Cell-dependent requirement of human immunodeficiency virus type 1 Vif protein for maturation of virus particles.

A highly sensitive single-round infection assay using a bacterial chloramphenicol acetyltransferase was developed to analyze an early stage of human immunodeficiency virus type 1 replication. By a combination of transfection and single-round infection assay, a virus with a vif mutation, depending on host cells from which the virus was derived, was demonstrated to be defective at the early phase of infection cycle. Analysis of viral proteins synthesized in cells indicated that incorporation of the Env surface protein into virions of the vif mutant, again in a cell-dependent way, was greatly restricted. Taken together, it is concluded that the Vif protein acts through modulation of the Env protein in the virions, directly or indirectly, to enhance viral infectivity in a certain cell type.     

Increased uniformity in the response of the coomassie blue G protein assay to different proteins

Coomassie blue G dye-based protein assays are exceptionally convenient because of their simplicity, sensitivity, speed, and resistance to interfering chemicals, notably reducing agents and most buffers. A major problem with the assay is the variation in response to different proteins. The addition of NaOH to the protein assay reagent reduced the variation in the response of this assay to different proteins. In addition, the sensitivity of the assay is increased. The NaOH can be added either in a separate step to solubilize cells or membranes or directly to the reagent. Linear standard curves were obtained when the log of the absorbance was plotted against the log of the protein quantity.     

Glutathione S-transferase fusion proteins as an affinity reagent for rapid isolation of specific sequence directly from genomic DNA.

We describe a DNA binding assay for isolation of specific sequence(s) recognized by protein of interest directly from genomic or cosmid DNA. In our assay, the protein is fused to the glutathione-S-transferase and bound to glutathione-Sepharose beads. Then the immobilized fusion protein can be used to search for DNA fragment(s) that interact specifically with the protein of interest. As an example of such an approach, we identified and cloned a few prokaryotic oriC regions using the initiator DnaA protein fused to the glutathione-S-transferase.     

Ionic inhibition of catalytic phosphorylation of histone by bovine brain protein kinase.

The effects of various ions commonly found in protein kinase assays upon the rate of histone phosphorylation catalyzed by the highly purified bovine brain enzyme, protein kinase I, have been investigated. Sodium, potassium, and magnesium were found to inhibit histone phosphorylation by protein kinase I in a similar manner. The degree of inhibition by any of these cations was demonstrated to be directly proportional to the square root of the ionic strength of the assay medium. The relationship between the ionic strength of the assay medium and the rate of histone phosphorylation catalyzed by protein kinase I was employed to correct the rate of histone phosphorylation at various magnesium acetate concentrations to a standard ionic strength. When this was done an analysis of the previously postulated rate law for histone phosphorylation c atalyzed by protein kinase I gave a binding constant for the magnesium-ATP complex which was in agreement with that expected for this complex on the basis of various binding constants available in the literature. These results demonstrate that it is unnecessary to postulate a specific ion inhibition process for protein kinase I by the ions employed in this study. They also support the reasonable assumption that magnesium ion binds to ATP at or prior to the rate-determining step in histone phosphorylation catalyzed by protein kinase I. The expression developed in this paper for the effect of ionic strength upon protein kinase I activity can now be used to correct activity measurements made under various assay conditions to a standard assay state, allowing facile comparisons of kinetic data. It should be possible to develop similar expressions for other protein kinases and substrates to permit useful interpretation of kinetic data.     

miR-9 在卵巢癌细胞上皮间质转化中的初步研究

目的:研究miR-9在卵巢癌细胞上皮间质转化(EMT)中的作用。方法:上调或者下调miR-9后,在RNA水平上通过RT-qPCR检测卵巢癌细胞系SKOV3和A2780中上皮指标E-cadherin表达变化;在蛋白水平,通过western blotting方法检测2株细胞系中上皮指标E-cadherin和间质指标vimentin蛋白表达变化。生物信息学预测可能靶向E-cadherin 3'UTR的miR NA,双荧光素酶报告系统进一步验证miR-9靶向结合E-cadherin的3'UTR区。结果:上调miR-9后,卵巢癌细胞系中E-cadherin表达受到明显抑制,vimentin表达明显增加;反之,下调miR-9后,E-cadherin表达明显增高,vimentin表达明显降低。通过生物信息学预测发现miR-9可以直接靶向E-cadherin的3'UTR区,荧光素酶报告系统验证预测结果正确。结论:miR-9促进卵巢癌细胞上皮间质转化。     

