Two-dimensional gel electrophoresis of actin-binding proteins isolated by affinity chromatography from human skeletal muscle

Obstructive azoospermia represents approximately 10 % of cases of male hypofertility. It is classified according to the volume of ejaculate. When the latter is normal a proximal obstruction is suspected. Scrotal sonography can help to detect dilation of the epididymal head when clinical findings are equivocal. Ejaculatory duct obstruction (EDO) is suspected when the volume of ejaculate is low. The use of transrectal ultrasonography (TRUS) plays a major role in the investigation of these patients, and endorectal MRI is a very useful adjunct in selected cases. The most common cause of EDO is congenital bilateral absence of vas deferens, which is now thought to be a genital form of cystic fibrosis in 80 % of cases. Consequently, a definitive diagnosis must be made before any attempt at in vitro fertilization. TRUS accurately visualizes abnormalities of the caudal junction of the vas deferens and seminal vesicles, yielding a definitive diagnosis without scrototomy. Other causes of EDO are congenital cysts compressing the distal part of the ejaculatory ducts and inflammatory distal stenosis. The former are accurately identified by TRUS, but the latter give more or less marked signs of obstruction which are only of value in azoospermic patients with a low-volume ejaculate. More invasive imaging is required to diagnose partial obstruction of the ED. Surgical vasography is still the reference, but puncture of the seminal vesicles under TRUS guidance is an attractive alternative, as it permits aspiration of seminal fluid (to seek motile sperm) and vasography without scrototomy. Lastly, endorectal MRI well assesses the relationships between the proximal prostatic urethra and the posterior wall of the ejaculatory ducts, which need to be precisely known when endoscopic resection of the ejaculatory ducts is planned.     

Two-dimensional separations of peptides and proteins by comprehensive liquid chromatography-capillary electrophoresis

Analysis of biological samples is problematic because of their complex composition. Reversed phase liquid chromatography (RPLC), size exclusion chromatography (SEC), and, more recently, capillary zone electrophoresis (CZE) are routinely used for the analysis of these samples, but are eventually limited because they are one-dimensional (1-D) methods. As sample complexity increases, the separation efficiency necessary to resolve a large number of sample components in one dimension becomes prohibitively high. A solution to this problem has been to use a two-dimensional (2-D) approach. Each dimension in a 2-D separation relies on a different separating mechanism. By expanding the separation into two dimensions, sample components unresolved in the first dimension can often be separated in the second. This circumvents the requirement for extremely high efficiencies in either dimension. Two-dimensional slab gel electrophoresis has been used successfully in this area, but a more instrumental approach is desired. In this paper we describe three coupled-column approaches to 2-D separations. First, microcolumn SEC-CZE is explored as a means of 2-D protein analysis. Next, RPLC-CZE is investigated for analysis of peptides in tryptic maps. Finally, RPLC is coupled with fast CZE (FCZE), a unique form of CZE analysis, for fast 2-D analysis of peptides. Details of the instrumentation used in these 2-D systems will be presented along with the results of some typical 2-D analyses.     

Two-dimensional gel electrophoresis for proteome projects: the effects of protein hydrophobicity and copy number

Two-dimensional (2-D) gel electrophoresis is often used in proteome projects to provide a global view of the proteins expressed in any cell or tissue type. Here we have investigated the effects of protein hydrophobicity and cellular protein copy number on a protein's presence or absence on a two-dimensional gel. The average hydropathy values of all known proteins from Bacillus subtilis, Escherichia coli and Saccharomyces cerevisiae were calculated, thus defining the range of protein hydrophobicity and hydrophilicity in these organisms. The average hydropathy values were then calculated for a total of 427 proteins from these species, which had been identified elsewhere on 2-D gels. Strikingly, it was seen that no highly hydrophobic proteins, as defined by average hydrophobicity values, have been found to date on 2-D gel separations of whole cell lysates. A clear hydrophobicity cutoff point was seen, above which current 2-D electrophoresis methods appear not to be useful for protein separation. The effect of cellular protein copy number on a protein's presence on a 2-D gel was investigated by means of a graphical model. This model showed how variations in protein loading and copy number per cell interact to determine the quantity of a protein that will be present on a 2-D gel. Considering the current maximum in 2-D gel loading capacity, it was found that 2-D probably can not visualize or produce analytical quantities of proteins present at less than 1000 copies per cell. We conclude that further developments of 2-D electrophoresis techniques are desirable to enable the visualization and analysis of all proteins expressed by a cell or tissue.     

