Coomassie Blue Research Articles

Icariin improves acute kidney injury and proteinuria in a rat model of pregnancy‑induced hypertension.

Icariin has previously been demonstrated to attenuate hyperglycemia-induced renal injury, however the renoprotective effects of icariin in a rat model of pregnancy-induced hypertension (PIH) remain to be elucidated. The present study aimed to investigate the effect of icariin on PIH-induced acute kidney injury (AKI) and proteinuria. Following 18 days of icariin treatment between day 1 and day 18 of gestation, which was combined with NG-nitro-L-arginine methyl ester (L-NAME) treatment between day 12 and day 18 of gestation to induce PIH, the 24 h urine protein level, blood urea nitrogen and serum creatinine were measured by using the Coomassie Brilliant Blue method, a commercial enzymatic kit and the picric acid method, respectively. Renal tissues were collected at day 18 of gestation for hematoxylin and eosin staining and immunohistochemistry. The mRNA expression of AGT and protein expression of angiotensin II (Ang II) in the kidneys of control and PIH rats was investigated by reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively, to determine the effect of icariin on components of the renin-angiotensin system. The results demonstrated that L-NAME treatment in pregnant rats resulted in significant increases in systolic blood pressure (SBP) and diastolic blood pressure, in addition to the induction of severe proteinuria. The significant increase in SBP and proteinuria in PIH rats was prevented by icariin. L-NAME-induced AKI resulted in profound renal histological alterations, including mesangial expansion and glomerular lesions. L-NAME administration exerted a marked decrease in the mRNA and protein expression levels of nephrin in the kidneys from PIH rats compared with control group. Furthermore, upregulation of circulating and renal Ang II levels in PIH rats was observed. However, icariin treatment significantly reversed the L-NAME-induced downregulation of nephrin and upregulation of circulating and renal Ang II levels in PIH rats. These results demonstrated that icariin administration improved urinary protein excretion levels and renal tissue damage in PIH rats, and the underlying mechanism was mediated in part, via upregulation of nephrin expression and downregulation of Ang II.

Phenoloxidase activity in humoral plasma, hemocyanin and hemocyanin separated proteins of the giant freshwater prawn Macrobrachium rosenbergii

Hemocyanin is a copper containing protein and its role in the immune function of phenoloxidase (PO) activity was investigated in the giant freshwater prawn Macrobrachium rosenbergii. Hemocyanin, sedimented by ultracentrifugation from the plasma appeared on polyacrylamide gel electrophoresis (PAGE 7%) on Coomassie Brilliant Blue and bathocuproine sulfonic acid stain as four copper containing proteins of molecular masses 50, 60, 114 and 325kDa. Accordingly, on diethylaminoethyl-cellulose anion exchange column hemocyanin separated into four proteins designated as MrHc1, MrHc2, MrHc3 and MrHc4 with electrophoretically (PAGE) determined molecular masses of 60, 114, 50 and 325kDa respectively. The reduction of proteins in sodium dodecyl sulphate (SDS)-PAGE revealed that MrHc1 and 3 were monomeric for 60and 50kDa respectively, MrHc2 dimeric of 56 and 58kDa subunits and MrHc4 appeared with three subunits of 74, 76 and 78kDa. The PO activity was determined in plasma, hemocyanin and the four separated hemocyanin proteins in vitro using L-3,4-dihydroxyphenylalanine (L-DOPA) at pH7.5, 25°C and appeared elicited by exogenous activators such as trypsin, SDS, cell wall components of bacteria and polysaccharide laminarin. This study clearly demonstrated hemocyanin as the major copper containing protein in the plasma of M. rosenbergii with potent PO activity.

Activity of xyloglucan endotransglucosylases/hydrolases suggests a role during host invasion by the parasitic plant Cuscuta reflexa.

