Slab Gel Research Articles

Toward a fluorescent single-strand conformation polymorphism technique that detects all mutations: F-DOVAM-S

Although DOVAM-S (detection of virtually all mutations–SSCP) in effect detects all mutations and is less costly than direct sequencing, the technique currently requires the use of radioactivity. F-DOVAM-S (fluorescent DOVAM-S) was developed to replace the isotopic label with fluorescence and to increase throughput via dye color multiplexing. As proof of principle, two multitemperature slab gel electrophoresis conditions were evaluated through the blinded analysis of mutations in the factor IX (FIX) genes of 88 hemophilia B (HB) patients and 7 wild-type controls. Using only two conditions, it was determined that F-DOVAM-S had a detection sensitivity of 97%. It is anticipated that when three or four optimized conditions are employed, F-DOVAM-S will detect all mutations. Three patient samples were multiplexed per well using three different fluorescent dyes (6FAM, VIC, and NED), demonstrating that it is possible to analyze up to 44 kb of diploid, color-coded amplification product per gel lane. This value corresponds to a throughput of approximately 4 Mb of DNA analyzed per 96-well gel, which is approximately triple that of conventional radiolabeled DOVAM-S. Throughput is further enhanced by the rapidity at which the fluorescent signal can be captured and the resultant multicolor chromatograms analyzed. Given these data, F-DOVAM-S has the potential to be a particularly powerful technology for clinical diagnosis because it allows the mutation analysis of multiple patients to be performed within 24 h.

Fast molecular diagnostics of canine T-cell lymphoma by PCR and capillary gel electrophoresis with laser-induced fluorescence detector

Lymphoma is the most common hematopoietic tumor in dogs and manifests as a proliferation of malignant lymphoid cells primarily affecting the lymph nodes or solid visceral organs. We describe the use of capillary gel electrophoresis (CGE) with a laser-induced fluorescence (LIF) detector based on polymerase chain reaction (PCR) to rapidly detect a disorder of the canine T-cell receptor γ (TCRγ) gene. After the PCR amplification of the specific TCR( gene in dogs, the 90-bp DNA fragment amplified was separated in a fused-silica capillary by CGE–LIF. Under an electric field of 375 V/cm and with a sieving matrix of 1.5% poly (ethyleneoxide) ( M r 600,000), the amplified PCR products were analyzed within 4 min by CGE separation. When the CGE–LIF method was applied to real clinical samples of the specific DNA fragment of the TCR( gene, the migration time and the corrected peak area showed relative standard deviations ( n = 5) of 0.29% and 0.58%, respectively. Both methods of CGE–LIF and slab gel electrophoresis showed same results for nine clinical samples. This PCR/CGE–LIF technique may prove to be a new fast and simple tool for the rapid diagnosis of the PCR-amplified DNA of canine T-cell lymphoma.

CIEF with hydrodynamic and chemical mobilization for the separation of forms of α‐1‐acid glycoprotein

Alpha-1-acid glycoprotein (AGP) is a protein that exists in different forms, which is due to variations in the amino acid sequence and/or in the glycosidic part of the protein. These differences confer to these forms, among other characteristics, diverse pIs. Changes in these forms of AGP have been correlated to modifications of the pathophysiological conditions of the individuals. One of the analytical techniques employed for their study has been IEF performed in slab gels. CIEF method with hydrodynamic and chemical mobilization, involving an isotachophoretic process, is developed in this work to separate up to 12 bands of forms of standard AGP, which is proposed as a more reproducible, quantitative, less sample-consuming, and more automated one than conventional IEF. The challenge of this work has been the development of a CIEF method for the separation of bands of a very acidic protein (pI range: 1.8-3.8) in a capillary. Intraday RSD values < or = 1.7% have been achieved for the relative migration time of the AGP bands to that of an internal standard. For intraday area precision, RSD (%) in the range of 2.70-22.71% for AGP zones accounting for more than 10% of total area of AGP sample has been obtained. As a proof of the potential of the methodology proposed, an AGP sample purified from a pool of sera of patients suffering from ovary cancer is analyzed by CIEF.