An 18K protein from ascites hepatoma cell depolymerizes actin filaments rapidly

Several actin binding proteins were isolated from ascites hepatoma cells AH7974 by DNase I affinity chromatography. Among them, a protein having a molecular weight of 18,000 was further purified by DEAE cellulose and hydroxyapatite column chromatographies and gel filtration on a Sephadex G-75 column. The 18K protein not only inhibits actin polymerization but also depolymerizes actin filaments. This conclusion was supported by viscosity and fluorescence intensity measurements and the DNase I inhibition assay. A chemical cross-linking experiment suggested that the 18K protein binds to monomeric actin and forms and 18K-actin 1:1 complex. The net depolymerization rate by the 18K protein measured by the DNase I inhibition assay was slower than the rapid reduction of the fluorescence intensity of pyrene-labeled F-actin upon addition of the 18K protein. This result suggests that the 18K protein not only binds to monomeric actin but also binds to actin filaments directly. The sedimentation assay showed that a part of the 18K protein was cosedimented with actin filaments. Electron microscopic observations demonstrated that the 18K protein decreased the amount of actin filaments and the remaining filaments appeared to be decorated and distorted by the 18K protein. The 18K protein had no Ca2+ ion sensitivity and exhibited the same effect on both this tumor actin and muscle actin.     

Affinity-tagged phosphorylation assay by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (ATPA-MALDI): application to calcium/calmodulin-dependent protein kinase

A matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)-based kinase assay using a peptide substrate tagged with a biotinyl group has been developed. The peptide moiety was designed to serve as an efficient substrate for calcium/calmodulin-dependent protein kinase II, based on the in vivo phosphorylation site of phosrestin I, a Drosophila homolog of arrestin. In the assay, the quantitative relationship was determined from the ratio of the peak areas between the two peaks respectively representing the unphosphorylated and the phosphorylated substrate. Attempts to assay phosphorylated peptides directly from the reaction mixture, gave inaccurate results because of the high noise level caused by the presence of salts and detergents. In contrast, after purifying the substrate peptides with the biotin affinity tag using streptavidin-coated magnetic beads, peak areas accurately represented the ratio between the unphosphorylated and phosphorylated peptide. By changing the substrate peptide to a peptide sequence that serves as a kinase substrate, it is expected that an efficient non-radioactive protein kinase assay using MALDI-TOF MS can be developed for any type of protein kinase. We call this technique "Affinity-Tagged Phosphorylation Assay by MALDI-TOF MS (ATPA-MALDI)." ATPA-MALDI should serve as a quick and efficient non-radioactive protein kinase assay by MALDI-TOF MS.     

A spectrophotometric assay of Zn(2+)-glycerophosphorylcholine phosphocholine phosphodiesterase using p-nitrophenylphosphorylcholine.

A direct spectrophotometric assay for the glycerophosphorylcholine phosphocholine phosphodiesterase requiring zinc ions for activity is described. This assay is based on the continuous measurement of p-nitrophenol produced from the enzymatic hydrolysis of p-nitrophenylphosphorylcholine. The assay method, which showed a good linearity with time and amount of protein, was found to be rapid, simple, and, at the same time, accurate and sensitive enough to allow the quantitation of nanomolar amounts of product. With an alkaline buffer containing Triton X-100, the Zn(2+)-glycerophosphorylcholine phosphocholine phosphodiesterase activity in the tissue homogenate can be directly and selectively measured by this technique. The specific activity of the phosphodiesterase in brain and kidney was determined to be 80 and 6.5 nmol/h mg protein, respectively, and much lower activity was present in other tissues.     

