Role of the glycosylphosphatidylinositol/inositol phosphoglycan system in human fibroblast proliferation

The involvement of the glycosylphosphatidylinositol/inositol phosphoglycan (gly-PtdIns/IPG) system in the stimulation of macromolecular syntheses in human fibroblasts has been investigated. The study demonstrates that an insulin sensitive gly-PtdIns/IPG system is present in human fibroblasts, that IPG can significantly stimulate DNA, RNA, and protein synthesis, and that the action of insulin on DNA synthesis as well as that of IPG can be significantly reduced by a specific anti-IPG antibody. These results strongly support the hypothesis that the gly-PtdIns/IPG system is involved in the signal transduction pathway leading to the stimulation of cell proliferation.     

A combination of immobilised pH gradients improves membrane proteomics

Membrane protein analyses have been notoriously difficult due to hydrophobicity and the general low abundance of these proteins compared to their soluble cytosolic counterparts. Shotgun proteomics has become the preferred method for analyses of membrane proteins, in particular the recent development of peptide immobilized pH gradient isoelectric focusing (IPG-IEF) as the first dimension of two-dimensional shotgun proteomics. Recently, peptide IPG-IEF has been shown to be a valuable shotgun proteomics technique through the use of acidic narrow range IPG strips, which demonstrated that small acidic p I increments are rich in peptides. In this study, we assess the utility of both broad range (BR) (p I 3-10) and narrow range (NR) (p I 3.4-4.9) IPG strips for rat liver membrane protein analyses. Furthermore, the use of these IPG strips was evaluated using label-free quantitation to demonstrate that the identification of a subset of proteins can be improved using NR IPG strips. NR IPG strips provided 2603 protein assignments on average (with 826 integral membrane proteins (IMPs)) compared to BR IPG strips, which provided 2021 protein assignments on average (with 712 IMPs). Nonredundant protein analysis demonstrated that in total from all experiments, 4195 proteins (with 1301 IMPs) could be identified with 1428 of these proteins unique to NR IPG strips with only 636 from BR IPG strips. With the use of label-free quantitation methods, 1659 proteins were used for quantitative comparison of which 319 demonstrated statistically significant increases in normalized spectral abundance factors (NSAF) in NR IPG strips compared to 364 in BR IPG strips. In particular, a selection of six highly hydrophobic transmembrane proteins was observed to increase in NSAF using NR IPG strips. These results provide evidence for the use of alternative pH gradients in combination to improve the shotgun proteomic analysis of the membrane proteome.     

Proteome resolution by two-dimensional gel electrophoresis varies with the commercial source of IPG strips

By facilitating reproducible first dimension separations, commercial immobilized pH gradient (IPG) strips enable high throughput and high-resolution proteomic analyses using two-dimensional gel electrophoresis (2DE). Amersham, Biorad, Invitrogen, and Sigma all market linear pH 3-10 IPG strips. We have applied optimized 2DE protocols with both membrane and soluble brain protein extracts to critically evaluate all four products. Resolved protein spots were quantitatively evaluated after carrying out these protocols using IPG strips from the four companies. Biorad and Amersham IPG strips resolved a high number of membrane and soluble proteins, respectively. Furthermore, Amersham IPG strips eluted the largest amount of protein into the second dimension gels and had the most protein remaining in the strip after 2DE. Biorad and Amersham IPG strips maintained a consistent linear pH 3-10 gradient, whereas those from Invitrogen appeared nonlinear or "compressed" within the central pH region. The gradient range within Sigma IPG strips appeared to be slightly less than pH 3-10, due to one extended pH unit within the gradient. Overall, all four commercially available IPG strips have the ability to resolve both membrane and soluble brain proteomes. The difference is that Amersham and Biorad do so more consistently and with better spot resolution. It appears that the physical/chemical nature of commercially available IPG strips can vary considerably, leading to marked differences in subsequent protein resolution in 2DE. These differences likely reflect variations in the uptake of proteins into the strips, and differences in the focusing and elution of proteins from the first to the second dimension. These differences would appear, in part, to underlie some inter-lab variations in the effective resolution of proteomes.     