Purification and Characterization of Three Galactose Specific Lectins from Sajna (Moringa Oleifera L.) Leaves

AbstractThree lectins designated as SLL‐1, SLL‐2 and SLL‐3 were purified from small sized Sajna (Moringa oleifera L.) leaves by gel filtration of 100% ammonium sulfate saturated crude protein extract on Sephadex G‐75 followed by ion‐exchange chromatography on DEAE and affinity chromatography on Sepharose‐4B. The molecular weight of the lectins SLL‐1, SLL‐2 and SLL‐3 were 1,55,000, 1,15,000 and 85,000, respectively, as determined by gel filtration on Sephadex G‐150 and 1,60,000; 1,20,000 and 85,500, respectively, by SDS‐polyacrylamide slab gel electrophoresis. SLL‐1 and SLL‐2 are dimer in nature held together by disulfide bond (s), while SLL‐3 is a monomer. The lectins agglutinated specifically rat red blood cells and the agglutination was inhibited specifically by methyl‐α‐D‐galactopyranoside, methyl‐β‐D‐galactopyranoside and D‐galactose. The lectins SLL‐1, SLL‐2 and SLL‐3 contain 3.9, 3.4 and 2.8% neutral sugar, respectively, and the sugar compositions were glucose for SLL‐1, mannose for SLL‐2 and SLL‐3 contained either N‐acetyl‐D‐glucosamine or N‐acetyl‐D‐galactosamine or both. The lectins exhibited cytotoxicity in brine shrimp lethality bioassay.

Comparative 2-DE Protein Analysis in a 3-D Geometry Gel

Two-dimensional gel electrophoresis (2-DE) separation has not been considered suitable for large-scale comparative protein expression studies due to its limited throughput. We present a high-throughput analysis method based on three-dimensional (3-D) geometry gel electrophoresis. Following conventional isoelectric focusing (IEF), up to 36 immobilized pH gradient (IPG) strips are arrayed on the top surface of a 3-D gel body, and the samples transferred electrokinetically to the gel. A specific thermal management ensures that sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) occurs under identical electrophoretic and thermal conditions, avoiding gel-to-gel variations and thereby providing immediate comparability of the separation patterns. Proteins are Cy3-labeled for online detection of laser-induced fluorescence (LIF). Images are acquired by a digital camera and recorded as a 3-D image stack during electrophoresis. Image processing software decomposes the 3-D image stack into vertical sections representing conventional 2-DE slab gels, making results immediately accessible without further gel processing. The large number of simultaneously analyzed samples (n = 36) allows treating the sample index as a quasi-continuous experimental parameter (e.g., concentration, time, dose). The method offers a wide range of applications in molecular discovery, clinical diagnosis, pharmacology, and toxicology, like protein monitoring during disease development and screening of drug candidates for their effect on protein expression.

Kinetics of binding and geometry of cells on molecular biochips

We examine how the shape of cells and the geometry of experiment affect the reaction–diffusion kinetics at the binding between target and probe molecules on molecular biochips. In particular, we compare the binding kinetics for the probes immobilized on surface of the hemispherical and flat circular cells, the limit of thin slab of analyte solution over probe cell as well as hemispherical gel pads and cells printed in gel slab over a substrate. It is shown that hemispherical geometry provides significantly faster binding kinetics and ensures more spatially homogeneous distribution of local (from a pixel) signals over a cell in the transient regime. The advantage of using thin slabs with small volume of analyte solution may be hampered by the much longer binding kinetics needing the auxiliary mixing devices. Our analysis proves that the shape of cells and the geometry of experiment should be included to the list of essential factors at biochip designing.

Shotgun electroelution: A proteomic tool for simultaneous sample elution from whole SDS‐polyacrylamide gel slabs

A high-throughput device has been constructed which allows parallel electroelution of separated SDS-protein bands directly from intact unsectioned polyacrylamide gel slabs as well as single electroelution of certain protein spots into a 384-well standard flat-bottom multiwell plate. The prototype provides complete, quick elution for proteomics from 1-D or from 2-D gels without gel sectioning. Since the elution chamber matrix requires no assembly, sample handling can be easily carried out by existing robotic workstations. The current design is a good candidate for automation of spot elution since there are no moving liquid containing components in the apparatus. Eight SDS-proteins were eluted in test runs and an average 70% sample recovery was achieved by re-electrophoresis of the electro-eluates.