The parasitic vines of the genus Cuscuta form haustoria that grow into other plants and connect with their vascular system, thus allowing the parasite to feed on its host. A major obstacle that meets the infection organ as it penetrates the host tissue is the rigid plant cell wall. In the present study, we examined the activity of xyloglucan endotransglucosylases/hydrolases (XTHs) during the host-invasive growth of the haustorium. The level of xyloglucan endotransglucosylation (XET) activity was found to peak at the penetrating stage of Cuscuta reflexa on its host Pelargonium zonale. In vivo colocalization of XET activity and donor substrate demonstrated XET activity at the border between host and parasite. A test for secretion of XET-active enzymes from haustoria of C. reflexa corroborated this and further indicated that the xyloglucan-modifying enzymes originated from the parasite. A known inhibitor of XET, Coomassie Brilliant Blue R250, was shown to reduce the level of XET in penetrating haustoria of C. reflexa. Moreover, the coating of P. zonale petioles with the inhibitor compound lowered the number of successful haustorial invasions of this otherwise compatible host plant. The presented data indicate that the activity of Cuscuta XTHs at the host-parasite interface is essential to penetration of host plant tissue.

Determination of Trace Amounts of Total Fe as Fe(II) in Environmental Samples by Catalytic Kinetic Spectrophotometry

The method is based on the selective catalytic effect of iron (II) ions in the presence of 1,10-phenanthroline as an activator, on the oxidation of Coomassie Brillant Blue 2R by bromate. The catalytic reaction was monitored spectrophotometrically at 520 nm by fixed time approach of 3 min. The optimization of the operating conditions are investigated. Obtained optimum conditions: 1.5 mL of Coomassie Brillant Blue 2R (1.0 x 10 -4 mol L -1 ), 0.6 mL of bromate (0.01 mol L -1 ), 1.5 mL of 1,10-phenanthroline (1.0 x 10 -3 mol L -1 ), reaction temperature 25 o C and time 3 min in pH 2.0 at 520 nm. The proposed method allows quantitatively determination of iron (II) in the range of 0.05-5 mg L -1 with a selectivity and quantification limit of 0.0141 and 0.047 mg L -1 . The relative standard deviations for five replicate determinations of 0.2 and 3 mg L -1 iron (II) are 3.8% and 2.3%, respectively. The method was applied to determination of total iron in some environmental surface waters such as lake, river and well water including pharmaceutical samples used in the treatment of iron deficiency (such as ferrosolanol and maltose) after pre-reduction of iron (III) to iron (II) with sulfite at 40 o C at pH 4.0, and quantitative percentages of retinas ranging from 98.7-102.7% were obtained by standard attachment-based analysis after wet acid dissolution for possible matrix effect.

Biological treatment of model dyes and textile wastewaters

Previous works conducted in our laboratory, reveled that Bacillus aryabhattai DC100 produce ligninolytic enzymes such as laccases and/or peroxidases, opening new applications in different bioprocesses, including the treatment of disposal residues such as dyestuffs from textile processing industries. This work described the degradation of three commercial model dyes Coomassie Brilliant Blue G-250 (CBB), Indigo Carmine (IC) and Remazol Brilliant Blue R (RBBR) under different culture media and operational conditions. The process was optimized using a Central Composite Rotatable Design, and the desirability predicted complete decolorization of 150 mg/L CBB at 37 °C, 304.09 rpm and salt concentration of 19.204 g/L. The model was validated with concentrations up to 180 mg/L CBB and IC, not being able to remove high amount of RBBR. The procedure here developed also allowed Chemical Oxygen Demands (COD) reductions in CBB of about 42%, meanwhile tests on real effluents from a local textile industry involved COD reductions of 50% in a liquid wastewater and 14% in semi-liquid sludge. Thus, allow the authorized discharge of wastewater into the corresponding treatment plant. Decolorization efficiencies and COD reductions open on the potential application of B. aryabhattai DC100 on the bioremediation of real effluents from textile industries.

Zymographic approach to determine the intrinsic enzyme specific activity of diamine oxidase in presence of interfering enzymes