Occlusive Impedance Plethysmography: A Noninvasive Method of Diagnosis of Proximal Deep Vein Thrombosis

The purpose of this study was to assess and confirm the accuracy of impedance plethysmography (IPG) by the occlusive cuff method, in detecting proximal (popliteal, femoral and iliac) deep vein thrombosis in patients with symptomatic limbs. In 27 patients 30 consecutive limbs were studied with concurrent venography and IPG. The IPG result was normal in 9 of 9 limbs which were normal on venography, and abnormal in 15 of 16 limbs which showed venographic evidence of proximal deep vein thrombosis (DVT). An abnormal IPG strongly suggests proximal DVT (predictive value 0.88). A normal IPG virtually rules out proximal DVT (predictive value 1.0).     

Characterization of citrate transport through the plasma membrane in a carrot mutant cell line with enhanced citrate excretion

The superior ability of citrate excretion in a carrot (Daucus carota L.) mutant cell line, namely IPG (insoluble phosphate grower) [Takita et al. (1999a) Plant Cell Physiol. 40: 489] cells has been characterized in terms of citrate transport at the plasma membrane. IPG cells released about a 20-fold increase in citrate in comparison with malate, while the concentration of malate was only 35% lower than that of citrate in the cell sap. Citrate excretion was sensitive to anion channel blockers, such as niflumic acid and anthracene-9-carboxylic acid. These results indicate that IPG cells release citrate through the plasma membrane using citrate specific anion channels. The rate of citrate release from IPG cells was not affected by the concentration of aluminum (0 and 50 micro M), soluble P(i) (0 or 2 mM) and the pH (4.5-5.6) of the medium, suggesting that anion channels would not be regulated by such external conditions. Citrate excretion correlated with the H(+) efflux, possibly from the action of H(+)-ATPase on the plasma membrane. The activity of plasma membrane H(+)-ATPase was about three times higher in IPG cells than in wild-type cells, and might be involved in the high citrate excretion ability.     

Serial isoelectric focusing as an effective and economic way to obtain maximal resolution and high-throughput in 2D-based comparative proteomics of scarce samples: proof-of-principle

In 2D-based comparative proteomics of scarce samples, such as limited patient material, established methods for prefractionation and subsequent use of different narrow range IPG strips to increase overall resolution are difficult to apply. Also, a high number of samples, a prerequisite for drawing meaningful conclusions when pathological and control samples are considered, will increase the associated amount of work almost exponentially. Here, we introduce a novel, effective, and economic method designed to obtain maximum 2D resolution while maintaining the high throughput necessary to perform large-scale comparative proteomics studies. The method is based on connecting different IPG strips serially head-to-tail so that a complete line of different IPG strips with sequential pH regions can be focused in the same experiment. We show that when 3 IPG strips (covering together the pH range of 3-11) are connected head-to-tail an optimal resolution is achieved along the whole pH range. Sample consumption, time required, and associated costs are reduced by almost 70%, and the workload is reduced significantly.     

Isoelectric point-based prefractionation of proteins from crude biological samples prior to two-dimensional gel electrophoresis

Two-dimensional gel electrophoresis (2-DE) is used to compare the protein profiles of different crude biological samples. Narrow pH range Immobilized pH Gradient (IPG) strips were designed to increase the resolution of these separations. To take full advantage of IPG strips, the ideal sample should be composed primarily of proteins that have isoelectric point (pI) values within the pH range of the IPG strip. Prefractionation of cell lysates from a human prostate cancer cell line cultured in the presence or absence of epigallocatechin-3-gallate was achieved in fewer than 30 min using an anion-exchange resin and two expressly designed buffers. The procedure was carried out in a centrifuge tube and standard instrumentation was used. The cell lysates were prefractionated into two fractions: proteins with pI values above 7 and between 4 and 7, respectively. The fractions were then analyzed by 2-DE, selecting appropriate pH ranges for the IPG strips, and the gels were compared with those of unprefractionated cell lysates. Protein loading capacity was optimized and resolution and visualization of the less abundant and differentially expressed proteins were greatly improved.     

Purification and characterization of insulin-mimetic inositol phosphoglycan-like molecules from grass pea (Lathyrus sativus) seeds.