Molecular Cloning and Characterization of a Novel β-Agarase, AgaB, from Marine Pseudoalteromonas sp. CY24

Agarases are generally classified into glycoside hydrolase families 16, 50, and 86 and are found to degrade agarose to frequently generate neoagarobiose, neoagarotetraose, or neoagarohexaose as the main products. In this study we have cloned a novel endo-type beta-agarase gene, agaB, from marine Pseudoalteromonas sp. CY24. The novel agarase encoded by agaB gene has no significant sequence similarity with any known proteins including all glycoside hydrolases. It degrades agarose to generate neoagarooctaose and neoagarodecaose as the main end products. Based on the analyses of enzymatic kinetics and degradation patterns of different oligosaccharides, the agarase AgaB appears to have a large substrate binding cleft that accommodates 12 sugar units, with 8 sugar units toward the reducing end spanning subsites +1 to +8 and 4 sugar units toward the non-reducing end spanning subsites -4 to -1, and enzymatic cleavage taking place between subsites -1 and +1. In addition, 1H NMR analysis shows that this enzyme hydrolyzes the glycosidic bond with inversion of anomeric configuration, in contrast to other known agarases that are retaining. Altogether, AgaB is structurally and functionally different from other known agarases and appears to represent a new family of glycoside hydrolase.

Toward an integrated microchip sized 2‐D polyacrylamide slab gel electrophoresis device for proteomic analysis

We describe a miniaturized instrument capable of performing 2-DE. Our miniaturized device is able to perform IEF and polyacrylamide slab gel electrophoresis (PASGE) in the same unit. It consists of a compartment for a first-dimensional IEF gel, which is connected to a second-dimensional PASGE gel. The focused samples are automatically transferred from the IEF gel to the PASGE gel by electromigration. Our preliminary experiments show that the device is able to focus and separate a mixture of proteins in approximately 1 h, excluding the time required for the staining procedure. On average, the gel-to-gel retardation factor (Rf) variation was 6.2% (+/-0.9%) and pI variation was 2.5% (+/-0.6%). Separated protein spots were excised from stained gels, digested with trypsin, and further identified by MS, thus enabling direct proteomic analysis of the separated proteins.

Circulating polymerase chain reaction chips utilizing multiple-membrane activation**The preliminary results of the current paper were presented at the 19th IEEE International Conference on Micro Electro Mechanical Systems (IEEE MEMS 2006), Istanbul, Turkey, 22–26 January, 2006.

This paper reports a new micromachined, circulating, polymerase chain reaction (PCR) chip for nucleic acid amplification. The PCR chip is comprised of a microthermal control module and a polydimethylsiloxane (PDMS)-based microfluidic control module. The microthermal control modules are formed with three individual heating and temperature-sensing sections, each modulating a specific set temperature for denaturation, annealing and extension processes, respectively. Micro-pneumatic valves and multiple-membrane activations are used to form the microfluidic control module to transport sample fluids through three reaction regions. Compared with other PCR chips, the new chip is more compact in size, requires less time for heating and cooling processes, and has the capability to randomly adjust time ratios and cycle numbers depending on the PCR process. Experimental results showed that detection genes for two pathogens, Streptococcus pyogenes (S. pyogenes, 777 bps) and Streptococcus pneumoniae (S. pneumoniae, 273 bps), can be successfully amplified using the new circulating PCR chip. The minimum number of thermal cycles to amplify the DNA-based S. pyogenes for slab gel electrophoresis is 20 cycles with an initial concentration of 42.5 pg µl−1. Experimental data also revealed that a high reproducibility up to 98% could be achieved if the initial template concentration of the S. pyogenes was higher than 4 pg µl−1.