The purpose of this investigation was to elaborate a fast zymographic assay of oxidase enzymes in the presence of interfering enzymes as catalase (which disturbs current dosages based on H2O2 detection). This method also allows the determination of intrinsic specific activity (ISA) of oxidases, such as diamine oxidase (DAO) or glucose oxidase (GOD). The SDS-PAGE gels with entrapped peroxidase have been obtained by polymerization of acrylamide and bis-acrylamide in the presence of horse-radish peroxidase. The entrapped peroxidase was uniformly distributed in the PolyacrylAmide (PAA) material and did not migrate during electrophoresis. The obtained PAA gels allow the electrophoretic separation of various oxidases from contaminating proteins. As an example, to reveal DAO, the resulting PAA-gel should be incubated after the electrophoretic run in the developing solution containing putrescine (a DAO substrate) and o-phenylenediamine (a HRP substrate) to give coloured bands on the gel in the presence of DAO-generated H2O2. The results showed that is possible to determine the DAO in the presence of interfering catalase because they migrate differently. Thus, the H2O2 released in situ by DAO is no more decomposed by catalase because of its different mobility. It was also found that the same electrophoretic gel, after zymography, can be restained by Coomassie Blue for quantitation of proteins corresponding to the zymographic bands. With the obtained enzyme units and protein concentration it is also possible to calculate the intrinsic specific activity of DAO directly from the intensities of enzyme bands in zymography and from those of protein bands (Coomassie Blue staining), quantified by densitometry.

Accounting for adjuvant-induced artifacts in the characterization of vaccine formulations by polyacrylamide gel electrophoresis.

Several vaccine adjuvants comprise complex nano- or micro-particle formulations, such as oil-in-water emulsions. In order to characterize interactions and compatibility of oil-in-water emulsion adjuvants with protein antigens in vaccines, effective protein characterization methods that can accommodate potential interference from high concentrations of lipid-based particles are needed. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is a standard protein characterization technique which is affected by the presence of adjuvants such as oil-in-water emulsions. In this article, we investigate variations in SDS-PAGE methods that result in a reduction of adjuvant-induced staining artifacts. We have investigated whether the SDS method or the adjuvant composition were the reason for these artifacts and succeeded in reducing the artifacts with a modified sample preparation and different staining procedures. The best results were obtained by using gold staining or silver staining instead of a Coomassie Blue staining procedure. Moreover, the replacement of the dilution buffer (20% SDS to disrupt emulsion) by alternative detergents such as Tween® 80 and Triton® X-100 removed adjuvant-induced streaking artifacts at the top of the gel. These methods may be useful for improving characterization approaches of antigen-adjuvant mixtures by SDS-PAGE.

SRY‐box 9 (SOX9): A Novel Binding Partner of Cardiac Muscle‐specific A‐Kinase Anchoring Protein (mAKAP) Signalosome

BackgroundBy definition, A‐kinase anchoring proteins (AKAPs) bind to protein kinase A and various other signaling proteins at specific subcellular compartments to regulate downstream signal transduction. Heart specific muscle AKAP (mAKAP) is present on the nuclear envelope of cardiomyocytes and regulates cardiac hypertrophy by forming a signalosome of its binding partners. It was previously reported that hypoxia‐inducible factor 1 alpha (HIF‐1α) physically binds to mAKAP. Also, it was previously published that HIF‐1α physically interacts with SRY‐box 9 (SOX9). Chromatin immunoprecipitation (ChIP) studies showed that HIF‐1α binds to SOX9 promoter region. Interestingly, both mAKAP and SOX9 have been demonstrated to play significant role in cardiac differentiation and endocardial cushion formation, respectively. Hence, we were interested to know whether SOX9 is a part of mAKAP scaffold on the nuclear envelope.ObjectiveThe aim of the current work is to confirm that SOX9 unambiguously interacts with mAKAP; either directly or indirectly.MethodsAs HEK 293T cells express SOX9 endogenously and not mAKAP, we chose these cells for immunoprecipitation (IP) studies. Myc‐DDK tagged human mAKAP cDNA was purchased from Origene and overexpressed in HEK 293T cells using Lipofectamine 2000, according manufacturer's instructions. IP was performed using M2 affinity gels (Sigma) and immunoblotting was performed using mAKAP and SOX9 antibodies. HepG2 cell lysate was used as positive control for SOX9 expression. In another set of experiment, a Coomassie Blue stained gel was used for in‐gel digestion to perform MALDI‐TOF Mass Spectrometry, followed by data analysis.Summary of ResultsIP samples of mAKAP transfected HEK cells displayed SOX9 expression; whereas, untransfected cells did not show expression. mAKAP expression was used as a loading control and remained unchanged in all samples. Mass Spectrometric data validated our findings by demonstrating the expected mass peak for SOX9 in the IP samples.Conclusion and Future ImplicationsSOX9 was identified as a novel binding partner of mAKAP. As mAKAP and SOX9 already have a role in cardiac developmental biology, we suggest that mAKAP‐SOX9 interaction has substantial role in cardiac physiology and pathophysiology. The significance of this mAKAP‐SOX9 interaction is a part of our ongoing investigations.Support or Funding InformationR15HL124458 Scaffolding Protein Interaction Network Regulates Cardiac Cell Signaling