BACKGROUND: Signal transduction through the hydrolysis of glycosyl-phosphatidylinositol (GPI) leading to the release of the water-soluble inositol phosphoglycan (IPG) molecules has been demonstrated to be important for mediating some of the actions of insulin and insulin-like growth factor-I (IGF-I). MATERIALS AND METHODS: In the present study, GPI from grass pea (Lathyrus sativus) seeds has been purified and partially characterized on the basis of its chromatographic properties and its compositional analysis. RESULTS: The results indicate that it shows similarities to GPI previously isolated from other sources such as rat liver. IPG was generated from L. sativus seed GPI by hydrolysis with a GPI-specific phospholipase D (GPI-PLD). This IPG inhibited protein kinase A (PKA) in an in vitro assay, caused cell proliferation in explanted cochleovestibular ganglia (CVG), and decreased 8-Br-cAMP-induced phosphoenolpyruvate carboxykinase (PEPCK) mRNA expression in cultured hepatoma cells. CONCLUSIONS: Our data indicate that L. sativus seed IPG possess insulin-mimetic activities. This may explain why L. sativus seeds have been used in some traditional medicines to ameliorate diabetic symptoms.     

Analytical isoelectric focusing with immobilized pH gradients of human apolipoprotein E from very low density lipoproteins and total plasma

A method for analytical isoelectric focusing (IEF) of apolipoprotein E (apoE) in immobilized pH gradients (IPG) and immunodetection of the separated isoforms has been developed for use with either very low density lipoproteins (VLDL) or whole plasma. Both VLDL and plasma were sequentially delipidated with 1,4-dioxane, acetone-ethanol, and ether. Neuraminidase treatment preceded the delipidation when required. Using preformed plates, pH 5.0-6.0 (LKB, Bromma) after rehydration with 6 M urea and dextran T-10, the IPG focusing pattern of the common isoforms (E2, E3, E4) was found to be equivalent to conventional IEF with the added resolution of the E4 disialo form. The use of self-poured narrower gradients permitted the further resolution of the E4 monosialo form, a previously unrecognized heterogeneity of the E2, E3, and E4 monosialo isoforms and differentiation of the apoE2** mutant; all of these forms comigrate with the common isoproteins in conventional IEF. Finally, the conditions for IPG of whole plasma using apoE monoclonal antibodies and enzyme-conjugated anti-mouse IgG for detection were established. Thus, IPG focusing is shown to be a powerful method for resolution of the apoE sialoforms and apoE mutant forms. The method has important implications in accurate and diagnostic phenotyping. Moreover, it is a convenient method for phenotyping which requires only very small volumes of plasma.     

Discovery of an Escherichia coli Esterase with High Activity and Enantioselectivity toward 1,2-O-Isopropylideneglycerol Esters

Escherichia coli has been widely used as an expression host for the identification of desired biocatalysts through screening or selection assays. We have previously used E. coli in growth selection and screening assays for identification of Bacillus subtilis lipase variants (located in the periplasm) with improved activity and enantioselectivity toward 1,2-O-isopropylideneglycerol (IPG) esters. In the course of these studies, we discovered that E. coli itself exhibits significant cytoplasmic esterase activity toward IPG esters. In order to identify the enzyme (or enzymes) responsible for this esterase activity, we analyzed eight E. coli knockout strains, in which single esterase genes were deleted, for their ability to hydrolyze IPG butyrate. This approach led to the identification of esterase YbfF as the major E. coli enzyme responsible for the hydrolytic activity toward IPG esters. The gene coding for YbfF was cloned and overexpressed in E. coli, and the corresponding protein was purified and characterized for its biocatalytic performance. YbfF displays a high level of activity toward IPG butyrate and IPG caprylate and prefers the R-enantiomer of these substrates, producing the S-enantiomer of the IPG product with high enantiomeric excess (72 to 94% ee). The enantioselectivity of YbfF for IPG caprylate (E = 40) could be significantly enhanced when using dimethylformamide (DMF) or dimethyl sulfoxide (DMSO) as cosolvents in kinetic resolution experiments. The enzyme also shows high enantioselectivity toward 1-phenylethyl acetate (E ≥ 200), giving the chiral product (R)-1-phenylethanol with >99% ee. The high activity and enantioselectivity of YbfF make it an attractive enzyme for organic synthesis.     

