis, HCV infection status(active or eradicated) was an independent factor significantly associated with the serum CETP level. TG concentrations in low-density lipoprotein(mean ± SD, 36.25 ± 15.28 μg/m L vs 28.14 ± 9.94 μg/m L, P = 0.001) and high-density lipoprotein(HDL)(mean ± SD, 25.9 ± 7.34 μg/m L vs 17.17 ± 4.82 μg/m L, P < 0.001) were significantly higher in patientswith active HCV infection than in those in whom HCV eradication was achieved. The CETP level was strongly correlated with HDL-TG in patients with active HCV infection(R = 0.557, P < 0.001), whereas CETP was not correlated with HDL-TG in patients in whom HCV eradication was achieved(R =-0.079, P = 0.56). CONCLUSION: Our results indicate that CETP plays a role in abnormalities of lipoprotein metabolism in patients with chronic HCV infection.展开更多
In plants,lipid transfer proteins(LTPs)transport pollen wall constituents from the tapetum to the exine,a process essential for pollen wall development.However,the functional cooperation of different LTPs in pollen wa...In plants,lipid transfer proteins(LTPs)transport pollen wall constituents from the tapetum to the exine,a process essential for pollen wall development.However,the functional cooperation of different LTPs in pollen wall development is not well understood.In this study,we have identified and characterized a grassspecific LTP gene,Os LTP47,an important regulator of pollen wall formation in rice(Oryza sativa).Os LTP47 encodes a membrane-localized LTP and in vitro lipid-binding assays confirms that Os LTP47 has lipidbinding activity.Dysfunction of Os LTP47 causes disordered lipid metabolism and defective pollen walls,leading to male sterility.Yeast two-hybrid and pull-down assays reveal that Os LTP47 physically interacts with another LTP,Os C6.These findings suggest that the plasma membrane-localized Os LTP47 may function as a mediator in a lipid transfer relay through association with cytosolic and/or locular Os C6 for pollen wall development and that various LTPs may function in a coordinated manner to transport lipid molecules during pollen wall development.展开更多
Several epidemiological studies have clearly shown that low plasma levels of high density lipoprotein cholesterol (HDL-C) represent a cardiovascular disease (CVD) risk factor. However, it is unclear if there is a caus...Several epidemiological studies have clearly shown that low plasma levels of high density lipoprotein cholesterol (HDL-C) represent a cardiovascular disease (CVD) risk factor. However, it is unclear if there is a causal association between HDL-C concentration and CVD. A recent study published in the Lancet, which performed two Mendelian randomization analyses, showed that increased HDL-C levels were not associated with a decreased risk of myocardial infarction. These findings, together with the termination of the niacin-based AIM-HIGH trial and the discontinuation of cholesteryl ester transfer protein inhibitor dalcetrapib, challenge the concept that raising of plasma HDL-C will uniformly translate into reductions in CVD risk. HDL particles exhibit several anti-atherosclerotic properties, such as anti-inflammatory and anti-oxidative activities and cellular cholesterol efflux activity. Furthermore, HDL particles are very heterogeneous in terms of size, structure, composition and metabolism. HDL functionality may be associated more strongly with CVD risk than the traditional HDL-C levels. More research is needed to assess the association of the structure of HDL particle with its functionality and metabolism.展开更多
Objective To develop retrovirus-mediated transfer of the fusion gene encoding EGFP-BMP_2 in mesenchymal stem cells.Methods Mesenchymal stem cells from New Zealand white rabbits were transduced with retroviral pLEGFP-B...Objective To develop retrovirus-mediated transfer of the fusion gene encoding EGFP-BMP_2 in mesenchymal stem cells.Methods Mesenchymal stem cells from New Zealand white rabbits were transduced with retroviral pLEGFP-BMP_2 vector by the optimized retroviral transduction protocol.Fluorescent microscopy's examination was to evaluate the results of the transduction,flow cytometer's analysis was to evaluate the transduction efficiency and the Fluorescence-activated cell sorting method was to sort the transduced cells.Bioactivity test from C_2C_12K_4 cells was to show the expression and bio-activity of the fusion gene.Results Fluorescent microscopy showed the success of the transduction.By flow cytometer's analysis,the mean efficiency of the transduction with EGFP was(42.8±6.1)% SD.Transduced cells were sorted efficiently by the fluorescence-activated cell sorting method and after sorting,almost of those showed the expression of BMP_2.Fluorescently and strongly bioactivity test for C_2C_12K_4 cells demonstrated that fluorescent materials were located the surface of cells and the activity of luciferase increased compared with the control.Analysis of long-term expression showed there was no difference between 2 week-time point and 3 month-time point of culture post-sorting.Conclusion Mesenchymal stem cells can be transduced efficiently by retrovirus-mediated transfer of the fusion gene encoding EGFP-BMP_2,the highly pure transduced cells are obtained by the fluorescence-activated cell sorting technique,the expressed chimeric protein embraced the double bioactivity of EGFP and BMP_2,and moreover,the expression had not attenuated over time.展开更多