Cholangiocyte-specific rat liver proteins identified by establishment of a two-dimensional gel protein database

The liver is composed of a variety of cells that form a functional unit involved in uptake, synthesis, metabolism, and secretion. Until recently, most studies examining liver function did not analyze the specific proteins expressed or functions performed by the multiple individual cell types that constitute the hepatic mass. In the last decade, novel isolation methods have been developed that allow the purification of liver cell populations highly enriched in one type of liver cell. Here, we present a detailed two-dimensional (2-D) protein map of rat bile duct epithelial cells (i.e., cholangiocytes) using a recently developed isolation procedure. In addition, we identify 27 major cholangiocyte proteins either by comparison to maps of known rat liver proteins (based on pI and Mr) or by tryptic digestion and microsequencing. Finally, we compare the relative abundance of individual proteins present in cholangiocytes to whole liver as well as hepatocyte-specific proteins. Our results show that cholangiocytes express a unique array of individual proteins. The cholangiocyte 2-D protein pattern is markedly different from that of isolated rat hepatocytes or whole rat liver, with high levels of proteins previously known to be expressed by cholangiocytes (e.g., cytokeratins, actins) as well as protein not previously demonstrated to be expressed at high levels (e.g., annexin V, selenium binding protein). We conclude that this cholangiocyte-derived, 2-D protein map will be a crucial resource for studies directed at our understanding of cholangiocyte physiology and pathobiology.     

Correlation of plasma protein separation patterns obtained by two-dimensional polyacrylamide gel electrophoresis and by capillary electrophoresis

We used three different electrophoretic techniques for the analysis of human plasma proteins: (i) two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), with sodium dodecyl sulfate (SDS) used only in slab gel electrophoresis; (ii) capillary isoelectric focusing (CIEF) with no denaturants; (iii) linear polyacrylamide (LPA)-filled capillary electrophoresis with SDS (SDS-CE). With technique (i), data on isoelectric point and molecular size of plasma proteins can be obtained. Techniques (ii) and (iii) are suited to obtain quantitative information on proteins. The separation principle used in technique (ii) is closely related to that used in the first dimension of technique (i), and that used in technique (iii) related to that in the second dimension of technique (i). Therefore, we could successfully correlate protein separation patterns obtained by 2-D PAGE and those obtained by capillary electrophoresis. The advantages of correlating data obtained by various electrophoretic techniques in the course of constructing a comprehensive database on human plasma proteins are discussed.     

Autonomic motor neuron migration and expression of nicotinamide adenine dinucleotide phosphate reduced diaphorase are dependent upon peripheral target

The mechanical behavior of calf pericardium employed in the manufacture of cardiac bioprostheses was assessed according to the region from which it was selected. For this purpose, selected samples of the tissue were sewn with different types of commercially available sutures and subjected to tensile testing, the results of which were compared with the findings in selected, but not sutured, tissue used as a control. The results confirm a loss of resistance--that is, a reduction of the capacity of sutured samples of the biomaterial to withstand breakage stress compared with control samples. Taking into account the marked resistance to breakage of the suture thread, this phenomenon can only be explained as a consequence of the deleterious mechanical interaction between the suture and chemically treated pericardium. This interaction is illustrated by the shearing force which is responsible for the loss of resistance in the tested samples. These trials demonstrate that the results can be improved and the deleterious interaction diminished, although not eliminated, when the pericardium is selected from a given region.     

Two-dimensional electrophoresis of proteins from breast cancer cells MCF-7. Changes in synthesis induced by FGF-2

Two-dimensional electrophoresis analysis of proteins from breast cancer cells MCF-7. Modifications of synthesis induced by FGF-2. Using high resolution two-dimensional electrophoresis, we have separated more than 1,000 proteins from the breast cancer cell line MCF-7. Computer assisted analysis of gels allowed us to classify these proteins in function of their isoelectric point, molecular weight and relative quantity. This data-base will now be used as a powerful tool to identified proteins which synthesis is regulated in various experimental or pathological situations. Thus we studied modifications induced by FGF-2. This growth factor induces the synthesis of four polypeptides which are not detected in cells not stimulated by this factor. In addition, intensity of nine other polypeptides was found increased in presence of FGF-2.     

