Two-dimensional SDS-polyacrylamide Gel Research Articles

The intermediate filament cytoskeleton of cutaneous neuroendocrine carcinoma (Merkel cell tumour). Immunohistochemical and biochemical analyses.

The presence and distribution of cytokeratins, neurofilament proteins, vimentin and glial fibrillary acidic protein were studied in 10 cutaneous neuroendocrine carcinomas (CNEC) by immunohistochemical techniques, using specific antibodies. In all cases tumour cells were specifically stained with antibodies to mouse liver cytokeratin component D in paraffin-embedded formalin-fixed and frozen sections. Moreover, one- and two-dimensional SDS-polyacrylamide gel electrophoresis of high salt/detergent resistant cytoskeletal residues from tumour material, isolated by microdissection from frozen sections, revealed the presence of cytokeratin components 8 and 18 which are characteristic constituents of cytokeratin filaments of simple epithelia. Neurofilament proteins were detected by immunocytochemistry in tumour cells from 2 patients, from whom frozen material was available, and their presence was also positively identified in cytoskeletal residues by immunoblotting using specific antibodies. Glial fibrillary acidic protein and vimentin could not be demonstrated in tumour cells. Our studies did not confirm the suggested origin of CNEC from epidermal Merkel cells.

Characterization of whole-cell and organelle protein synthesis in normal and aposymbiotic strains of Crithidia oncopelti and Blastocrithidia culicis

1. 1. Normal (bacterial endosymbiont-containing) and aposymbiotic strains of Crithidia oncopelti and Blastocrithidia culicis were metabolically labelled with [ 3H]leucine and [ 35S]methionine. 2. 2. Autoradiography of labelled proteins separated by one- or two-dimensional SDS-polyacrylamide gel electrophoresis failed to detect differences between normal and aposymbiotic strains, although interspecific differences were evident. 3. 3. Whole cells and symbiont and mitochondrial fractions were separated by differential centrifugation; each synthesized a distinct and different array of electrophoretically separable, [ 35S]methionine-labelled proteins. 4. 4. Proteins synthesized by the host cell were eliminated by cycloheximide inhibition but were unchanged in the presence of chloramphenicol; proteins synthesized by the symbiont and mitochondrial fractions were eliminated by chloramphenicol but not by cycloheximide.

Molecular cloning and biosynthetic regulation of cry1 gene of Saccharomyces cerevisiae.

The cryptopleurine resistance gene, cry1, of Saccharomyces cerevisiae has been molecularly cloned using genetic complementation of cryptopleurine sensitivity by the cryptopleurine resistance gene contained in a clone library prepared from DNA of a cryptopleurine resistant strain. Analysis of RNA transcripts indicated that the cry1 gene is the template for a transcript of approximately 900 bases and that the primary transcript contains an intron of approximately 300 bases. In vitro hybrid selection translation experiments indicated that this transcript encodes a protein of molecular weight 17 kilodaltons which on two-dimensional SDS polyacrylamide gels exactly coincides with ribosomal protein rp59. Further analysis showed that when the gene was present on a plasmid of about five copies per cell the amount of messenger RNA was elevated approximately five-fold compared to a cell that had only a single chromosomal copy. The rate of synthesis of ribosomal protein rp59 was not detectably elevated. These data suggest that the cry1 gene is regulated, at least in part, post-transcriptionally.

Mutant strains of Chlamydomonas reinhardtii that move backwards only.

Mutations at three independent loci in Chlamydomonas reinhardtii result in a striking alteration of cell motility. Mutant cells representing the three mbo loci move backwards only, propelled by a symmetrical "flagellar" type of bending pattern. The characteristic asymmetric "ciliary" type of flagellar bend pattern responsible for forward movement that predominates in wild-type cells is seldom seen in the mutants. This defect in motility was found to be a property of the mutant axonemes themselves: the isolated axonemes, reactivated by addition of ATP, showed exclusively the symmetrical wave form, and the protein composition of these axonemes differed from the wild-type composition. Axonemes obtained from mbo1 , mbo2 , and mbo3 cells were found to be deficient in six polypeptides regularly present in wild type. The mbo2 axonemes were deficient in two additional polypeptides. The polypeptides were identified in autoradiograms of two-dimensional SDS polyacrylamide gel electrophoretograms of 35S- or 32P-labeled axonemes. One of the six polypeptides has previously been identified; it is a component missing in a mutant deficient for inner dynein arms. Of the five axonemal polypeptides newly identified by the mbo mutants, four were shown to be present as phosphoproteins in wild-type axonemes. One of the additional polypeptides deficient in mbo2 axonemes was also shown to be phosphorylated in wild-type axonemes. Detailed ultrastructural analysis of the mbo1 flagella and the mbo1 , mbo2A , and mbo3 axonemes revealed that the mutants specifically lack the beak-like projections found within the B-tubules of outer doublets 5 and 6.

Aluminum effect on slow axonal transport: a novel impairment of neurofilament transport