Lipoproteins are protein-lipid macromolecular assemblies which are used to transport lipids in circulation and are key targets in cardiovascular disease （CVD）. The highly dynamic lipoprotein molecules are capable of...Lipoproteins are protein-lipid macromolecular assemblies which are used to transport lipids in circulation and are key targets in cardiovascular disease （CVD）. The highly dynamic lipoprotein molecules are capable of adopting an array of conformations that is crucial to lipid transport along the cholesterol transport pathway, among which high-density lipopro- tein （HDL） and low-density lipoprotein （LDL） are major players in plasma cholesterol metabolism. For a more detailed illustration of cholesterol transport process, as well as the development of therapies to prevent CVD, here we review the functional mechanism and structural basis of lipoproteins in cholesterol transport, as well as their structural dynamics in the plasma lipoprotein （HDL and LDL） elevations, in order to obtain better quantitative understandings on structure-function relationship of lipoproteins. Finally, we also provide an approach for further research on the lipoprotein in cholesterol transport.展开更多
Western blotting is a popular technique for examining expression levels of proteins using gel-based electrophoretic fractionation followed by blotting and antibody reactions. Although this is a mature technique, one o...Western blotting is a popular technique for examining expression levels of proteins using gel-based electrophoretic fractionation followed by blotting and antibody reactions. Although this is a mature technique, one of the major limitations is the need to prepare an individual electrophoretic gel for each of the protein species to be analyzed. Since most analyses require the detection of multiple protein species, a procedure that allows utilization of a single gel for detecting multiple protein species should significantly save time and resources. In this paper, we developed a novel multiprotein detection device, which enabled simultaneous detection of several proteins species from a single electrophoretic gel. In this device, a protein transfer unit utilized a multi-anode plate that generated a non-uniform voltage profile. This voltage profile enabled uniform transfer regardless of molecular mass of proteins. In vitro experiments using samples, isolated from boneforming osteoblast cells, showed that the expression levels of 5 - 7 different proteins were detectable in the presence and absence of mechanical stimulation that activated genes necessary for bone formation. The result supports the notion that through simultaneous detection of multiple protein species, the described device contributes to reduction in procedural time and sample amounts, as well as a removal of variations among multiple gels.展开更多
Blotting is a common technique widely used for molecular analysis in life sciences. The Western blot, in particular, is a process of transferring protein samples from a polyacrylamide gel to a blotting membrane and de...Blotting is a common technique widely used for molecular analysis in life sciences. The Western blot, in particular, is a process of transferring protein samples from a polyacrylamide gel to a blotting membrane and detecting the levels of specific proteins through reactions with primary and secondary antibodies. The state-of-the-art of Western blotting usually generates one blotting membrane per gel. However, multiple copies of blots are useful in many applications. Two blotting copies from a single protein gel, for instance, can be used for identifying a total amount of proteins of interest as well as its specific subpopulation level such as a phosphorylated isoform. To achieve this multi-blotting operation from a single gel, we modified a blotting procedure and developed a novel blotting device. The device consisted of a multi-anode plate and a microcontroller. It was designed to generate a well-controlled electrophoretic voltage profile, which allowed a quasi-uniform transfer of proteins of any size. The prototype device was built and its operation procedure was described. The experimental results clearly supported the notion that the described device was able to achieve multiple blotting from a single gel and reduce time and cost for protein analysis.展开更多
