A sensitive and rapid method for identification and characterization of low abundance receptors

An improved method for detection of low intensity radioligand-receptor complexes resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is described. [3H]Azidopine-labeled 1,4-dihydropyridine (DHP) receptor from skeletal muscle resolved by SDS-PAGE was transferred to nitrocellulose and cut into strips and individual slices were analyzed for radioincorporation by liquid scintillation counting. [3H]Azidopine-labeled DHP binding subunit migrated as a single entity with a mass of 170 kDa and was confirmed using conventional methods. Results were obtained within 4 h after resolution by SDS-PAGE compared to 3-40 days using conventional methods. In addition, detection of extremely low signals (less than 50 cpm/lane), otherwise overwhelmed by background noise using conventional methods, was possible due to removal of free ligand during electro-transfer to nitrocellulose. This technique offers a rapid sensitive, cost effective alternative to fluorography or other conventional gel slice analysis methods for detecting low intensity radiolabeled complexes resolved by SDS-PAGE.     

Anti-HIV Activity in Cervical-Vaginal Secretions from HIV-Positive and -Negative Women Correlate with Innate Antimicrobial Levels and IgG Antibodies

Background

We investigated the impact of antimicrobials in cervicovaginal lavage (CVL) from HIV(+) and HIV(−) women on target cell infection with HIV. Since female reproductive tract (FRT) secretions contain a spectrum of antimicrobials, we hypothesized that CVL from healthy HIV(+) and (−) women inhibit HIV infection.

Methodology/Principal Findings

CVL from 32 HIV(+) healthy women with high CD4 counts and 15 healthy HIV(−) women were collected by gently washing the cervicovaginal area with 10 ml of sterile normal saline. Following centrifugation, anti-HIV activity in CVL was determined by incubating CVL with HIV prior to addition to TZM-bl cells. Antimicrobials and anti-gp160 HIV IgG antibodies were measured by ELISA. When CXCR4 and CCR5 tropic HIV-1 were incubated with CVL from HIV(+) women prior to addition to TZM-bl cells, anti-HIV activity in CVL ranged from none to 100% inhibition depending on the viral strains used. CVL from HIV(−) controls showed comparable anti-HIV activity. Analysis of CH077.c (clone of an R5-tropic, mucosally-transmitted founder virus) viral inhibition by CVL was comparable to laboratory strains. Measurement of CVL for antimicrobials HBD2, trappin-2/elafin, SLPI and MIP3α indicated that each was present in CVL from HIV(+) and HIV(−) women. HBD2 and MIP3α correlated with anti-HIV activity as did anti-gp160 HIV IgG antibodies in CVL from HIV(+) women.

Conclusions/Significance

These findings indicate that CVL from healthy HIV(+) and HIV(−) women contain innate and adaptive defense mechanisms that inhibit HIV infection. Our data suggest that innate endogenous antimicrobials and HIV-specific IgG in the FRT can act in concert to contribute toward the anti-HIV activity of the CVL and may play a role in inhibition of HIV transmission to women.     

A beta-galactose-specific lectin isolated from the marine worm Chaetopterus variopedatus possesses anti-HIV-1 activity

A 30 kDa beta-galactose-specific lectin named CVL was isolated from the polychaete marine worm Chaetopterus variopedatus (Annelida) and its anti-HIV-1 activity in vitro was determined. Results showed that CVL inhibited cytopathic effect induced by HIV-1 and the production of viral p24 antigen. The EC(50) values were 0.0043 and 0.057 microM, respectively. Time-of-addition analysis of anti-HIV-1 activity indicated its action was at the early stage of virus replication. CVL could blocked the cell-to-cell fusion process of HIV infected and uninfected cells with an EC(50) of 0.073 microM. The inhibition of HIV-1 entry into host cells was demonstrated by using fluorescence-based real-time quantify PCR. At CVL concentration of 0.33 microM and 0.07 microM, 86% and 21% virus attachment were blocked, respectively. The anti-HIV-1 action of CVL might relate to blockade of HIV-1 entry into cells.     

Solubilization of the binding protein from Ehrlich ascites cells and erythrocytes to Pseudomonas aeruginosa cytotoxin.