Physicochemical properties and cell-based bioactivity of Pu’erh tea polysaccharide conjugates

Polysaccharide conjugates were prepared from Pu’erh tea and fractionated by DEAE-cellulose DE-52 column chromatography to yield one unexplored polysaccharide-conjugate fraction termed TPC-P with a molecular weight of 251,200Da. DVS (dynamic vapour sorption) result discovered that the humidity condition of long-term preservation for TPC-P is below 70% RH. Although it contained proteins, TPC-P could not bind to the Coomassie Brilliant Blue dyes G250 and R250. The “shoulder-shaped” ultroviolet absorption peak in TPC-P UV–vis scanning spectum ascribe theabrownins that inevitably adsorbed the polysaccharide conjugate. Zeta potential results demonstrated TPC-P aqueous solution merely presented the negative charge properties of polysaccharides instead of acid-base property of its protein section, and had more stability in greater than pH 5.5. No precipitation or haze occurred in the three TPC-P/EGCG aqueous mixtures during their being stored for 12h. The phase separation was observed in aqueous mixtures of TPC-P and type B gelatin. TPC-P possessed the fine stability as a function of temperature heating and cooling between 0 and 55°C. It is proposed that some properties of the covalent binding protein of TPC-P were “shielded” by its polysaccharide chains.

Coomassie Brilliant Blue G-250 Dye: An Application for Forensic Fingerprint Analysis.

The Bradford reagent, comprised of the Coomassie Brilliant Blue G-250 dye, methanol, and phosphoric acid, has been traditionally used for quantifying proteins. Use of this reagent in the Bradford assay relies on the binding of the Coomassie Blue G-250 dye to proteins. However, the ability of the dye to react with a small group of amino acids (arginine, histidine, lysine, phenylalanine, tyrosine, and tryptophan) makes it a viable chemical assay for fingerprint analysis in order to identify the biological sex of the fingerprint originator. It is recognized that the identification of biological sex has been readily accomplished using two other methods; however, both of those systems are reliant upon a large group of amino acids, 23 to be precise. The Bradford assay, described here, was developed specifically to aid in the transition from targeting large groups of amino acids, as demonstrated in the previous studies, to targeting only a single amino acid without compromising the intensity of the response and/or the ability to differentiate between two attributes. In this work, we aim to differentiate between female fingerprints and male fingerprints.

The Effect of Hydrophobic Alkyl Silane Self-Assembled Monolayers on Adult Barnacle Adhesion

AbstractAlthough there exists a wide range of nonbiocidal and environmentally friendly surface coatings to reduce biofouling on marine structures, there is still not a fundamental understanding of barnacle adhesion upon reattachment. The purpose of this study is to assess the effect of hydrophobicity on adhesion in the barnacle Amphibalanus amphitrite, an abundant and widespread biofouler. Self-assembled monolayers were made on glass slides from alkyl silanes with methylated and fluorinated terminal groups to produce hydrophobic surfaces. Coated and uncoated glass slides underwent a 2-week barnacle reattachment assay. Barnacles were removed using a force gauge, and critical shear stress was calculated for each substrate. Following reattachment assays, a Coomassie Blue G250 protein stain was used to quantify the amount of glue remaining on substrates by measuring pixel density with ImageJ software on glue scans. Critical shear stress was found to be significantly higher for both hydrophobic surfaces as compared to the hydrophilic uncoated glass, and correspondingly, the density of residual glue was higher on hydrophobic surfaces. Given that hydrophobic substrates can exclude water from the surface, they may provide a protected environment for glue release that is favorable for adhesive bond formation with the substrate as well as inter- and intramolecular bonding within the glue layer. Critical shear stress showed a strong positive correlation with residual glue density, suggesting that barnacle release occurs primarily via cohesive failure. Scanning Electron Microscope (SEM) micrographs confirm morphological differences in the glue remnants, depending on the substrate coating. Among the hydrophobic substrates tested, results suggest that contact angle alone is not enough to predict the critical shear stress of barnacles. The chemical and physical properties of the coating become important parameters to consider in antifouling coating design.