Human lymphocyte cDNA ordered library analyzed by 2D gel electrophoresis. 1. Pooling strategy and matching of gel patterns

beta-Citryl-L-glutamate-hydrolysing enzyme (beta-CGHE) was purified from rat testis particulate fraction 13,000-fold, at a yield of 7%. The enzyme was purified by ammonium sulfate fractionation, hydroxyapatite, chelating Sepharose, beta-CG-Sepharose affinity chromatography and Sephacryl S-300 gel filtration. The purified enzyme usually migrated as two periodic acid Schiff's-stained bands on native polyacrylamide gel-electrophoresis (PAGE) with molecular weights of 350 and 420 kDa. Both bands hydrolyzed beta-citryl-L-glutamate (beta-CG) to citrate and glutamate. The 420 kDa band was changed by digestion with N-glycosidase F, into a 350 kDa band on native PAGE. The purified enzyme was composed of 90, 100, 115 and 130 kDa subunits on SDS-PAGE under non-reduced conditions. The purified enzyme was pharmacologically similar to the beta-CGHE activity partially purified from rat testis. This enzyme required manganese ions for full activity and it was strongly inhibited by nucleotides such as ATP or GTP and phosphate ions. beta-CGHE was also potently inhibited by an excitatory amino acid agonist, L-quisqualate, but not by another agonists, N-methyl-D-aspartate and kinate. It had high substrate specificity for beta-CG. The antibodies against the purified enzyme reacted mainly to the 115 kDa band on the SDS-PAGE and precipitated the enzyme activity from the crude and purified enzyme solution.     

Analysis of photoreceptor proteins of microorganisms by gradient gel electrophoresis and other biochemical separation methods

Photoreceptor proteins for photoorientation in microorganisms are usually membrane bound and can be isolated by standard biochemical methods. Three examples are shown: the flagellates Euglena gracilis, Peridinum gatunense and the slime mold Dictyostelium discoideum. The photoreceptor of Euglena is attached to the basis of the flagellum and is composed of at least four chromoproteins which can be separated by gradient sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) fast protein liquid chromatography (FPLC) and isoelectric focusing (IEF); it contains pterins and a flavin as chromophoric groups. The photoreceptor of Peridinium absorbs in the red wavelength band. Though not yet identified in detail, multiple receptors are probably involved, as indicated by fluorescence spectroscopy. Dictyostelium shows positive and negative phototaxis in its amoebal form and exclusively positive phototaxis in its pseudoplasmodial form. It is still open to discussion whether the two stages use separate photoreceptors. From amoebae two photoreceptor pigments have been isolated, showing an absorption which resembles the action spectrum, one membrane bound with a molecular mass of 45 kDa and one cytoplasmic fraction with a molecular mass of 27 kDa.     

Hypoxia induces vascular endothelial growth factor gene and protein expression in cultured rat islet cells

The formation of new microvasculature by capillary sprouting at the site of islet transplantation is crucial for the long-term survival and function of the graft. Vascular endothelial growth factor (VEGF), an endothelial cell-specific mitogen with potent angiogenic and vascular permeability-inducing properties, may be a key factor in modulating the revascularization of islets after transplantation. In this study, we examined the gene expression of VEGF mRNA in three tumor cell lines and in isolated whole and dispersed rat islets in vitro by Northern blot hybridization in normoxic (5% CO2, 95% humidified air) and hypoxic (1% O2, 5% CO2, 94% N2) culture conditions. Increased expression of VEGF mRNA was observed in beta-TC3, RAW 264.7, and IC-21 tumor cell lines when subjected to hypoxia. With isolated whole islets in normoxic culture, a threefold increase in VEGF mRNA (P < 0.001) was seen at 48 h as compared with freshly isolated islets. This response was similar to the 3.8-fold increase observed with islets subjected to hypoxia. Dispersed rat islet cell clusters cultured on Matrigel for 24 h under hypoxic conditions showed a 3.4-fold increase (P < 0.01) in VEGF mRNA compared with those cultured in normoxia. This correlated with increased VEGF secretion as determined by enzyme-linked immunosorbent assay. Immunohistochemical studies revealed the presence of increased expression of VEGF protein near the center of islets after 24 h of normoxic culture. Islet cell clusters on Matrigel showed intense cellular localization of VEGF in both beta-cells and non-beta-cells. These findings suggest that rat islet cells, when subjected to hypoxia during the first few days after transplantation, may act as a major source of VEGF, thereby initiating revascularization and maintaining the vascular permeability of the grafted islets.     