Protein purification by Off-Gel electrophoresis

A novel free-flow protein purification technique based on isoelectric electrophoresis is presented, where the proteins are purified in solution without the need of carrier ampholytes. The gist of the method is to flow protein solutions under an immobilised pH gradient gel (IPG) through which an electric field is applied perpendicular to the direction of the flow. Due to the buffering capacity of the IPG gel, proteins with an isoelectric point (pI) close to pH of the gel in contact with the flow chamber stay in solution because they are neutral and therefore not extracted by the electric field. Other proteins will be charged when approaching the IPG gel and are extracted into the gel by the electric field. Both a demonstration experiment with pI markers and a simulation of the electric field distribution are presented to highlight the principle of the system. In addition, an isoelectric fractionation of an Escherichia coli extract is shown to illustrate the possible applications.     

Web-based two-dimensional database of Saccharomyces cerevisiae proteins using immobilized pH gradients from pH 6 to pH 12 and matrix-assisted laser desorption/ionization-time of flight mass spectrometry

An image based two-dimensional (2-D) reference map of very alkaline yeast cell proteins was established by using immobilized pH gradients (IPG) up to pH 12 (IPG 6-12, IPG 9-12 and IPG 10-12) for 2-D electrophoresis and by using matrix-assisted laser desorption/ionization-time of flight mass spectrometry peptide mass fingerprinting for spot identification. Up to now 106 proteins with theoretical isoelectric points up to pH 11.15 and molecular mass between 7.5 and 115 kDa were localized and identified. Additionally, due to the improved resolution of steady-state isoelectric focussing with IPGs, even low copy number proteins with codon bias below 0.02 were detected and identified.     

ACTH stimulates the release of alkaline phosphatase through Gi-mediated activation of a phospholipase C and the release of inositolphosphoglycan

We have previously reported that ACTH activates a phospholipase C that hydrolyzes glycosylphosphatidylinositol (GPI), which would release inositolphosphoglycan (IPG) to the extracellular medium, and that an IPG purified from Trypanosoma cruzi is able to inhibit ACTH-mediated steroid production in adrenocortical cells. In the present paper, it was found that anti-inositolphosphoglycan antibodies (anti-CRD) increased ACTH-mediated corticosterone production, which indicates that an endogenous IPG is a physiological inhibitor of ACTH response. On the other hand, we investigated the release to the extracellular medium of the GPI-anchored enzyme, alkaline phosphatase, by ACTH. We found that: (a) the released enzyme appeared in the aqueous phase after Triton X-114 partitioning, consistent with loss of the GPI, (b) the phospholipase C inhibitor, U73122, impaired the release of the enzyme by the hormone and (c) two inhibitors of IPG uptake, inositol 2-monophosphate and 2 M NaCl, increased the amount of alkaline phosphatase in the extracellular medium. These results suggest that ACTH releases alkaline phosphatase by activation of a phospholipase C. Dibutyryladenosine-3',5'-cyclic monophosphate (db-cAMP) was able to increase the release of alkaline phosphatase from adrenocortical cells and this effect was inhibited by U73122, suggesting that cAMP is involved in the activation of phospholipase C. In addition, it was found that a pertussis-toxin sensitive G-protein is required for ACTH- and db-cAMP-mediated release of alkaline phosphatase and that incorporation of anti-Gi antibodies in adrenocortical cells inhibited the release of alkaline phosphatase by ACTH. Our results suggest that ACTH increases the release of alkaline phosphatase by activation of a phospholipase C through cAMP and Gi which would contribute to produce IPG It was also found that the two inhibitors of IPG uptake, inositol-2-monophosphate and 2 M NaCl, increased the amount of alkaline phosphatase in the extracellular medium of ACTH-treated cells more than in control cells, indicating that ACTH also stimulates the uptake of IPG These data support a role of GPI and the involvement of Gi in ACTH action.     

Synthesis of an hydrophilic, pK 8.05 buffer for isoelectric focusing in immobilized pH gradients

The synthesis of a new, pK 8.05 acrylamido weak base for isoelectric focusing in immobilized pH gradients (IPG) is here reported. This compound N,N-bis(2-hydroxyethyl)-N'-acryloyl-1,3-diaminopropane is strongly hydrophilic, and thus inhibits any potential hydrophobic interaction among proteins and the grafted basic groups in an IPG matrix. In addition, this novel buffer represents a step ahead towards the goal of closing the 'gap' between the commercially available Immobilines, pK 7.0 and 8.5. Owing to the large distance between these two neighboring pK values, it is difficult to arrange for linear narrow pH gradients in this region. IPG compositions obtained with this new buffer give highly linear pH gradients and protein profiles identical to those obtained with commercial Immobilines.     