Effects of nicotine on zebrafish: A comparative response between a newly established gill cell line and whole gills

A novel cell line, Danio rerio gill (DrG), derived from the gill tissue of zebrafish, was established and characterized. The cells were able to grow at a wide range of temperatures from 25°C to 32°C in Leibovitz's L-15 medium. The DrG cell line consists of epithelial-like cells with a diameter of 18–22μm. The cell line was characterized by mitochondrial 12S rRNA gene. Acute toxicity tests were conducted on D. rerio by exposing them to nicotine for 96h under static conditions. In vitro cytotoxicity of nicotine was assessed in DrG cell line using multiple endpoints such as 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), Neutral Red assay, Alamar Blue assay and Coomassie Blue protein assay. Linear correlations between each in vitro cytotoxicity assay and the in vivo mortality data were highly significant. Nicotine induced intracellular reactive oxygen species generation in DrG cell line in a concentration dependent manner. DrG cell line and zebrafish exposed to nicotine significantly increased the elevation of lipid peroxidation (LPO) while depletion of reduced glutathione (GSH), manganese superoxide dismutase (MnSOD), catalase (CAT), glutathione S-transferase (GST) and glutathione peroxidise(GPx1a) was observed. In nicotine treated fish and cells a negative correlation between reduced glutathione and LPO was observed. In addition, the production of ROS and the resulting oxidative stress resulted in increased expression of apoptosis related genes p53 and cas3.Collectively, our result suggests that nicotine has the potential to induce reactive oxygen species (ROS) production, oxidative stress and apoptosis in DrG cell line and zebrafish.

In-situ protein determination to monitor contamination in a centrifugal partition chromatograph

Centrifugal partition chromatography (CPC) works with biphasic liquid systems including aqueous two-phase systems. Metallic rotors are able to retain an aqueous stationary phase able to purify proteins. But the adhesion of proteins to solid surface may pose a cross-contamination risk during downstream processes. So it is of utmost importance to ensure the cleanliness of the equipment and detect possible protein contamination in a timely manner. Thereby, a direct method that allows the determination of the effective presence of proteins and the extent of contamination in the metallic CPC rotors was developed. This in-situ method is derived from the Amino Density Estimation by Colorimetric Assay (ADECA) which is based on the affinity of a dye, Coomassie Brillant Blue (CBB), with protonated N+ groups of the proteins. In this paper, the ADECA method was developed dynamically, on a 25 mL stainless-steel rotor with various extents of protein contaminations using bovine serum albumin (BSA) as a fouling model. The eluted CBB dye was quantified and found to respond linearly to BSA contamination up to 70 mg injected. Limits of detection and quantification were recorded as 0.9 mg and 3.1 mg, respectively. While the non-specific interactions between the dye and the rotor cannot currently be neglected, this method allows for in situ determination of proteins contamination and should contribute to the development of CPC as a separation tool in protein purification processes.

Analysis of protein profile of tomato root infected with Fusarium oxysporum f. sp lycopersici.

Fusarium wilt caused by Fusarium oxysporum f. sp lycopersici (Fol) is one of the main diseases affecting tomatoes. The BHRS 2,3 genotype of tomato is, however, resistant to this disease. A proteomic approach was used to understand the defense mechanisms of this genotype using the tomato root, the first tissue that interacts with the fungus, as a target. Protein was extracted and separated by two-dimensional electrophoresis followed by staining with Coomassie brilliant blue. The proteins were identified by MALDI-TOF/TOF mass spectrometry. A total of 22 proteins were identified, 21 of which showed differential expression with 12 proteins being upregulated and nine being downregulated. Plants responded to the pathogen with increased expression of pathogenesis-related proteins. We noted the induction of proteins involved in hypersensitivity reaction and other defense mechanisms. The expression of proteins of primary metabolism related to energy production, however, decreased, as did the expression of two proteins related to defense against abiotic stress. These results demonstrate the presence of important mechanisms for defense against Fol in the tomato genotype BHRS 2,3.