The binding protein for pore-forming Pseudomonas aeruginosa cytotoxin was solubilized from Ehrlich ascites cell plasma membranes and rabbit and bovine erythrocyte ghosts using nonionic and zwittergent detergents. Analysis of solubilized plasma membranes from Ehrlich cells by a ligand-blot technique after separation by SDS-PAGE/electrophoretic transfer to nitrocellulose or affinity chromatography showed a protein of 70 kDa molecular mass, which binds to cytotoxin. The binding protein solubilized from rabbit erythrocyte ghosts showed a molecular mass of 50 kDa and that from bovine ghosts 55 kDa according to the former test. The binding proteins could be characterized as acidic. They contain a glycan moiety which is, however, not involved in the interaction of cytotoxin with the binding site.     

Analysis of electroblotted proteins by mass spectrometry: protein identification after Western blotting

We describe a new approach for the identification and characterization by mass spectrometry of proteins that have been electroblotted onto nitrocellulose. Using this method (Blotting and Removal of Nitrocellulose (BARN)), proteins can be analyzed either as intact proteins for molecular weight determination or as peptides generated by on-membrane proteolysis. Acetone is used to dissolve the nitrocellulose and to precipitate the adsorbed proteins/peptides, thus removing the nitrocellulose which can interfere with MS analysis. This method offers improved protein coverage, especially for membrane proteins, such as uroplakins, because the extraction step after in-gel digestion is avoided. Moreover, removal of nitrocellulose from the sample solution allows sample analysis by both MALDI- and (LC) ESI-based mass spectrometers. Finally, we demonstrate the utility of BARN for the direct identification of soluble and membrane proteins after Western blotting, obtaining comparable or better results than with in-gel digestion.     

Identification of plasma membrane glycoproteins involved in the contact-dependent inhibition of growth of diploid human fibroblasts

Growth of normal, nontransformed cells is regulated by the interplay between growth stimulating compounds and growth inhibiting cell-cell contacts. We have previously shown that the growth of normal diploid human fibroblasts is mainly regulated by a specific class of plasma membrane glycoproteins (R. J. Wieser and F. Oesch (1986) J. Cell Biol. 103, 361-367). Because it was found that immobilization of the glycoproteins involved in contact-dependent inhibition of growth is an essential step in the recovery of the biological activity of the glycoproteins, we developed a technique for a first characterization of the active compounds. After SDS-PAGE separation of plasma membrane glycoproteins, they were transferred onto nitrocellulose. The nitrocellulose was cut along the separation track into circles which fit into wells of a 96-well microtiter plate. Culturing human diploid fibroblasts on the nitrocellulose circles resulted in characteristic growth patterns, which were dependent upon the source and the treatment of the plasma membrane proteins which had been separated. Five major inhibitory fractions with apparent molecular masses of 300, 170, 90, 50, and 25 kDa have been identified in plasma membranes from confluent fibroblast cultures.     

Direct hybridization to DNA from small numbers of flow-sorted nucleated newborn cells

A technique is described that allows direct hybridization to the DNA of cells flow sorted onto nitrocellulose filters, which obviates an intervening DNA isolation step. The feasibility of this technique for studying small numbers of cells is demonstrated with human cord blood, which has a high proportion of nucleated cells. The cells are stained with fluorescein-conjugated anti-HLe-l, a monoclonal antibody that recognizes mature leucocytes. Anti-HLe-l-positive cells are all nucleated, and a controlled, precise number of them may be sorted directly onto a nitrocellulose membrane. In cord blood, a small percentage of anti-HLe-l-negative cells are nucleated erythrocytes, which may also serve as a source of DNA. Studies were performed on male or female newborn cells flow sorted onto nitrocellulose membranes and hybridized with either a non-specific human repeat DNA probe or a Y chromosome-specific probe. Importantly, the sex of the newborn could be determined at the DNA level from as few as 50 sorted cord blood leucocytes or 5,000 HLe-l-negative cells. Since nucleated erythrocytes are common in fetal blood but rarely found in the peripheral circulation of adults, the method has potential application for the determination of fetal sex from analysis of flow-sorted nucleated erythrocytes present in the maternal circulation during pregnancy.     