Sunset Yellow: A Food Color for Protein Staining with SDS-PAGE

This study shows that Sunset Yellow FCF is a fast-reacting dye for food proteins and may be used as a fast staining agent for all types of resolved proteins with PAGE, which is important because Coomassie Brilliant Blue R 250 and Amido Black 10 B take at least 24 hr for visualization of proteins bands with PAGE. Sunset Yellow FCF is highly sensitive, rapid, lasting, and provides reproducible results with a variety of proteins. It produces sharp, nondiffuseable, clear, distinct, and stable bands on gel. It requires just 1 hr for staining and destaining to visualize the known proteins separated on the acrylamide gels. intestinal anaerobes (5). The dye may be purified by a chemometric method (6) and, even in its crude form, is equally effective for protein staining on acrylamide and for use after blotting (7). Apart from Coomassie Brilliant Blue, the other standard protocols of dyes include silver and gold staining (8–10) and Amido Black 10 B (11); iodine is less commonly used (12). Although the spectrophotometric determination and estimation of Sunset Yellow FCF at 485 nm is common, capillary electrophoresis has recently been used as a sophisticated technique to estimate colors in foods, including this dye in beverages (13,14). The nanomaterials in foods, such as micronutrients and synthetic colors like Sunset Yellow FCF, may be analyzed by the above separation technique. The dyebinding assays are widely used in protein analysis. Our data show that Sunset Yellow FCF is a sensitive, reliable, and competent dye for binding proteins to make them visible with PAGE. MATERIALS AND METHODS Chemicals N,N′-methylene-bis-acrylamide was purchased from Scharlau, while Tris (hydroxymethyl) aminomethane was obtained from Research Organics. Sodium dodecyl sulfate (SDS), acrylamide, ammonium peroxodisulfate (APS), glycine, Coomassie Brilliant Blue, bovine serum albumin (BSA), N,N,N′,N′-tetramethylenediamine (TEMED), bromophenol blue, and trypsin were supplied from Merck, while nisin was purchased from Suzhou Hengliang Co. Ltd., Suzhou, China. The 2-mercaptoethanol and HCl were supplied from Riedel-deHaen, Seeize, Germany. Sunset Yellow FCF No. 6 was a gift from coauthor Asad Sayeed (National Foods Ltd., Karachi, Pakistan). Glycerol and takadiastase from Aspergillus oryzae were purchased from Fluka, Steinhein, Denmark. Preparation of Slab Gel The following solutions were first prepared. For solution A, acrylamide (30 g) and bis-acrylamide (0.80 g) were dissolved in 100 mL of double-distilled, deionized water and filtered through Whatman No. 1 filter paper. For solution B, Tris-HCl buffer (1.5M) was prepared by mixing 18.02 g of Tris dissolved in 80 mL of double-distilled, deionized water, and the pH was adjusted to 8.8 by using 1M HCl; the volume was made up to 100 mL. For solution C, Tris (12.114 g) was added to 80 mL of doubledistilled, deionized water; the pH was adjusted to 6.8 with 1M HCl; and the volume was made up to 100 mL with double-distilled, deionized water. For solution D, SDS (10 g) was dissolved in double-distilled, deionized water, and the volume was made up to 100 mL. For solution E, APS (1 g) was dissolved in double-distilled, deionized water, and the volume was made up to 10 mL, while TEMED was used as supplied. The 12.5% slab gel (thickness 0.75 mm) was prepared by using 2.083 mL of solution A, 0.666 mL of solution B, 0.500 mL of solution D, and 0.250 mL of solution E. The mixture was degassed, and 1.66 μL of TEMED was added before use. The gel was left for 15 min to settle. Staking Gel Solutions A, C, D, and E were added in the ratio of 0.416:0.830:0.333:0.166, respectively. The mixture was immediately degassed, and 1.66 μL of TEMED was added before the stacking gel was poured. The wells were prewashed with reservoir buffer after the gel had settled. For the reservoir buffer, Tris (0.9 g), glycine (3.6 g), RESEARCH

On-chip pumping for pressure mobilization of the focused zones following microchip isoelectric focusing

Isoelectric focusing (IEF), traditionally accomplished in slab or tube gels, has also been performed extensively in capillary and, more recently, in microchip formats. IEF separations performed in microchips typically use electroosmotic flow (EOF) or chemical treatment to mobilize the focused zones past the detection point. This report describes the development and optimization of a microchip IEF method in a hybrid PDMS-glass device capable of controlling the mobilization of the focused zones past the detector using on-chip diaphragm pumping. The microchip design consisted of a glass fluid layer (separation channels), a PDMS layer and a glass valve layer (pressure connections and valve seats). Pressure mobilization was achieved on-chip using a diaphragm pump consisting of a series of reversible elastomeric valves, where a central diaphragm valve determined the volume of solution displaced while the gate valves on either side imparted directionality. The pumping rate could be adjusted to control the mobilization flow rate by varying the actuation times and pressure applied to the PDMS to actuate the valves. In order to compare the separation obtained using the chip with that obtained in a capillary, a serpentine channel design was used to match the separation length of the capillary, thereby evaluating the effect of diaphragm pumping itself on the overall separation quality. The optimized mIEF method was applied to the separation of labeled amino acids.