Soft immobilized pH gradient gels in proteome analysis: a follow-up

As a follow-up of a previous work on two-dimensional map analysis utilizing soft (< 4%T) immobilized pH gradient (IPG) matrices in the first dimension (Candiano et al., Electrophoresis 2002, 23, 292-297), we have further optimized the preparation of such dilute IPG gels. One important step for obtaining an even reswelling of the entire IPG strip along the pH 3-10 interval is a washing step in 100 mM citric acid. It appears as though after rinsing off the excess acid in distilled water, a gradient of this tricarboxylic acid remains trapped into the IPG matrix, from almost nil at the acidic gel region to substantially higher amounts in its basic counterpart. This gradient helps in obtaining a uniform reswelling of the IPG strip, since carboxyl groups are more heavily hydrated than amino groups. The combined effects of uniform reswelling and of diluting the gel matrix favor penetration of large macromolecules (> 200 kDa) and allow for better spot resolution and for the display of a substantially higher number of spots also in the 30-60 000 Da region. A delipidation step in tri-n-butylphosphate:acetone:methanol (1:12:1) appears to substantially improve spot focusing and greatly diminish streaking and smearing of spots in all regions of the pH gradient.     

Organic Acid Metabolism in Aluminum-Phosphate Utilizing Cells of Carrot (Daucus carota L.)

Carbohydrate metabolism in Al-phosphate utilizing cells of carrot[designated as IPG, Koyama et al. (1992) Plant Cell Physiol.33: 171], which grow normally in Al-phosphate medium accompaniedby citrate excretion, was investigated. The excretion of citratewas strongly related to the availability of sucrose in medium,indicating that citrate excretion was severely limited by sucrosein medium. The ratio of the amount of carbon in the excretedcitrate to the consumed sucrose, was significantly higher inIPG cells than in wild-type cells. When 50% of the sucrose inthe medium was consumed, the ratio was 0.6% for the IPG cellsand 0.2% the wild-type cells. Under these conditions, IPG cellsshowed altered citrate synthesis metabolism, which resultedin increased citrate production. Specific activity of mitochondrialcitrate synthase was higher in IPG cells than in wild-type cells,whereas the activity of cytosolic NADP-specific isocitrate dehydrogenasewas lower in IPG cells than in wild-type cells. (Received August 27, 1998; Accepted February 21, 1999)     

G-electrode-loading method for isoelectric focusing, enabling separation of low-abundance and high-molecular-mass proteins

The G-electrode-loading method (GELM) is a technique enabling a large number of proteins from rat liver to enter an immobilized pH gradient (IPG) gel strip for isoelectric focusing (IEF). In this method, three slips containing the sample solution are placed on the cathodic edge of an IPG gel strip and a slip containing Chaps solution, a filtration membrane, and an electrode slip are placed on top. Finally, a G-electrode is placed on these slips. The Chaps solution (an amphoteric compound) is supplied gently to the sample solution during IEF and helps the proteins in the sample solution to enter the IPG gel strips with a high solubilization capacity. This method was compared with traditional slip-loading and in-gel rehydration, and it showed the best results for protein separation, including high-molecular-mass proteins.     

Immobilized pH gradients for isoelectric focusing. III. Preparative separations in highly diluted gels

A further improvement on the preparative aspects of immobilized pH gradients (IPG) (J. Biochem. Biophys. Methods (1983) 8, 135–172) is described, based on the use of soft (highly diluted) polyacrylamide gels. While in conventional IPGs in 5%T gels an upper load limit of 40–45 mg protein/ml gel volume is found, in 2.5%T gels, containing the same amount of Immobiline, as much as 90 mg protein/ml gel can be applied, without overloading effects. This is an extraordinary amount of material to ba carried by a gel phase, and renders IPG by far the leading technique in any electrophoretic fractionation. A new, two-step casting technique, based on the formation of a %T step and a pH plateau around the application trench, is described. A new method for electrophoretic protein recovery from IPG gel strips, based on embedding on low-gelling agarose (37°C), is reported. The physico-chemical properties of highly diluted gels, in relation to their protein loading ability, are evaluated and discussed. It is recommended that diluted gels (e.g. 3.5%T) be used also in analytical runs, since sharper protein zones are obtained, due to the increased charge density on the polymer coil.