Demonstration of a high density lipoprotein (HDL)-binding protein in Hep G2 cells using colloidal gold-HDL conjugates

Solubilized membrane proteins of Hep G2 cells were electrophoretically separated on polyacrylamide gels and electrotransferred onto nitrocellulose paper. Overlaying the nitrocellulose with human high density lipoproteins conjugated to colloidal gold revealed the presence of a single protein band with an apparent molecular mass of 80 kDa. Binding of the conjugates to this protein was specific for high density lipoproteins in as much as it was effectively displaced by an excess of unlabelled high density lipoproteins but not by a similar excess of unlabelled low density lipoproteins. Binding was not dependent on Ca²⁺ as 10 mM EDTA had no effect. The binding activity of the solubilized membranes was increased by incubating the cells with non-lipoprotein cholesterol. This was detected on electroblots and quantified with a new dot blot assay using the colloidal gold-high density lipoprotein conjugates.     

Replication complex of tick-borne encephalitis complex. I. Identification of a nuclear fraction protein responsible for the initiation of RNA synthesis using affinity labeling

The pig embryo kidney cells infected by tick-borne encephalitis virus were fractionated into nuclear-associated, cytoplasmic and membrane fractions. The main part of the virus replicase activity was associated with the nuclei. The replication complex is able to synthesize full-length viral RNAs in vitro. To identify proteins involved in the initiation of the replication at the late stages of the infection, the highly specific affinity labelling technique was used. It was shown that with aldehyde-containing derivatives of ATP, ADP and AMP and [alpha-32P]GTP the target of labelling is a polypeptide having molecular weight about 69 kDa. The same protein is immunostained with TBE virus specific antibodies after blotting onto nitrocellulose. The conclusion is made that nonstructural protein NS3 takes part in virus replication at the late stage of the infection.     

大麦条纹花叶病毒新疆分离物是三组分RNA病毒

大麦条纹花叶病毒新疆分离物(BSMV-XJ)的RNA在乙二醛,二甲亚砜中进行变性电泳,电泳图谱可重复的显示三个组分。将电泳分离的BSMV-RNA各组分在高盐浓度下吸附转移到硝酸纤维素薄膜上,经固定后,与~(32)P标记的病毒总RNA的cDNA行分子杂交。在X光放射自显影图谱上,进一步证实BSMV-XJ为三组分RNA病毒。本文同时就BSMV-RNA不经变性电泳也可以部分吸附转移到硝酸纤维素薄膜上的现象讨论了可能的分子基础。     

A nuclear protein associated with cell divisions in plants

A nuclear protein, present in carrot meristems and rapidly proliferating cultured cells of carrot (Daucus carota L.) has been identified by the use of a monoclonal antibody (MAb 21D7). By combining the techniques of two-dimensional polyacrylamide gel analysis and blotting separated proteins onto nitrocellulose sheets, it was shown that the antibody detected a single polypeptide of apparent molecular mass (M _r) of 45000 and an isoelectric focusing point (pI) of 6.7. This protein was found by subcellular fractionation and immunofluorescence to be highly concentrated in the nucleoli of somatic and zygotic embryos of a wide range of plants. It was not detectable in logarthmically growing cells ofEscherichia coli, yeast, embryos ofDrosophila melanogaster or cultured C3H mouse cells. These data indicate that this protein is a highly conserved non-histone protein associated with nuclei of rapidly dividing plant cells.Abbreviations M _r apparent molecular mass - Da dalton - Ig immunoglobulins - MAb monoclonal antibody - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - 2-D gel two-dimensional gel electrophoresis - 2,4-D 2,4-dichlorophenoxyacetic acid     

Asia1型口蹄疫病毒胶体金免疫层析检测方法的建立

为建立一种快速、简便、灵敏检测Asia1型口蹄疫病毒的胶体金免疫层析方法(GICA)。本研究采用柠檬酸三钠还原法制备胶体金颗粒,标记纯化的抗Asia1型口蹄疫病毒的单克隆抗体,将该标记物与羊抗豚鼠IgG分别包被在硝酸纤维素膜(Nitrocellulose membrane)上,作为检测带和质控带。经条件优化,组装成检测Asia1型口蹄疫的诊断试纸条。用该试纸条分别对A、O、C和Asia1型口蹄疫病毒抗原以及猪水泡病病毒抗原等87份样品进行了检测,发现该试纸条不与口蹄疫病毒A、O、C型以及猪水泡病病毒抗原发生反应,特异性良好。用该试纸条对口蹄疫细胞毒(TCID50为6.25)的10倍系列稀释液进行了检测,最低可以检测到大约10?4。该试纸条与其他传统诊断方法的符合率为98.8%。初步实验确定该试纸条在4oC下可保存3个月、37oC和室温下大概可保存1周左右。该试纸条是一种快速、灵敏、特异的FMD抗原检测方法,对现场检测具有一定实用价值。     