The Hemolymph of the Ascidian Styela plicata (Chordata-Tunicata) Contains Heparin inside Basophil-like Cells and a Unique Sulfated Galactoglucan in the Plasma

The hemolymph of ascidians (Chordata-Tunicata) contains different types of hemocytes embedded in a liquid plasma. In the present study, heparin and a sulfated heteropolysaccharide were purified from the hemolymph of the ascidian Styela plicata. The heteropolysaccharide occurs free in the plasma, is composed of glucose ( approximately 60%) and galactose ( approximately 40%), and is highly sulfated. Heparin, on the other hand, occurs in the hemocytes, and high performance liquid chromatography of the products formed by degradation with specific lyases revealed that it is composed mainly by the disaccharides DeltaUA(2SO(4))-1-->4-beta-d-GlcN(SO(4)) (39.7%) and DeltaUA(2SO(4))-1-->4-beta-d-GlcN(SO(4))(6SO(4)) (38.2%). Small amounts of the 3-O-sulfated disaccharides DeltaUA(2SO(4))-1-->4-beta-d-GlcN(SO(4))(3SO(4)) (9.8%) and DeltaUA(2SO(4))-1-->4-beta-d-GlcN(SO(4))(3SO(4))(6SO(4)) (3.8%) were also detected. These 3-O-sulfated disaccharides were demonstrated to be essential for the binding of the hemocyte heparin to antithrombin III. Electron microscopy techniques were used to characterize the ultrastructure of the hemocytes and to localize heparin and histamine in these cells. At least five cell types were recognized and classified as univacuolated and multivacuolated cells, amebocytes, hemoblasts, and granulocytes. Immunocytochemistry showed that heparin and histamine co-localize in intracellular granules of only one type of hemocyte, the granulocyte. These results show for the first time that in ascidians, a sulfated galactoglucan circulates free in the plasma, and heparin occurs as an intracellular product of a circulating basophil-like cell.

Allozyme Diversity and Population Genetic Structure of Three Medicinal Epimedium Species from Hubei

Three Epimedium species, E. pubescens Maxim., E. sagittatum (Sieb. & Zucc.) Maxim., and E. wushanense T. S. Ying, which are sympatrically distributed in the western Hubei Province, have been used in traditional Chinese medicine (TCM) for about 2,000 years. Genetic variability and population genetic structure of 11 natural populations of these Epimedium species were investigated using isoelectric focusing in thin-layer polyacrylamide slab gels. Of the 22 enzyme systems prescreened, six coding for 13 loci and 45 alleles were resolved, which were used for analyzing genetic diversity and population structure at both intraspecific and interspecific levels. The results showed that: 1) high levels of genetic diversity were observed in all three species ( A = 2.6-3.2, P = 69.2%-84.6%, H O = 0.274-0.377, H E = 0.282-0.369), which were higher than that of other herbaceous and animal-pollinated species with similar life-history characteristics; 2) there was significant deviation from Hardy-Weinberg Equilibrium, with one half of the loci showing heterozygote excess and the other homozygote excess, in all populations, suggesting the complicated breeding system of Epimedium species; 3) the low level of intraspecific and interspecific genetic differentiation ( G ST = 0.0246-0.0409 and 0.0495-0.1213, respectively) indicated a high level of gene flow among populations and close genetic relationship among the three species; and 4) UPGMA cluster analysis further showed that E. pubescens was more closely related to E. sagittatum than to E. wushanense, which was in good agreement with the morphological characters and the recent phylogenetic analysis of these species. On the basis of these results, it was concluded that the mixed breeding system, long-lived perennial life form, ancient evolutionary history, and seed dispersal by ants in Epimedium are responsible for the genetic variation and population structure of these species.