Objective: To investigate the feasibility of marking the human tumor cells with enhanced green fluorescent protein (EGFP) in vitro. Methods: The retroviral vector LGSN encoding EGFP was constructed and three human tum...Objective: To investigate the feasibility of marking the human tumor cells with enhanced green fluorescent protein (EGFP) in vitro. Methods: The retroviral vector LGSN encoding EGFP was constructed and three human tumor cell lines were infected with LGSN amphotropic virus. Tumor cell lines that stably express EGFP were selected with G418. The integration and expression of EGFP gene were analyzed by polymerase chain reaction, and flow cytometry (FCM). Results: After gene transfection and ping-pong transduction, amphotropic producer line Am12/LGSN was generated with a stable green fluorescence signal readily detectable by FCM in up to 97% of examined cells. The viral titer in the supernatants was up to 8.2×105CFU/ml. After transduction and selection, G418-resistant leukemia K562, mammary carcinoma MCF-7, and bladder cancer 5637 cells were developed, in which the integration of both EGFP and neomycin resistance gene was confirmed by DNA amplification. In comparison with uninfected cells, FCM analysis revealed EGFP expression in up to 90% (range 85.5%–90.0%) of tumor cells containing LGSN provirus. Conclusion: The retroviral vector LGSN can effectively mark the human tumor cells with a stably EGFP expression which may be in studying tumor growth, metastasis and angiogenesis.展开更多
The green fluorescence of bioluminescent jellyfish Aequorea victoria is due to the presence of the green fluorescent protein (GFP). To examine whether the GFP gene can be applied as a reporter gene in insect cells...The green fluorescence of bioluminescent jellyfish Aequorea victoria is due to the presence of the green fluorescent protein (GFP). To examine whether the GFP gene can be applied as a reporter gene in insect cells, a baculovirus transfer vector containing the neomycin resistance gene (neo) was established. The GFP gene was subcloned into the vector downstream of the polyhedrin gene (ocu) promoter. In the presence of G418, the recombinant virus can be purified. Expression of the GFP gene in the recombinant virus should give rise to synthesis of the GFP with a molecular weight of 30×10 3 dalton, and is observable by the strong green light irradiated by ultraviolet or blue light in viable intact insect cells. The GFP produced in insect cells has typical fluorescent spectra indistinguishable from those of the purified native GFP. The GFP gene as a good reporter gene can be applied to the baculovirus insect cell expression system.展开更多
Exogenous DNA expressing green fluorescent protein( GFP) and labeled with fluorescein isothiocyanate( FITC) was used to transform the Chinese oak silkmoth Antheraea pernyi( A. pernyi)via sperm-mediated gene transfer( ...Exogenous DNA expressing green fluorescent protein( GFP) and labeled with fluorescein isothiocyanate( FITC) was used to transform the Chinese oak silkmoth Antheraea pernyi( A. pernyi)via sperm-mediated gene transfer( SMGT). Sperms entry into the female reproductive system and eggs were observed using fluorescence microscopy. The ability of A. pernyi sperms to uptake exogenous DNA was confirmed,and transfer of the exogenous DNA was shown by GFP expression in the transgenic eggs. Our result suggested that SMGT could also be used to directly generate transgenic A. pernyi expressing functional genes of interest.展开更多
文摘BlotMan is a protein blotting device that allows generating multiple membranes from a single polyacrylamide gel. To transfer all proteins uniformly with the same efficiency regardless of protein size, BlotMan employs pulse-width-modulated (PWM) voltage that applies a higher average voltage to a larger protein species. BlotMan can be controlled not only by its custom-made interface but also by a smart phone via Bluetooth technology. In this study, we examined effects of PWM signals (50%, 60%, and 80% duty cycle) on transfer efficiency and signal intensity in comparison to a constant voltage signal (100% duty cycle). The result revealed that in response to the same average voltage of 150 V, a lower duty cycle with a higher maximum voltage increased transfer efficiency as well as sharpness of transferred proteins. We validated BlotMan’s capability using a chondrosarcoma cell line (SW1353 cells) and a breast cancer cell line (MDA-MB231 cells) in response to antitumor chemical agents. BlotMan successfully generated 5 membranes from a single gel and detected 5 protein species such as c-Src, eukaryotic translation initiation factor 2 alpha (eIF2), phosphorylated eIF2, lamin B, and actin. Collectively, we demonstrated herein that BlotMan reduces an amount of protein samples by generating multiple membranes from a single gel and improving signal intensity with PWM voltage signals.