萌芽菊芋块茎对盐碱土壤胁迫的生理响应

PDF HTML阅读 XML下载 导出引用 引用提醒 萌芽菊芋块茎对盐碱土壤胁迫的生理响应 DOI: 10.5846/stxb201610262181 作者: 作者单位: 东北林业大学,东北林业大学盐碱地生物资源环境研究中心,东北林业大学,东北林业大学盐碱地生物资源环境研究中心 作者简介: 通讯作者: 中图分类号: 基金项目: 国家自然科学基金项目（31470467） Physiological response of sprouting Jerusalem artichoke tubers to saline-alkali stress Author: Affiliation: Northeast Forestry University,,Northeast Forestry University,Alkali Soil Natural Environmental Science Center,Northeast Forestry University Fund Project: 摘要 | 图/表 | 访问统计 | 参考文献 | 相似文献 | 引证文献 | 资源附件 | 文章评论 摘要:土壤盐碱化是影响全球农业生产和生态环境的重要问题。在农田、轻度盐碱草地和重度盐碱草地设置样地以块茎种植菊芋，次年5月块茎萌发阶段取块茎样品测定丙二醛、游离脯氨酸、可溶性糖含量以及抗氧化酶活性并进行蛋白质组学分析，分析了萌芽菊芋块茎对盐碱土壤胁迫的生理响应。0-20 cm土层的电导率（表征土壤可溶盐含量）表明从农田到轻度、重度盐碱草地土壤盐碱胁迫逐渐增强，丙二醛含量变化反映出菊芋块茎受害程度逐渐增加，并且基于游离脯氨酸的渗透调节能力也在逐渐增强。蛋白质组学分析结果显示与遗传信息加工相关的差异蛋白数量最多（占28.75%）且多为表达上调，意味着DNA复制和转录、蛋白质合成和折叠的相关蛋白在响应盐碱胁迫中发挥关键作用。碳水化合物及多糖代谢（占15%）、氨基酸代谢（占11.25%）以及能量代谢（占7.5%）相关的差异蛋白数量也较多，说明调节物质代谢平衡在萌芽菊芋块茎应对盐碱土壤胁迫过程中有重要作用。这些结果为揭示萌芽菊芋块茎适应盐胁迫的生理机制奠定了基础。 Abstract:Soil salinization is one of the most common abiotic stresses affecting plant growth and is becoming an important issue, owing to its impact on agricultural production and the environment. Saline-alkali soil is becoming particularly widespread and may cover more than 50% of all arable lands by the year 2050. The combination of soil salinization and high pH conditions represents a major impediment for plant growth and crop productivity. The study area was a typical alkalinized grassland in northeastern China. Jerusalem artichoke (Helianthus tuberosus) is an annual flowering plant that has been cultivated as a vegetable, fodder crop, and bioenergy material in many countries, owing to its high levels of polysaccharides, especially inulin. The ability to survive in the saline-alkali soils of semiarid areas is one of the most important characters of Jerusalem artichoke. Jerusalem artichoke tubers were sown in farmland, light saline-alkali, or severe saline-alkali soil and sprouting tubers were collected from the sample in May of the following year. The malondialdehyde, free proline, and soluble sugar contents, antioxidant enzyme activity, and protein profile were quantified, in order to assess the physiological response of Jerusalem artichoke to saline-alkali stress. The soil electrical conductivity (0-20 cm) indicated that the soluble salt contents of the three soil types were significantly different, with the lowest soluble salt content in the farmland soil and the greatest content in the severe saline-alkali soil. With increasing soluble salt content, the malondialdehyde content increased, obviously indicating higher levels of stress, and increased free proline content indicated that Jerusalem artichoke could enhance its resistance to salt stress via osmotic adjustment. To investigate the proteomic response of the sprouting tubers to saline-alkali stress, two-dimensional gel electrophoresis (2-DE) gels from three biological experiments were analyzed. Then, using ImageMaster 2D Platinum Software, we identified more than 1000 highly reproducible protein spots on the coomassie brilliant blue (CBB)-stained 2D gels. A total of 80 differentially expressed proteins were successfully identified using MALDI-TOF/TOF analysis, according to the peptide matching results provided by MASCOT. Among these proteins, 42 were detected in the sprouting H. tuberosus tubers from the light saline-alkali soil and 38 of the proteins accumulated differentially in the tubers grown in the severe saline-alkali soil. KEGG pathway analysis attributed these proteins to eleven different metabolic pathways, which included carbohydrate and polysaccharide metabolism (15%), energy metabolism (7.5%), genetic information processing (28.75%), amino acid metabolism (11.25%), nucleotide metabolism (2.5%), biosynthesis of secondary metabolites (3.75%), signal transduction (17.5%), transport and catabolism (2.5%), cell motility (2.5%), and unknown (8.75%). The differentially expressed proteins were mainly involved in genetic information processing, which might indicate that the regulation of proteins involved in DNA replication, transcription, protein synthesis, and protein folding are responsive to saline-alkali stress and play a pivotal role in salinity tolerance. Carbohydrate, energy, and amino acid metabolism-related proteins constituted one-third of the differentially expressed proteins, which suggests that metabolism homeostasis is important for the survival of seedlings exposed to saline-alkali stress. These findings provide new insight into the underlying molecular mechanisms of saline-alkali resistance in Jerusalem artichoke. 参考文献 相似文献 引证文献