MicroRNA-320 Enhances Radiosensitivity of Glioma Through Down-Regulation of Sirtuin Type 1 by Directly Targeting Forkhead Box Protein M1

Glioma is the most common cancer in human brain system and seriously threatens human health. miRNA-320 has been demonstrated to be closely correlated with the development of glioma. However, its effect and molecular mechanism underlying radioresistance have not been fully elucidated in glioma. Here, RT-qPCR assay was used to assess the expressions of miR-320 and forkhead box protein M1 (FoxM1) mRNA in glioma tumor tissues and cells. The effects of miR-320, FoxM1 and sirtuin type 1 (Sirt1) on radiosensitivity in glioma cells were evaluated by clone formation assay, apoptosis assay, histone H2AX phosphorylation level (γH2AX) detection and caspase 3 activity analysis, respectively. The direct interaction between miR-320 and FoxM1 was detected by luciferase assay. The protein levels of FoxM1, Sirt1 and γH2AX were measured by western blot assay. We found that miR-320 expression was down-regulated and FoxM1 expression was up-regulated in radioresistant glioma tissues and IR-treated glioma cells. miR-320 overexpression dramatically enhanced radiosensitivity, promoted apoptosis, and improved γH2AX expression and caspase 3 activity in glioma cells. Luciferase reporter assay and western blot assay further validated that miR-320 suppressed FoxM1 expression by directly targeting 3’ UTR region of FoxM1. Moreover, miR-320 inhibited Sirt1 expression via targeting FoxM1 in glioma cells. Furthermore, overexpression of FoxM1 and Sirt1 strikingly attenuated miR-320-induced increase of radiosensitivity, apoptosis and γH2AX expression in glioma cells. In conclusion, miR-320 enhanced radiosensitivity of glioma cells through down-regulation of Sirt1 by directly targeting FoxM1.     

Using aptamers as capture reagents in bead-based assay systems for diagnostics and hit identification

Most applications of xMAP (Luminex) bead-based assay technology in diagnostics and drug discovery use immobilized antigens or antibodies. Here the authors describe the development of novel assay systems in which synthetic oligonucleotides that specifically bind and inhibit other biomolecules--so-called aptamers--are directly immobilized on beads. The robustness, specificity, and sensitivity of aptamer-based assays were demonstrated in a test system that detected human alpha-thrombin in serum samples. xMAP technology was also adapted to competitive screening formats where an aptamer/protein complex was disrupted by a functionally analogous competitor. The results indicate that such assays are excellently suited for diagnostic applications or drug screening, where aptamers serve as competitive binding probes for the identification of small-molecule hits. These methods should be transferable to a large number of applications because specific aptamers can be rapidly generated for almost any protein target.     

Microassay for proteins on nitrocellulose filter using protein dye-staining procedure

A simple and rapid microassay for proteins utilizing the protein dye-staining procedure with a nitrocellulose (NC) filter is described. Proteins were directly bound to an NC filter using the "BIO DOT" microfiltration apparatus to ensure their uniformity. The proteins were then stained with a dye (Ponceau Red 3R or amido black 10B), and the optical density of the stained protein spots was directly measured by a densitometer. A good linearity between the optical density and the amount of protein was obtained in the range 0.05 to 10 micrograms. A larger number of samples (up to 96 samples) could be assayed within 1.5 h simultaneously. Contaminating chemicals, such as amino acids, sugars, reducing agents, chelating agents, tris(hydroxymethyl)aminomethane, deoxyribonucleic acid, and nucleotides, did not interfere with the assay. The reproducibility, pH dependency, and application of the assay to the quantitation of a small amount of proteins in body fluids are discussed.     

Evaluating the compatibility of three colorimetric protein assays for two-dimensional electrophoresis experiments

To evaluate compatibility of commonly used colorimetric protein assays for 2-DE experiments, we investigated the interfering mechanisms of major 2-DE component(s) in the Lowry-based assay, the Bradford assay and the bicinchoninic acid (BCA) assay. It was found that some 2-DE components did not directly interfere with the assays' color development reaction, but possibly influenced the quantitation results by interacting with proteins. Generally, simultaneous presence of 2-DE components in the samples demonstrated a cooperative rather than additive interference. Interference by reductants in the Lowry-based assay and the BCA assay were too prominent and could not be completely eliminated by either the reported alkylation procedure or the water dilution procedure. The Bradford assay however, presented a more suitable method for quantitating 2-DE samples because it was less interfered by most 2-DE components. Furthermore, despite slightly compromising protein solubility, utilization of reductant free 2-DE sample buffers conferred application of the Lowry-based and BCA assays in the 2-DE experiments.     