Agarose gel electrophoresis of cerebrospinal fluid proteins and analysis of the pherogram profiles by analog computer

A micro-method of agarose gel electrophoresis requiring 1--3 ml of cerebrospinal fluid for quantitative analysis of cerebrospinal fluid proteins is described. After concentration of CSF to about 50 microliter by ultrafiltration and refractometric determination of protein, approximately 20--40 microgram of total protein are used for electrophoresis. Photometric scanning of the electrophoretic pattern at two different wavelengths permits quantitative evaluation. The pherograms are analysed by means of a modified DU-PONT analog computer. Factors which influence quantitative electrophoresis are examined. In cerebrospinal fluid of normal children 15 protein fractions are demonstrated: 2 prealbumins, albumin, 5 alpha-, 3 beta- and 4 gamma-globulins.     

The band areas of proteins determined by fluorescent scanning in the commercial automated gel electrophoresis apparatus

An automated gel electrophoresis apparatus, recently available commercially, allows one to follow the band during electrophoresis in real time, and lends itself therefore to an evaluation of bandwidth as a function of migration time (the dispersion coefficient), resolution and band shape. These determinations assume the constancy of band area with migration time and at various gel concentrations. The purpose of the present study was to verify these assumptions. Representative proteins and sodium dodecyl sulfate (SDS)-proteins, either natively fluorescent or fluorescein carboxylate labeled, were found to exhibit band areas which approach constancy as a function of migration time in both agarose and polyacrylamide gel electrophoresis, provided that (i) the protein concentration under the band was low enough to obviate self-quenching of fluorescence; (ii) the separation of the protein of interest from contaminants had progressed sufficiently during the time at which band areas were measured; (iii) the baseline under the peak was sufficiently well defined. However, band areas decrease with increasing gel concentration. Protein peaks exhibited leading and trailing tails. The ratio of the combined tail area to total area appeared to be near-constant at varying migration times. However, that ratio increases with increasing gel concentration. The tail area does not appear to be an artifact of fluorometric detection since it is reproduced upon fluorimetric analysis of the protein eluted from gel slices after electrophoresis. However, it may be due to photochemical destruction under the conditions of repetitive fluorometric peak detection.     

ATP potentiates interleukin-1 beta-induced MMP-9 expression in mesangial cells via recruitment of the ELAV protein HuR

Renal mesangial cells express high levels of matrix metalloproteinase 9 (MMP-9) in response to inflammatory cytokines such as interleukin (IL)-1 beta. We demonstrate here that the stable ATP analog adenosine 5'-O-(thiotriphosphate) (ATP gamma S) potently amplifies the cytokine-induced gelatinolytic content of mesangial cells mainly by an increase in the MMP-9 steady-state mRNA level. A Luciferase reporter gene containing 1.3 kb of the MMP-9 5'-promoter region showed weak responses to ATP gamma S but conferred a strong ATP-dependent increase in Luciferase activity when under the additional control of the 3'-untranslated region of MMP-9. By in vitro degradation assay and actinomycin D experiments we found that ATP gamma S potently delayed the decay of MMP-9 mRNA. Gel-shift and supershift assays demonstrated that three AU-rich elements (AREs) present in the 3'-untranslated region of MMP-9 are constitutively bound by complexes containing the mRNA stabilizing factor HuR. The RNA binding of these complexes was markedly increased by ATP gamma S. Mutation of each ARE element strongly impaired the RNA binding of the HuR containing complexes. Reporter gene assays revealed that mutation of one ARE did not affect the stimulatory effects by ATP gamma S, but mutation of all three ARE motifs caused a loss of ATP-dependent increase in luciferase activity without affecting IL-1 beta-inducibility. By confocal microscopy we demonstrate that ATP gamma S increased the nucleo cytoplasmic shuttling of HuR and caused an increase in the cytosolic HuR level as shown by cell fractionation experiments. Together, our results indicate that the amplification of MMP-9 expression by extracellular ATP is triggered through mechanisms that likely involve a HuR-dependent rise in MMP-9 mRNA stability.     

Formyl peptide receptors are coupled to multiple mitogen-activated protein kinase cascades by distinct signal transduction pathways: role in activation of reduced nicotinamide adenine dinucleotide oxidase