应用斑点金免疫渗滤试验快速同步检测抗HIV-1，HIV-2 IgG抗体

应用斑点金免疫渗滤试验(dotimmunogoldfiltrationassay,DIGFA)建立了一种同步快速检测四种抗HIV-1/2IgG抗体的HIV诊断试纸。通过基因工程技术在大肠杆菌中表达了5种HIV抗原蛋白片段(P24,GP41,GP36,GP120V3,GP120C)。这5种抗原蛋白首先被固定在硝酸纤维素膜上,然后滴加待测血清,其中的病毒抗体通过免疫反应与抗原结合,再加胶体金标记的葡萄球菌蛋白A(SPA),待其渗过膜片后,洗涤,即可形成肉眼可见的红色斑点。用已确证的21份HIV阳性血清(其中包括1份HIV-1标准阳性血清和1份HIV-2标准阳性血清)和30份阴性血清进行了试验,结果表明该快速检测方法与ELISA方法无显著差异。该检测方法不需任何仪器,仅凭肉眼即可判定结果,整个检测过程不超过5分钟。与传统的的ELISA法相比,具有方便快速,成本低廉,应用范围广等优点。同时,此HIV快速诊断试纸可以同步检测并区分针对HIV-1和HIV-2感染的不同检测标志物(抗P24、GP41、GP120和GP36抗体),这对提高快速检测的灵敏度和准确性,以及对判断HIV感染者是否临近或已进入AIDS期有着较高的应用价值。     

Peptide mass fingerprinting by matrix-assisted laser desorption ionization mass spectrometry of proteins detected by immunostaining on nitrocellulose

We have developed an approach that allows peptide mass mapping by matrix-assisted laser desorption ionization-mass spectrometry of proteins visualized on a nitrocellulose membrane by immunochemical detection. Proteins are separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), electroblotted onto a nitrocellulose membrane and after blocking with a nonprotein-containing polymer such as polyvinylpyrrolidone 40 (PVP-40) or Tween 20, the proteins are stained with fount India ink. After incubation with primary and, if required, secondary peroxidase-coupled antibodies, immunochemically reactive proteins can be visualized using conventional enhanced chemiluminescence detection and assigned to the India ink-stained membrane by simple superposition. The proteins of interest are excised, submitted to "on-membrane" cleavage and the peptides are analyzed by mass spectrometry. Protein-based blocking reagents normally used in standard immunodetection protocols, such as skimmed milk, can be employed. We have obtained high-quality mass spectra of bovine serum albumin (BSA) detected on an immunoblot with an estimated amount of 100 fmol applied onto the gel, indicating the sensitivity of the present method. In addition, the approach is demonstrated with two other commercially available proteins, a serum protein, the successful identification of a tyrosine phosphorylated protein from total rat liver homogenate and serine phosphorylated proteins from an EcR 293 nuclear extract separated by two-dimensional (2-D) SDS-PAGE.     

Autoradiographic quantitation of radiolabeled proteoglycans.

Radiolabeled proteoglycans or glycosaminoglycans were precipitated with the cationic dye safranin O onto a sheet of nitrocellulose filter using a dot-blot apparatus. An autoradiography film was exposed against the nitrocellulose sheet. The developed film and the nitrocellulose sheet were separately digitized in a flat-bed-gray-scale scanner connected to a microcomputer. An image analysis program of the microcomputer was used to quantify the density of the radioactivity dots produced in the film, and the intensity of the dye spots on nitrocellulose. With this procedure, a single sample containing the minimum of about 20 ng uronic acid and 5 dpm of incorporated 35SO4 was quantified for both total glycosaminoglycan content and radioactivity. Unincorporated 35SO4 and low molecular mass radioactivity (e.g. products of glycosaminoglycan degrading enzymes) did not interfere since they were quantitatively washed through the membrane before the assay.     