Enzyme assay for protein kinase using micellar electrokinetic chromatography with laser-induced fluorescence detection

Micellar electrokinetic chromatography (MEKC) with laser-induced fluorescence (LIF) detection has been developed for a protein kinase assay. This protein kinase assay could readily determine the phosphorylation activity of substrate peptide kemptide using cAMP-dependent protein kinase (PKA) as a model enzyme. Kemptide and phosphorylated kemptide could be reacted with 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-F) as a fluorescence derivatization reagent for LIF detection by directly adding NBD-F into the PKA enzymatic reaction mixture. These derivatives of substrate and product were separated and detected within the analysis time of 5 min by micellar electrokinetic mode using a mixture of sodium dodecylsulfate and methanol as a running buffer. Good linearity of the peak response of the phosphorylated kemptide was obtained over the range of 1-20 mU/tube of PKA in the assay. The relative standard deviation of the peak areas of the phosphorylated kemptide using 2, 5 and 10 mU/tube of PKA were calculated to <10.4%, indicating that the assay was reproducible. Also, IC(50) values of six PKA inhibitors, the K(i) value and the inhibition pattern of one inhibitor, which were calculated to estimate by the variation of the peak area of the phosphorylated kemptide using 5 mU/tube of PKA, were consistent with the published data. The sensitivity of the assay was higher than that of enzyme-linked immunosorbent assay (ELISA) for PKA phosphorylation activity, as IC(50) values, K(i) value, and the inhibition mechanism of inhibitors could be estimated using one-tenth amounts of PKA, compared with that of ELISA. The MEKC-LIF is expected to be very useful for protein kinase assay and its application to the estimation of inhibitors because this method does not entail experimentally troublesome procedures such as the preparation of antibody or fluorescence-labeled substrate.     

马动脉炎病毒核蛋白基因的克隆与表达

马病毒性动脉炎是危害世界养马业的重要传染病之一,是由动脉炎病毒科动脉炎病毒属的马动脉炎病毒(Equinearteritisvirus,EAV)引起的一种以病马发热,步态僵直,躯干及眼周围水肿,并出现粘液脓性鼻炎、结膜炎,外生殖道水肿为特征的传染病,对妊马能引起流产,使易感怀孕母马的流产率     

鼠疫菌cAMP受体蛋白对毒力相关基因yopD调控的初步研究

目的研究鼠疫菌cAMP受体蛋白（CRP）对毒力相关基因yopD的调控情况。方法利用芯片表达谱和实时定量RT-PCR初步判断CRP对yopD的调控,在获得纯化的his-CRP蛋白后进行体外凝胶阻滞实验（EMSA）证明CRP能否直接结合yopD启动子区,DNase I足迹实验（DNase I footprinting assay）确定yopD启动子区CRP结合位点序列精确序列。结果芯片表达谱和实时定量RT-PCR实验一致证实CRP可能直接负调控yopD,EMSA证明CRP能直接结合yopD启动子区,DNase I足迹实验确定yopD的启动子区上CRP位点的精确序列。结论CRP可能直接负调控毒力相关基因仰D。     

Antibody quantitation in seconds using affinity Perfusion Chromatography

An extremely rapid assay technique for antibodies has been developed utilizing protein A or protein G bound to Perfusion Chromatography support matrices. Either dilute or concentrated samples are directly injected on a column that selectively binds antibody, which is quantitated directly by elution and UV absorbance. Due to the unique mass transport characteristics of the supports, total assay cycle times are typically 1 minute or less, with assays as short as 15 seconds possible. The assay system can accurately quantitate a 100,000:1 or greater dynamic range in sample concentration without sample dilution, is extremely repeatable and is easy to automate with conventional HPLC systems. Assay of antibodies in a wide range of sample types has been demonstrated.     