基金This work was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences(XDB11050200)the National Science Foundation of China(31970274)+1 种基金CAS Youth Innovation Prom otion Association(2018426)the International Partnership Program of the Chinese Academy of Sciences(151853KYSB20170025).
文摘Cuscuta species(dodders)are holoparasites that totally rely on host plants to survive.Although various mobile proteins have been identified to travel within a plant,whether and to what extent protein transfer between Cuscuta and host plants remain unclear.We found that hundreds to more than 1500 proteins were transferred between Cuscuta and the host plants Arabidopsis and soybean,and hundreds of interplant mobile proteins were even detected in the seeds of Cuscuta and the host soybean.Different hosts bridge-connected by dodder were also found to exchange hundreds of proteins.Quantitatively,the mobile proteins represent a few to more than 10%of the proteomes of foreign plants.Using Arabidopsis plants expressing different reporter proteins,we further showed that these reporter proteins could travel between plants and,importantly,retained their activity in the foreign plants.Comparative analysis between the interplant mobile proteins and mRNAs indicated that the majority of mobile proteins were not de novo synthesized from the translocated mRNAs,but bona fide mobile proteins.We propose that large-scale inter-plant protein translocation may play an important role in the interactions between host plants and dodder and even among the dodder bridge-connected hosts.
基金F.Hoffmann-La Roche Ltd Switzerland and the National High Technology ResearchDevelopment Program of China (863 Program), No. 2006AA02A411
文摘AIM: To identify the two polymorphisms of microsomal triglyceride transfer protein (MTP) gene in the Chinese population and to explore their correlation with both hepatitis B virus (HBV) self-limited infection and persistent infection. METHODS: A total of 316 subjects with self-limited HBV infection and 316 patients with persistent HBV infection (195 subjects without familial history), matched with age and sex, from the Chinese Han population were enrolled in this study. Polymorphisms of MTP at the promoter region -493 and at H297Q were determined by the allele specific polymerase chain reaction (PCR). RESULTS: The ratio of males to females was 2.13:1 for each group and the average age in the self-limited and chronic infection groups was 38.36 and 38.28 years, respectively. None of the allelic distributions deviated significantly from that predicted by the Hardy-Weinberg equilibrium. There was a linkagedisequilibrium between H297Q and -493G/T (D’ = 0.77). As the χ2 test was used, the genotype distribution of MTP -493G/T demonstrated a significant difference between the self-limited infection group and the entire chronic group or the chronic patients with no family history (χ2 = 8.543, P = 0.015 and χ2 = 7.199, P = 0.019). The allele distribution at the MTP-493 position also demonstrated a significant difference between the study groups without family history (χ2 = 6.212, P = 0.013). The T allele emerged as a possible protective factor which may influence the outcomes of HBV infection (OR: 0.59; 95% CI: 0.389-0.897). CONCLUSION: The polymorphism of the MTP gene, T allele at -493, may be involved in determining the HBV infection outcomes, of which the mechanism needs to be further investigated.
文摘Cholesteryl ester transfer protein （CETP） is a plasma glycoprotein that plays an important role in decreasing high-density lipoprotein cholesterol （HDL-C） levels and increasing low-density lipoprotein cholesterol （LDL-C） levels. Inhibition of CETP may be a new therapy for treating atherosclerosis. Herein, we report the development of a ligand-based pharmacophore model and pharmacophore-based virtual screening of the ZINC/big-n-greasy database, leading to the identification of compound H-10 as a potential CETP inhibitor in vitro. Based on H-10, a series of 3-（（3,4-dichlorophenyl）（4-substituted benzyl）amino） propanamides were designed, synthesized, and evaluated against CETP. Compound 41 was found to have the best activity, resulting in 85.0% inhibition of CETP at l0 μmol/L.