Pericardial Parietal Mesothelial Cells: Source of the Angiotensin-Converting-Enzyme of the Bovine Pericardial Fluid.

BackgroundAngiotensin II (Ang II), the primary effector hormone of the renin-angiotensin system (RAS), acts systemically or locally, being produced by the action of angiotensin-converting-enzyme (ACE) on angiotensin I. Although several tissue RASs, such as cardiac RAS, have been described, little is known about the presence of an RAS in the pericardial fluid and its possible sources. Locally produced Ang II has paracrine and autocrine effects, inducing left ventricular hypertrophy, fibrosis, heart failure and cardiac dysfunction. Because of the difficulties inherent in human pericardial fluid collection, appropriate experimental models are useful to obtain data regarding the characteristics of the pericardial fluid and surrounding tissues.ObjectivesTo evidence the presence of constituents of the Ang II production paths in bovine pericardial fluid and parietal pericardium.MethodsAlbumin-free crude extracts of bovine pericardial fluid, immunoprecipitated with anti-ACE antibody, were submitted to electrophoresis (SDS-PAGE) and gels stained with coomassie blue. Duplicates of gels were probed with anti-ACE antibody. In the pericardial membranes, ACE was detected by use of immunofluorescence.ResultsImmunodetection on nitrocellulose membranes showed a 146-KDa ACE isoform in the bovine pericardial fluid. On the pericardial membrane sections, ACE was immunolocalized in the mesothelial layer.ConclusionsThe ACE isoform in the bovine pericardial fluid and parietal pericardium should account at least partially for the production of Ang II in the pericardial space, and should be considered when assessing the cardiac RAS.

Addressing the distribution of proteins spotted on μPADs.

Adsorption is the most common approach to immobilize biorecognition elements on the surface of paper-based devices. Adsorption is also the route selected to coat the substrate with albumin, therefore minimizing the interaction of other proteins. While similar in nature, the structure of the selected proteins as well as the conditions selected from the immobilization have a significant effect on the amount and distribution of the resulting composites. To illustrate these differences and provide general guidelines to efficiently prepare these devices, this article explores the interaction (adsorption and desorption) of BSA with 3MM chromatography paper. The experimental conditions investigated were the protein concentration, the interaction time, the number of times the protein was spotted, the pH of buffer solution, and the ionic strength of the buffer solution. The proposed approach mimics the steps involved in the fabrication (adsorption) and use (rinsing induced by the sample) of paper-based microfluidic devices. To identify the protein location following the rinsing step, the protein was fixed by dehydration in a convection oven and then stained using Coomassie Blue. The color intensity, which was found to be proportional to the amount of protein immobilized, was determined using a desktop scanner. To highlight the importance of understanding the adsorption process to the rational development of μPADs, results were complemented by experiments performed with lysozyme and immunoglobulin G.