Human spermatozoa produce C16-platelet-activating factor.

Recent data indicate that human spermatozoa produce platelet-activating factor as determined by the rabbit platelet [3H]serotonin release bioassay. In this report, we examined by fast atom bombardment/mass spectrometry the molecular species of platelet-activating factor generated by these germ cells. Extracted spermatozoal samples that contained platelet-activating factor bioactivity underwent straight-phase high-performance liquid chromatography, and fractions which coeluted with authentic C16- and C18-platelet-activating factor standards were subjected to fast atom bombardment/mass spectrometry. Our mass spectral data indicate that human spermatozoa synthesize C16-platelet-activating factor but not C18-platelet-activating factor.     

Serological survey with PCR validation for canine visceral leishmaniasis in northern Palestine

Leishmania infantum is the causative agent of human and canine visceral leishmaniasis (CVL) in the Mediterranean region. A seroprevalence study for CVL was conducted in northern Palestine. Domestic dogs (n = 148) were screened for antileishmanial antibodies by enzyme-linked immunosorbent assay (ELISA). Ten dogs (6.8%) were seropositive. Promastigotes were isolated from one seropositive dog and identified as L. infantum by excreted factor (EF) serotyping, isozyme electrophoresis, and polymerase chain reaction (PCR). In addition to the ELISA, the internal transcribed spacer 1 (ITS1)-, modified ITS1 (mITS1)-, and kinetoplast DNA (kDNA)-PCRs were used to validate this technique as a diagnostic tool for CVL using blood; each assay was performed on 60 blood samples. kDNA-PCR (13/60 positives, 21.7%) was the most sensitive of the assays examined followed by mITS1-PCR (9/60, 15.0%), ELISA (5/60, 8.3%), and ITS1-PCR (3/60, 5%). However, ITS1-PCR and mITS1-PCR were also capable of identifying the parasite species and indicated they belong to L. infantum. In view of its higher sensitivity, kDNA-PCR is recommended for the routine diagnosis of CVL.     

Rapid measurement of protein kinase and phosphatase activities by slot-filtration.

A slot-filtration method has been developed for the detection and quantitation of protein kinase and phosphatase activities. In this technique, after kinase-dependent phosphorylation or phosphatase-dependent dephosphorylation of different substrates, samples are transferred under vacuum onto nitrocellulose using a slot-blotting apparatus. Non-incorporated or released radioactivity is then removed by filtration and washing under vacuum. Quantitation is performed by scintillation or Cerenkov counting of the excised membrane slots. Application of the method to the assay of four different protein kinases (protein kinase N, cyclic AMP-dependent protein kinase and calcium/calmodulin-dependent protein kinases type I and type III) and one phosphatase is presented. A number of protein substrates with varying molecular masses and isoelectric points were found suitable for the slot-filtration technique. The method is applicable to impure as well as purified kinase and phosphatase preparations, can be used over a wide range of concentrations of substrates, has a very low background of nonspecific ATP binding and provides highly reproducible data. The slot-filtration method can also be adapted for use with ion-exchange paper, particularly for assays using peptides as substrates. The technique, with either nitrocellulose or ion-exchange paper, can be used to rapidly process large numbers of samples and can be simultaneously applied to direct comparison of different kinases, phosphatases and/or substrates in the same experiment.     

Multimembrane dot-blotting: a cost-effective tool for proteome analysis

The molecular profiles of protein expression from hundreds of cell lysates can be determined in a high-throughput manner by using fluorescent bead technologies, enzyme-linked immunosorbent assays (ELISAs), and protein microarrays. Although powerful, these tools are costly and technically challenging and thus have limited accessibility for many research groups. We propose a modification of traditional dot blotting that increases throughput of this approach and provides a simple and cost-effective technique for profiling multiple samples. In contrast to traditional blotting that uses a single membrane, we introduce blotting onto a stack of novel, thin, sieve-like membranes. These membranes have a high affinity for binding proteins, but have a lower capacity of protein binding compared to traditional (nitrocellulose) membranes. We compare the linear binding capacity and variability of these novel membranes with nitrocellulose membranes. Also, we describe the use of these membranes in a multilayer dot blot format for profiling mitogen-mediated signal transduction pathways in T cells.