基金Project supported by the National Natural Science Foundation of China(Nos. 30900526 and 81070250)the Fundamental Research Funds for the Central Universitiesthe China Postdoctoral Science Foundation(No. 20100471616)
文摘Objective: To assess whether the TaqIB polymorphism of cholesteryl ester transfer protein (CETP) is associated with coronary artery disease (CAD) in Chinese population, we performed a meta-analysis in this paper. Methods: We searched PubMed, Embase, the Science Citation Index (SCI), the China Biological Medicine database (CBM), the China National Knowledge Infrastructure (CNKI), and the Wanfang database for relevant articles. Data were extracted, and pooled odds ratios (ORs) and 95% confidence intervals (CIs) were calculated. Results: The lit- erature search yielded 448 studies, in which 10 case-control studies including 1 694 cases and 1 456 controls matched the selection criteria. The combined B1 and B2 allele frequencies were 0.587 and 0.413, respectively. The pooled OR was 1.10 (95% CI, 0.89-1.34) for comparing the B1B1 or B1B2 carriers with B2B2 carriers, and was 1.27 (95% CI, 1.09-1.49) in the B1B1 carriers versus B2B2 or B1B2 carriers. Conclusions: In the present study, the TaqIB poly- morphism of CETP was found to be associated with CAD in the Chinese population.
基金supported by funds from the National Key Research and Development Program of China (2016YFD0100903)
文摘Non-specific lipid transfer proteins(nsLTPs) are small, basic proteins that are characterized by an eight-cysteine motif. The biological functions of these proteins have been reported to involve plant reproduction and biotic or abiotic stress response. With the completion of the barley genome sequence, a genome-wide analysis of nsLTPs in barley(Hordeum vulgare L.)(HvLTPs) will be helpful for understanding the function of nsLTPs in plants. We performed a genome-wide analysis of the nsLTP gene family in barley and identified 70 nsLTP genes,which can be divided into five types(1, 2, C, D, and G). Each type of nsLTPs shares similar exon and intron gene structures. Expression analysis showed that barley nsLTPs have diverse expression patterns, revealing their various roles. Our results shed light on the phylogenetic relationships and potential functions of barley nsLTPs and will be useful for future studies of barley development and molecular breeding.
基金supported by grants from Natural Science Foundation of China(31501018,31760310,and 81660224)the Social Development Project of Hainan Province(SF201401)
文摘Objective: To investigate the relationship between polymorphisms of rs1532624 and rs289741 loci in cholesteryl ester transfer protein(CETP) genes and atherosclerotic cerebral infarction(ACI). Methods: The CETP gene rs1532624 and rs289741 in 95 patients with ACI and 177 healthy subjects were genotyped by Mass ARRAY mass spectrometry. Each locus genotype and allele frequency distributions were compared. Results: The difference of allele frequency distribution between the rs1532624(χ~2=1.723, P=0.189) and rs289741(χ~2=2.466, P=0.116) were not statistically significant. The frequency distribution of rs1532624 genotype between the cerebral infarction group and healthy control group was statistically significant(χ~2=7.096, P=0.029), while rs289741 genotype frequency distribution between the two groups was not statistically significant(χ~2=2.906, P=0.234). Conclusion: ACI have a positive correlation with rs1532624 polymorphism, and AA genotype may be susceptible factors of ACI.
基金Natural Science Foundation of Guangdong Province,China(No.5001160)
文摘Cholesteryl ester transfer protein （CETP）, an important regulatory factor in reverse cholesterol transport, has been investigated in relation to plasma HDL cholesterol, which is associated with restenosis after percutaneous transluminal coronary angioplasty （PTCA） and stent implantation. We examined the effect of plasma CETP activity and the Taq1B polymorphism on restenosis in Chinese HaM population. Methods and Results Our study included 228 patients with symptomatic coronary artery disease （CAD）. The genotype of the subjects for Taq1B polymorphism of CETP was analyzed by using polymerase chain reaction-restriction fragment length poly- morphism （PCR-RFLP）. In-stent restenosis （ISR） was observed in 93 patients （40.8 %）. Baseline clinical, angiographic,and procedural characteristics data were not significantly different in patients with and without restenosis,except previous history of myocardial infarction （MI）, left ventricular ejection fraction （LVEF）, statin medication, and stent type. Taq1B polymorphism and plasma activity of CETP were not associated with the incidence of ISR （P = 0.68, P = 0.30, respectively）. Linear regression analysis revealed that the risk of ISR was statistically significant correlation with stent type, LVEF, statin medication, and lesion length （P 〈 0.05）. Conclusions Useful markers for risk of ISR were stent type, LVEF, statin medication and lesion length but not the Taq1B polymorphism and plasma activity of CETP.