Integration of isoelectric focusing with multi-channel gel electrophoresis by using microfluidic pseudo-valves

Two-dimensional (2D) protein separation is achieved in a plastic microfluidic device by integrating isoelectric focusing (IEF) with multi-channel polyacrylamide gel electrophoresis (PAGE). IEF (the first dimension) is carried out in a 15 mm-long channel while PAGE (the second dimension) is in 29 parallel channels of 65 mm length that are orthogonal to the IEF channel. An array of microfluidic pseudo-valves is created for introducing different separation media, without cross-contamination, in both dimensions; it also allows transfer of proteins from the first to the second dimension. Fabrication of pseudo-valves is achieved by photo-initiated, in situ gel polymerization; acrylamide and methylenebisacrylamide monomers are polymerized only in the PAGE channels whereas polymerization does not take place in the IEF channel where a mask is placed to block the UV light. IEF separation medium, carrier ampholytes, can then be introduced into the IEF channel. The presence of gel pseudo-valves does not affect the performance of IEF or PAGE when they are investigated separately. Detection in the device is achieved by using a laser induced fluorescence imaging system. Four fluorescently-labeled proteins with either similar pI values or close molecular weight are well separated, demonstrating the potential of the 2D electrophoresis device. The total separation time is less than 10 minutes for IEF and PAGE, an improvement of 2 orders of magnitude over the conventional 2D slab gel electrophoresis.

A typical N-terminal Extensions Confer Novel Regulatory Properties on GTP Cyclohydrolase Isoforms in Drosophila melanogaster

The cofactor tetrahydrobiopterin plays critical roles in the modulation of the signaling molecules dopamine, serotonin, and nitric oxide. Deficits in cofactor synthesis have been associated with several human hereditary diseases. Responsibility for the regulation of cofactor pools resides with the first enzyme in its biosynthetic pathway, GTP cyclohydrolase I. Because organisms must be able to rapidly respond to environmental and developmental cues to adjust output of these signaling molecules, complex regulatory mechanisms are vital for signal modulation. Mammalian GTP cyclohydrolase is subject to end-product inhibition via an associated regulatory protein and to positive regulation via phosphorylation, although target residues are unknown. GTP cyclohydrolase is composed of a highly conserved homodecameric catalytic core and non-conserved N-terminal domains proposed to be regulatory sites. We demonstrate for the first time in any organism that the N-terminal arms of the protein serve regulatory functions. We identify two different modes of regulation of the enzyme mediated through the N-terminal domains. The first is end-product feedback inhibition, catalytically similar to that of the mammalian enzyme, except that feedback inhibition by the cofactor requires sequences in the N-terminal arms rather than a separate regulatory protein. The second is a novel inhibitory interaction between the N-terminal arms and the active sites, which can be alleviated through the phosphorylation of serine residues within the N termini. Both mechanisms allow for acute and highly responsive regulation of cofactor production as required by downstream signaling pathways.

Decorin Modulates Fibrin Assembly and Structure

Emerging evidence indicates that fibrin clotting is regulated by different external factors. We demonstrated recently that decorin, a regulator of collagen fibrillogenesis and transforming growth factor-beta activity, binds to the D regions of fibrinogen (Dugan, T.A., Yang, V. W.-C., McQuillan, D.J., and Höök, M. (2003) J. Biol. Chem. 278, 13655-13662). We now report that the decorin-fibrinogen interaction alters the assembly, structure, and clearance of fibrin fibers. Relative to fibrinogen, substoichiometric amounts of decorin core protein modulated clotting, whereas an excess of an active decorin peptide was necessary for similar activity. These concentration-dependent effects suggest that decorin bound to the D regions sterically modulates fibrin assembly. Scanning electron microscopy images of fibrin clotted in the presence of increasing concentrations of decorin core protein showed progressively decreasing fiber diameter. The sequestration of Zn(2+) ions from the N-terminal fibrinogen-binding region abrogated decorin incorporation into the fibrin network. Compared with linear thicker fibrin fibers, the curving thin fibers formed with decorin underwent accelerated tissue-type plasminogen activator-dependent fibrinolysis. Collectively, these data demonstrate that decorin can regulate fibrin organization and reveal a novel mechanism by which extracellular matrix components can participate in hemostasis, thrombosis, and wound repair.