Formyl peptide receptor activation of three mitogen-activated protein kinase (MAPK) cascades, extracellular signal-regulated kinases (ERKs), N-terminal kinases (JNKs), and p38 MAPK was examined in differentiated HL-60 granulocytes. FMLP stimulated a concentration- and time-dependent increase in ERK, JNK, and p38 MAPK activities, all of which were dependent on a pertussis toxin-sensitive G protein. Pharmacologic inhibitors were used to examine the roles of tyrosine kinases, phosphatidylinositol 3-kinase, protein kinase C, and phospholipase C. FMLP-stimulated ERK activity was dependent on tyrosine kinases, phosphatidylinositol 3-kinase, protein kinase C, and phospholipase C; p38 MAPK activation was dependent on phosphatidylinositol 3-kinase and phospholipase C; while JNK activation was independent of all of these signaling components. The mitogen-activated protein kinase/ERK kinase inhibitor PD098059 reduced ERK activation by 90%, while an inhibitor of p38 MAPK, SB203580, inhibited p38 MAPK activation by 80%. Both PD098059 and SB203580 inhibited FMLP-stimulated superoxide release, as did inhibitors directed against protein kinase C, tyrosine kinases, and phosphatidylinositol 3-kinase. We conclude that formyl peptide receptors are coupled to three MAPK cascades by Gi proteins. ERKs, p38 MAPK, and JNKs are each activated by distinct proximal signal transduction pathways. Activation of p38 MAPK is necessary for FMLP stimulation of respiratory burst activity; however, a second signal that may involve ERK is also required for this activity.     

Effect of protein application mode and acrylamide concentration on the resolution of protein spots separated by two-dimensional gel electrophoresis

Two-dimensional gel electrophoresis separates large numbers of proteins in two steps on the basis of differences in their pIs and molecular masses. The separation is usually performed on immobilized pH gradient strips, followed by gradient polyacrylamide gels separating proteins with molecular masses between 5-200 kDa. For the first-dimensional separation the protein samples are usually applied near one end of the strip. Using total soluble protein extracts of the bacterium Haemophilus influenzae, we found that simultaneous sample application at both the basic and the acidic ends of the strip resulted in detection of more and stronger protein spots in comparison with sample application at one end only. Because many proteins of an organism have similar pI and Mr values, an overlapping of protein spots is frequently observed in the second-dimensional separation. The soluble protein fraction of H. influenzae was further separated on gels of constant acrylamide concentration between 7.5% and 15.0%. We found that for proteins of molecular mass within certain ranges, the gels of homogeneous acrylamide concentration provided more efficient spot separation than the gradient gels. The observed improvements in spot resolution may be useful in the characterization of proteins from other organisms or cell lines.     

Analysis of the low molecular weight nuclear polypeptide by isoelectrofocusing of nuclear proteins first separated by gel electrophoresis in SDS

Some of the nuclear proteins, especially those tightly bound with nucleic acids, show a tendency to aggregate and some of them are also hydrophobic. Thus, SDS-containing buffers are useful for their analysis. The method of isoelectrofocusing of proteins separated by polyacrylamide gel electrophoresis in the presence of SDS was used for the analysis of the nuclear proteins. First, SDS-polyacrylamide gel electrophoresis of total chromatin proteins was conducted. The sample of the examined nuclear proteins after SDS-gel electrophoresis was isolated as a gel slice (thus corresponding to the particular molecular weight range) and used for isoelectrofocusing. This approach has been used for the analysis of about 19 kD nuclear polypeptide, tightly bound with DNA. The isoelectric point of this polypeptide has been estimated as 5.3.     

Purification by reflux electrophoresis of whey proteins and of a recombinant protein expressed in Dictyostelium discoideum

Protein purification that combines the use of molecular mass exclusion membranes with electrophoresis is particularly powerful as it uses properties inherent to both techniques. The use of membranes allows efficient processing and is easily scaled up, while electrophoresis permits high resolution separation under mild conditions. The Gradiflow apparatus combines these two technologies as it uses polyacrylamide membranes to influence electrokinetic separations. The reflux electrophoresis process consists of a series of cycles incorporating a forward phase and a reverse phase. The forward phase involves collection of a target protein that passes through a separation membrane before trailing proteins in the same solution. The forward phase is repeated following clearance of the membrane in the reverse phase by reversing the current. We have devised a strategy to establish optimal reflux separation parameters, where membranes are chosen for a particular operating range and protein transfer is monitored at different pH values. In addition, forward and reverse phase times are determined during this process. Two examples of the reflux method are described. In the first case, we described the purification strategy for proteins from a complex mixture which contains proteins of higher electrophoretic mobility than the target protein. This is a two-step procedure, where first proteins of higher mobility than the target protein are removed from the solution by a series of reflux cycles, so that the target protein remains as the leading fraction. In the second step the target protein is collected, as it has become the leading fraction of the remaining proteins. In the second example we report the development of a reflux strategy which allowed a rapid one-step preparative purification of a recombinant protein, expressed in Dictyostelium discoideum. These strategies demonstrate that the Gradiflow is amenable to a wide range of applications, as the protein of interest is not necessarily required to be the leading fraction in solution.