基金supported by grants from the Ministry of Education and Science of Ukraine(0116U003593)grant from cieA3(Campus de Excelencia Internacional Agroalimentario)-UCO,Spain
文摘Plant nonspecific lipid transfer proteins (nsLTPs) are widely distributed through plant kingdom and are characterized by the presence of a central hydrophobic cavity, suitable for binding various hydrophobic molecules. Despite extensive research on nsLTP in different plant species, mostly angiosperm, and the great diversity of physiological processes in which they seem to be involved, their exact functions still remain unclear. Also, very limited experimental data are available on nsLTP in gymnosperm. In this study, we report for the first time on the molecular cloning of nsLTP, from Pinus sylvestris L.(PsLTP1, GenBank accession JN980402.1) and the expression pattern of PsLTP1 during ontogenesis and in response to environmental stress conditions. Total RNA from roots of 7-day old pine seedlings was used to isolate the cDNA clone, corresponding to Scots pine lipid transfer protein. The open reading frame of PsLTP1 consists of 372 bp encoding a protein of 123 amino acids. Amino acid sequence alignment revealed that mature PsLTP1 shares high level of similarity with nsLTP from other conifers and with well-studied nsLTPs from angiosperms. The PsLTP1 contains a 27-amino-acid N-terminal signal sequence and presents all the features of a plant nsLTP. Amino acid comparison analysis and 3D structure prediction showed that PsLTP1 is a type 1 nsLTP. The results of the expression analysis of Scots pine PsLTP1 gene revealed that its transcripts accumulate in actively growing tissues. Furthermore, transcription of PsLTP1 was upregulated in response to cold and salt treatments, and downregulated during acidic, osmotic and water stresses.
文摘AIM: To determine the significance of cholesteryl ester transfer protein(CETP) in lipoprotein abnormalities in chronic hepatitis C virus(HCV) infection.METHODS: We evaluated the significance of the serum concentration of CETP in 110 Japanese patients with chronic HCV infection. Fifty-five patients had active HCV infection, and HCV eradication had been achieved in 55. The role of CETP in serum lipoprotein abnormalities, specifically, in triglyceride(TG) concentrations in the four major classes of lipoproteins, was investigated using Pearson correlations in conjunction with multiple regression analysis and compared them between those with active HCV infection and those in whom eradication had been achieved. RESULTS: The serum CETP levels of patients with active HCV infection were significantly higher than those of patients in whom HCV eradication was achieved(mean ± SD, 2.84 ± 0.69 μg/m L vs 2.40 ± 1.00 μg/m L, P = 0.008). In multiple regression analysis, HCV infection status(active or eradicated) was an independent factor significantly associated with the serum CETP level. TG concentrations in low-density lipoprotein(mean ± SD, 36.25 ± 15.28 μg/m L vs 28.14 ± 9.94 μg/m L, P = 0.001) and high-density lipoprotein(HDL)(mean ± SD, 25.9 ± 7.34 μg/m L vs 17.17 ± 4.82 μg/m L, P < 0.001) were significantly higher in patientswith active HCV infection than in those in whom HCV eradication was achieved. The CETP level was strongly correlated with HDL-TG in patients with active HCV infection(R = 0.557, P < 0.001), whereas CETP was not correlated with HDL-TG in patients in whom HCV eradication was achieved(R =-0.079, P = 0.56). CONCLUSION: Our results indicate that CETP plays a role in abnormalities of lipoprotein metabolism in patients with chronic HCV infection.
基金supported by grants from the Guangdong Natural Science Funds for Distinguished Young Scholars (2021B1515020089)the National Natural Science Foundation of China (32030080)the Major Program of Guangdong Basic and Applied Research(2019B030302006)
文摘In plants,lipid transfer proteins(LTPs)transport pollen wall constituents from the tapetum to the exine,a process essential for pollen wall development.However,the functional cooperation of different LTPs in pollen wall development is not well understood.In this study,we have identified and characterized a grassspecific LTP gene,Os LTP47,an important regulator of pollen wall formation in rice(Oryza sativa).Os LTP47 encodes a membrane-localized LTP and in vitro lipid-binding assays confirms that Os LTP47 has lipidbinding activity.Dysfunction of Os LTP47 causes disordered lipid metabolism and defective pollen walls,leading to male sterility.Yeast two-hybrid and pull-down assays reveal that Os LTP47 physically interacts with another LTP,Os C6.These findings suggest that the plasma membrane-localized Os LTP47 may function as a mediator in a lipid transfer relay through association with cytosolic and/or locular Os C6 for pollen wall development and that various LTPs may function in a coordinated manner to transport lipid molecules during pollen wall development.