Quantitative‐competitive polymerase chain reaction coupled with slab gel and capillary electrophoresis for the detection of roundup ready soybean and maize

The aim of the present study was to develop a quantitative-competitive PCR (QC-PCR) method to detect DNA from transgenic herbicide-resistant (roundup ready, RR) soybean and maize. Since no QC-PCR system for the quantification of RR maize had been published at the time of writing, a specific competitor DNA for transgenic event was developed. For the QC-PCR of RR-soybean, a commercially available competitor was employed. These internal standards were calibrated by coamplifying with mixtures containing RR-soybean and maize DNAs. The calibrated QC-PCR systems were applied to certified RR-soybean and maize flour mixtures in order to demonstrate their suitability not only for the quantification of the glyphosate resistance traits in DNA matrices, but also in practically relevant samples. In addition, a special focus of the present work was to compare the detection of QC-PCR products by slab gel and CGE with UV detection. CGE permitted the precise detection of transgenic events also below the equivalence points; while in slab gel electrophoresis, due to the low sensitivity the quantification of genetically modified DNA was allowed only at the equivalence point.

Both Cleavage Products of the mCLCA3 Protein Are Secreted Soluble Proteins

Members of the chloride channels, calcium-activated (CLCA) family of proteins and in particular the murine mCLCA3 (alias gob-5) and its human ortholog hCLCA1 have been identified as clinically relevant molecules in diseases with secretory dysfunctions including asthma and cystic fibrosis. Initial studies have indicated that these proteins evoke a calcium-activated chloride conductance when transfected into human embryonic kidney cells 293 cells. However, it is not yet clear whether the CLCA proteins form chloride channels per se or function as mediators of other, yet unknown chloride channels. Here, we present a systematic biochemical analysis of the posttranslational processing and intracellular trafficking of the mCLCA3 protein. Pulse-chase experiments after metabolic protein labeling of mCLCA3-transfected COS-1 or human embryonic kidney 293 cells revealed cleavage of a primary 110-kDa mCLCA3 translation product in the endoplasmic reticulum into a 75-kDa amino-terminal and a 35-kDa carboxyl-terminal protein that were glycosylated and remained physically associated with each other. Confocal fluorescent analyses identified both cleavage products in vesicles of the secretory pathway. Neither cleavage product was associated with the cell membrane at any time. Instead, both subunits were fully secreted into the extracellular environment as a soluble complex of two glycoproteins. These results suggest that the two mCLCA3 cleavage products cannot form an anion channel on their own but may instead act as extracellular signaling molecules. Furthermore, our results point toward significant structural differences between mCLCA3 and its human ortholog, hCLCA1, which is thought to be a single, non-integral membrane protein.

Mouse Ornithine Decarboxylase-like Gene Encodes an Antizyme Inhibitor Devoid of Ornithine and Arginine Decarboxylating Activity

Ornithine decarboxylase (ODC), a key enzyme in the biosynthesis of polyamines, is a labile protein that is regulated by interacting with antizymes (AZs), a family of polyamine-induced proteins. Recently, a novel human gene highly homologous to ODC, termed ODC-like or ODC-paralogue (ODCp), was cloned, but the studies aimed to determine its function rendered contradictory results. We have cloned the mouse orthologue of human ODCp and studied its expression and possible function. mRNA of mouse Odcp was found in the brain and testes, showing a conserved expression pattern with regard to the human gene. Transfection of mouse Odcp in HEK 293T cells elicited an increase in ODC activity, but no signs of arginine decarboxylase activity were evident. On the other hand, whereas the ODCp protein was mainly localized in the mitochondrial/membrane fraction, ODC activity was found in the cytosolic fraction and was markedly decreased by small interfering RNA against human ODC. Co-transfection experiments with combinations of Odc, Az1, Az2, Az3, antizyme inhibitor (Azi), and Odcp genes showed that ODCp mimics the action of AZI, rescuing ODC from the effects of AZs and prevented ODC degradation by the proteasome. A direct interaction between ODCp and AZs was detected by immunoprecipitation experiments. We conclude that mouse ODCp has no intrinsic decarboxylase activity, but it acts as a novel antizyme inhibitory protein (AZI2).