Interaction of HRC (histidine-rich Ca(2+)-binding protein) and triadin in the lumen of sarcoplasmic reticulum

HRC (histidine-rich Ca(2+) binding protein) has been identified from skeletal and cardiac muscle and shown to bind Ca(2+) with high capacity and low affinity. While HRC resides in the lumen of the sarcoplasmic reticulum, the physiological function of HRC is largely unknown. In the present study, we have performed co-immunoprecipitation experiments and show that HRC binds directly to triadin, which is an integral membrane protein of the sarcoplasmic reticulum. Using a fusion protein binding assay, we further identified the histidine-rich acidic repeats of HRC as responsible for the binding of HRC to triadin. These motifs may represent a novel protein-protein interaction domain. The HRC binding domain of triadin was also localized by fusion protein binding assay to the lumenal region containing the KEKE motif that was previously shown to be involved in the binding of triadin to calsequestrin. Notably, the interaction of HRC and triadin is Ca(2+)-sensitive. Our data suggest that HRC may play a role in the regulation of Ca(2+) release from the sarcoplasmic reticulum by interaction with triadin.     

Imaging Beads-Retained Prey Assay for Rapid and Quantitative Protein-Protein Interaction

Conventional Western blot based pull-down methods involve lengthy and laborious work and the results are generally not quantitative. Here, we report the imaging beads-retained prey (IBRP) assay that is rapid and quantitative in studying protein-protein interactions. In this assay, the bait is immobilized onto beads and the prey is fused with a fluorescence protein. The assay takes advantage of the fluorescence of prey and directly quantifies the amount of prey binding to the immobilized bait under a microscope. We validated the assay using previously well studied interactions and found that the amount of prey retained on beads could have a relative linear relationship to both the inputs of bait and prey. IBRP assay provides a universal, fast, quantitative and economical method to study protein interactions and it could be developed to a medium- or high-throughput compatible method. With the availability of fluorescence tagged whole genome ORFs in several organisms, we predict IBRP assay should have wide applications.     

ELISA measurement of LDL receptors

An enzyme-linked immunosorbent assay was developed for measurement of low density lipoprotein (LDL) receptors. A monospecific polyclonal antibody to LDL receptor purified from rat liver that reacted with rat, mouse, canine, and human LDL receptor was used. With this assay, LDL receptors could be measured on 2-4 x 10(5) adherent cells and 1.0 x 10(5) cells in suspension, although results were more variable with cell suspensions. Membranes from a variety of receptor-rich and receptor-poor tissues could be assayed directly after adherence of the membranes to the ELISA plate by an overnight incubation. In some instances, the quality of the assay was improved by first solubilizing the membranes. The sensitivity of the assay is such that between 0.15 and 2 micrograms of membrane protein is required. This could be obtained from leukocytes in a modest (20-30 ml) quantity of human blood. The assay was used to demonstrate the rapid down-regulation of LDL receptors in human mononuclear leukocytes in response to a cholesterol-containing meal. Overall, the results support the use of ELISA technology to measure LDL receptors, particularly for physiologic studies.     

A silver-binding assay for measuring nanogram amounts of protein in solution

We recently reported a highly sensitive assay for measuring protein in solution based on the capacity of glutaraldehyde-treated protein to bind silver. This assay has now been made more sensitive, with a lower limit of detection of 5 ng, and more reproducible by supplementing protein samples with sodium dodecyl sulfate (SDS) to reduce protein loss to glassware. Two procedures have been developed. In one, protein samples are supplemented with both SDS and Tween 20 to yield very steep protein dose-response curves, which allow for more precise protein determinations, and very stable color formation, permitting OD measurements to be made several hours after the assay has been completed. In the second procedure, protein samples are supplemented with SDS alone which results in a less steep dose-response curve and less stable color formation but makes the assay substantially more tolerant of interfering substances. Thus, proteins in most commonly used buffers can be assayed directly with the second procedure without the need for buffer exchange. The procedure of choice, therefore, depends on the type and concentration of interfering substance. Proteins in buffers totally incompatible with either assay procedure (e.g., those containing reducing agents) can be easily buffer exchanged by centrifugation through 0.2% SDS equilibrated, drained Bio-Gel P-2 beads. The clinical utility of this improved assay is demonstrated by the accurate quantitation of protein in 0.5 μl of samples of human cerebral spinal fluid. This assay should therefore prove especially useful when a limited amount of protein is available for quantitation.