文摘Several epidemiological studies have clearly shown that low plasma levels of high density lipoprotein cholesterol (HDL-C) represent a cardiovascular disease (CVD) risk factor. However, it is unclear if there is a causal association between HDL-C concentration and CVD. A recent study published in the Lancet, which performed two Mendelian randomization analyses, showed that increased HDL-C levels were not associated with a decreased risk of myocardial infarction. These findings, together with the termination of the niacin-based AIM-HIGH trial and the discontinuation of cholesteryl ester transfer protein inhibitor dalcetrapib, challenge the concept that raising of plasma HDL-C will uniformly translate into reductions in CVD risk. HDL particles exhibit several anti-atherosclerotic properties, such as anti-inflammatory and anti-oxidative activities and cellular cholesterol efflux activity. Furthermore, HDL particles are very heterogeneous in terms of size, structure, composition and metabolism. HDL functionality may be associated more strongly with CVD risk than the traditional HDL-C levels. More research is needed to assess the association of the structure of HDL particle with its functionality and metabolism.
基金This work was supported by the National Natural Science Foundation of China(No.30400163).
文摘Objective To develop retrovirus-mediated transfer of the fusion gene encoding EGFP-BMP_2 in mesenchymal stem cells.Methods Mesenchymal stem cells from New Zealand white rabbits were transduced with retroviral pLEGFP-BMP_2 vector by the optimized retroviral transduction protocol.Fluorescent microscopy's examination was to evaluate the results of the transduction,flow cytometer's analysis was to evaluate the transduction efficiency and the Fluorescence-activated cell sorting method was to sort the transduced cells.Bioactivity test from C_2C_12K_4 cells was to show the expression and bio-activity of the fusion gene.Results Fluorescent microscopy showed the success of the transduction.By flow cytometer's analysis,the mean efficiency of the transduction with EGFP was(42.8±6.1)% SD.Transduced cells were sorted efficiently by the fluorescence-activated cell sorting method and after sorting,almost of those showed the expression of BMP_2.Fluorescently and strongly bioactivity test for C_2C_12K_4 cells demonstrated that fluorescent materials were located the surface of cells and the activity of luciferase increased compared with the control.Analysis of long-term expression showed there was no difference between 2 week-time point and 3 month-time point of culture post-sorting.Conclusion Mesenchymal stem cells can be transduced efficiently by retrovirus-mediated transfer of the fusion gene encoding EGFP-BMP_2,the highly pure transduced cells are obtained by the fluorescence-activated cell sorting technique,the expressed chimeric protein embraced the double bioactivity of EGFP and BMP_2,and moreover,the expression had not attenuated over time.
基金Project supported by the National Natural Science Foundation of China(Grant Nos.11504287 and 11774279)
文摘Lipoproteins are protein-lipid macromolecular assemblies which are used to transport lipids in circulation and are key targets in cardiovascular disease （CVD）. The highly dynamic lipoprotein molecules are capable of adopting an array of conformations that is crucial to lipid transport along the cholesterol transport pathway, among which high-density lipopro- tein （HDL） and low-density lipoprotein （LDL） are major players in plasma cholesterol metabolism. For a more detailed illustration of cholesterol transport process, as well as the development of therapies to prevent CVD, here we review the functional mechanism and structural basis of lipoproteins in cholesterol transport, as well as their structural dynamics in the plasma lipoprotein （HDL and LDL） elevations, in order to obtain better quantitative understandings on structure-function relationship of lipoproteins. Finally, we also provide an approach for further research on the lipoprotein in cholesterol transport.