A Transdermal Measurement Platform Based on Microfluidics

The Franz diffusion cell is one of the most widely used devices to evaluate transdermal drug delivery. However, this static and nonflowing system has some limitations, such as a relatively large solution volume and skin area and the development of gas bubbles during sampling. To overcome these disadvantages, this study provides a proof of concept for miniaturizing models of transdermal delivery by using a microfluidic chip combined with a diffusion cell. The proposed diffusion microchip system requires only 80 μL of sample solution and provides flow circulation. Two model compounds, Coomassie Brilliant Blue G-250 and potassium ferricyanide, were successfully tested for transdermal delivery experiments. The diffusion rate is high for a high sample concentration or a large membrane pore size. The developed diffusion microchip system, which is feasible, can be applied for transdermal measurement in the future.

Therapeutic Effect of Dendrobium candidum on Lupus Nephritis in Mice.

Context:Dendrobium candidum (D. candimum) widely is a functional drug. The curative effect of D. candidum on lupus nephritis has been studied in vivo.Materials and Method:The DBA/2 and B6D2F1 mice were used for this in vivo experiment. The 50% effective dose (ED50) was used to check the effective concentration for this study. Then the SCr, BUN, TC, TG, IL-6, IL-12, TNF-α, and IFN-γ levels were determined by kits. The output of urine protein was determined by means of Coomassie Brilliant Blue, and the auto-antibody dsDNA was determined with titer plate technology and indirect immunofluorescence. The NF-κB, IκB-α, TGF ‘β1, Fas, and FasL expressions were measured by RT-PCR and western blot assay. The component analysis of D. candidum was determined by nuclear magnetic resonance.Results:Based on the ED50 result at 329 mg/kg, 200 and 400 mg/kg doses were chosen for this study. SCr, BUN, TC and TG levels of 400 mg/kg D. candidum mice were lower than control mice, TP and ALB levels were higher than control mice. The control and 400 mg/kg treated mice tested positive for dsDNA at the end of sixth and tenth week after the experiment began. The glomerular number of 400 mg/kg treated mice was more than control group. Treatment with 400 mg/kg D. candidum reduced IL-6, IL-12, TNF-α and IFN-γcytokine levels as compared to control mice. D. candidum decreased NF-κb, TGF ‘β1, Fas, FasL and increased IκB-α expressions in kidney tissue. There were 11 compounds in dry D. candidum, these compounds might make the curative effects of lupus nephritis.Conclusion:D. candidum showed a potential curative effect on lupus nephritis. It could be used as a health medicine on lupus nephritis.SUMMARY D. candidum reduced the SCr, BUN, TC, TG serum levels and raised the TP, ALB levels compared to control group.The glomerular number of D. candidum treated mice was more than control group.D. candidum treated mice showed lower IL-6, IL-12, TNF-α and IFN-γ cytokine levels than control mice.D. candidum decreased NF-κb, TGF-β1, Fas, FasL and increased IκB-α expressions in kidney tissue. Abbreviations used: LN: Lupus nephritis, SLE: systemic lupus erythematosus, D. candidum: Dendrobium candidum; IL-6: interleukin-6, IL-12: interleukin-12, TNF-α: tumor necrosis factor alpha, IFN-γ: Interferon-gamma, SCr: serum creatinine, BUN: blood urea nitrogen, TC: total cholesterol, TG: triglyceride, TP: total protein, ALB: albumin.

Blue Native PAGE: Applications to Study Peroxisome Biogenesis.

Blue native polyacrylamide gel electrophoresis (BN-PAGE) is one of the useful methods to isolate protein complexes including membrane proteins under native conditions. In BN-PAGE, Coomassie Brilliant Blue G-250 binds to proteins and provides a negative charge for the electrophoretic separation without denaturing at neutral pH, allowing the analysis of molecular mass, oligomeric state, and composition of native protein complexes. BN-PAGE is widely applied to the characterization of soluble protein complexes as well as isolation of membrane protein complexes from biological membranes such as the complexes I-V of the mitochondrial respiratory chain and subcomplexes of the mitochondrial protein import machinery. BN-PAGE has also been introduced in the field of peroxisome research, for example, analysis of translocation machinery for peroxisomal matrix proteins embedded in the peroxisomal membrane. Here, we describe a basic protocol of BN-PAGE and its application to the study of peroxisome biogenesis.