文摘Western blotting is a popular technique for examining expression levels of proteins using gel-based electrophoretic fractionation followed by blotting and antibody reactions. Although this is a mature technique, one of the major limitations is the need to prepare an individual electrophoretic gel for each of the protein species to be analyzed. Since most analyses require the detection of multiple protein species, a procedure that allows utilization of a single gel for detecting multiple protein species should significantly save time and resources. In this paper, we developed a novel multiprotein detection device, which enabled simultaneous detection of several proteins species from a single electrophoretic gel. In this device, a protein transfer unit utilized a multi-anode plate that generated a non-uniform voltage profile. This voltage profile enabled uniform transfer regardless of molecular mass of proteins. In vitro experiments using samples, isolated from boneforming osteoblast cells, showed that the expression levels of 5 - 7 different proteins were detectable in the presence and absence of mechanical stimulation that activated genes necessary for bone formation. The result supports the notion that through simultaneous detection of multiple protein species, the described device contributes to reduction in procedural time and sample amounts, as well as a removal of variations among multiple gels.
文摘Blotting is a common technique widely used for molecular analysis in life sciences. The Western blot, in particular, is a process of transferring protein samples from a polyacrylamide gel to a blotting membrane and detecting the levels of specific proteins through reactions with primary and secondary antibodies. The state-of-the-art of Western blotting usually generates one blotting membrane per gel. However, multiple copies of blots are useful in many applications. Two blotting copies from a single protein gel, for instance, can be used for identifying a total amount of proteins of interest as well as its specific subpopulation level such as a phosphorylated isoform. To achieve this multi-blotting operation from a single gel, we modified a blotting procedure and developed a novel blotting device. The device consisted of a multi-anode plate and a microcontroller. It was designed to generate a well-controlled electrophoretic voltage profile, which allowed a quasi-uniform transfer of proteins of any size. The prototype device was built and its operation procedure was described. The experimental results clearly supported the notion that the described device was able to achieve multiple blotting from a single gel and reduce time and cost for protein analysis.
文摘Objective: To investigate the feasibility of marking the human tumor cells with enhanced green fluorescent protein (EGFP) in vitro. Methods: The retroviral vector LGSN encoding EGFP was constructed and three human tumor cell lines were infected with LGSN amphotropic virus. Tumor cell lines that stably express EGFP were selected with G418. The integration and expression of EGFP gene were analyzed by polymerase chain reaction, and flow cytometry (FCM). Results: After gene transfection and ping-pong transduction, amphotropic producer line Am12/LGSN was generated with a stable green fluorescence signal readily detectable by FCM in up to 97% of examined cells. The viral titer in the supernatants was up to 8.2×105CFU/ml. After transduction and selection, G418-resistant leukemia K562, mammary carcinoma MCF-7, and bladder cancer 5637 cells were developed, in which the integration of both EGFP and neomycin resistance gene was confirmed by DNA amplification. In comparison with uninfected cells, FCM analysis revealed EGFP expression in up to 90% (range 85.5%–90.0%) of tumor cells containing LGSN provirus. Conclusion: The retroviral vector LGSN can effectively mark the human tumor cells with a stably EGFP expression which may be in studying tumor growth, metastasis and angiogenesis.
文摘The green fluorescence of bioluminescent jellyfish Aequorea victoria is due to the presence of the green fluorescent protein (GFP). To examine whether the GFP gene can be applied as a reporter gene in insect cells, a baculovirus transfer vector containing the neomycin resistance gene (neo) was established. The GFP gene was subcloned into the vector downstream of the polyhedrin gene (ocu) promoter. In the presence of G418, the recombinant virus can be purified. Expression of the GFP gene in the recombinant virus should give rise to synthesis of the GFP with a molecular weight of 30×10 3 dalton, and is observable by the strong green light irradiated by ultraviolet or blue light in viable intact insect cells. The GFP produced in insect cells has typical fluorescent spectra indistinguishable from those of the purified native GFP. The GFP gene as a good reporter gene can be applied to the baculovirus insect cell expression system.
基金Scientific Research Project for High Schools of the Educational Department of Liaoning Province,China(No.2008643)
文摘Exogenous DNA expressing green fluorescent protein( GFP) and labeled with fluorescein isothiocyanate( FITC) was used to transform the Chinese oak silkmoth Antheraea pernyi( A. pernyi)via sperm-mediated gene transfer( SMGT). Sperms entry into the female reproductive system and eggs were observed using fluorescence microscopy. The ability of A. pernyi sperms to uptake exogenous DNA was confirmed,and transfer of the exogenous DNA was shown by GFP expression in the transgenic eggs. Our result suggested that SMGT could also be used to directly generate transgenic A. pernyi expressing functional genes of interest.