Administration of aluminum (Al) produces accumulation of neurofilaments (NF), called neurofibrillary tangles (NFT), in neuronal cell bodies and proximal axonal segments. This study was undertaken to investigate whether these changes are associated with impairment of the slow axonal transport. Local administration of AlCl3 induced the formation of NFT in 90 to 100% of the rabbit hypoglossal neurons. [35S]Methionine was then administered to the hypoglossal nerve nuclei. The hypoglossal nerves were processed 18 or 28 days later for one- and two-dimensional SDS-polyacrylamide gel electrophoresis and fluorography. Labeled NF polypeptides and a polypeptide of 57 kilodaltons (Kd) were not detectable beyond the proximal 9-mm segment of the hypoglossal nerve in Al-treated rabbits 18 days after labeling, whereas they were present up to 27 mm from the medulla in controls. Tubulin and polypeptides migrating with slow component b were not significantly affected. In rabbits sacrificed 28 days after labeling, accumulation of NF subunits within the proximal 9 mm of hypoglossal nerve was less dramatic, and labeled NF were present up to 30 mm from the medulla whereas they were detectable up to 45 mm in controls. Morphological studies demonstrated the presence of enlarged axons filled with NF in the proximal 9 mm of the hypoglossal nerve. In nerve segments immediately distal, axons were markedly reduced in size and contained no NF but an apparently normal number of microtubules and other organelles. Transport of NF and of a 57-Kd polypeptide is markedly but reversibly slowed down or blocked within the proximal 9-mm segments of the hypoglossal nerve following Al administration to the hypoglossal nucleus. It is suggested that NF transport is maintained distally, resulting in lack of NF in axonal segments immediately distal to the block. Local Al intoxication provides a novel model of impairment of NF transport.

Characterization of the DNA-cellulose-binding proteins from Escherichia coli K 12

The various [ 35S]DNA-binding proteins present in lysates of Escherichia coli K 12 cells have been analyzed by means of two-dimensional SDS-polyacrylamide gel electrophoresis. The proteins were isolated by the DNA-cellulose technique and eluted by increasing concentrations of NaCl (0.15, 0.4, 0.6 and 2 M). Only 2% of the total 35S radioactivity in the lysate became bound to the DNA-cellulose column. A total of 237 polypeptides were detected and the distribution among the salt eluates were 85, 83, 40 and 29 polypeptides, respectively. The 40 major polypeptides with regard to concentrations were also identified from gels stained with a protein-specific reagent. The polypeptides could be divided into two main groups according to p I values, namely, acidic polypeptides (total number, 174) and basic polypeptides (total number, 63). The ratio between acidic and basic polypeptides descreased with increasing salt concentrations in the eluates. The majority of the basic polypeptides had molecular weights in the range 10 000–30 000, whereas the acidic polypeptides had molecular weights from 10 000 to 165 000.

Photoaffinity labelling of the serotinin carrier protein in platelets and brain synaptosomes

Azidoimipramine, a photoaffinity labelling reagent for the serotonin transport protein, was synthesized. This reagent, upon irradiation, binds covalently to brain synaptosomes preparation and to gel-filtered platelets. Two-dimensional SDS-polyacrylamide gel electrophoresis-isoelectric focussing and tritium fluorography analysis indicate that two synaptosomal proteins and four platelets proteins were labelled by [ 3H]azidoimipramine.

Analysis of the glycoprotein and protein composition of Bernard-Soulier platelets by single and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Previous reports have described conflicting results concerning the glycoprotein (GP) and protein composition of Bernard-Soulier platelets. In view of this controversy we have analyzed the platelets of four Bernard-Soulier patients using improved single and two-dimensional sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis procedures. An absence of staining for carbohydrate of membrane GP Ib was characteristic for the platelets of each patient. Major periodate-Schiff staining bands corresponding to membrane GP IIb, IIIa, and IIIb were clearly detected and their presence was confirmed by two-dimensional SDS-polyacrylamide gel electrophoresis. The protein content of the Bernard-Soulier platelets was increased two- to fourfold. However, analysis of their protein composition using 7-12% acrylamide gradient gels showed normal polypeptide profiles. Lactoperoxidase-catalyzed 125I-labeling of the Bernard-Soulier platelet surface proteins was followed by SDS-polyacrylamide gel electrophoresis and autoradiography. No labeling in the Ib position was detected whereas the other major membrane GP, including Ia and IIa, were normally located. In contrast, GP Ib was clearly detected by periodate-Schiff staining and autoradiography when normal human platelets that had been exhaustively treated with neuraminidase before the lactoperoxidase-catalyzed iodination were analysed. No abnormalities were detected in the GP patterns of membranes isolated from the patients' erythrocytes. Only a severe molecular abnormality or possible deletion of GP Ib could account for this major platelet lesion in the Bernard-Soulier syndrome.

Biochemical analysis of actin in crane-fly gonial cells: evidence for actin in spermatocytes and spermatids--but not sperm.

A biochemical assay employing DNase-I affinity chromatography, two-dimensional peptide analysis and SDS polyacrylamide gel electrophoresis was used to isolate, identify, and assess the amount of actin from gonial cells of the crane fly, Nephrotoma suturalis. Based on the analysis of cell homogenates under conditions in which all cellular actin is converted to the monomeric DNase-binding form, actin comprises approximately 1% of the total protein in homogenates of spermatocytes and spermatids. SDS gel analysis of mature sperm reveals no polypeptides with a molecular weight similar to that of actin. Under conditions that preserve native supramolecular states of actin, approximately 80% of the spermatocyte actin is in a sedimentable form whereas only approximately 30% of the spermatid actin is sedimentable. These differences could be meaningful with regard to strutural changes that occur during spermiogenesis. A comparative analysis of two-dimensional peptide maps of several radioiodinated actins reveals similarities among spermatocyte, spermatid, and human erythrocyte actins. The results suggest the general applicability of this approach to other cell types that contain limited amounts of actin.

Localization and biosynthesis of NADH-cytochrome b5 reductase, an integral membrane protein, in rat liver cells. II. Evidence that a single enzyme accounts for the activity in its various subcellular locations.

NADH-cytochrome b5 reductases of rat liver microsomes, mitochondria, and heavy and light Golgi fractions (GF3 and GF 1+2) were compared by antibody inhibition and competition experiments, by peptide mapping, and by CNBr fragment analysis. The water-soluble portion of the microsomal enzyme, released by lysosomal digestion and purified by a published procedure, was used to raise antibodies in rabbits. Contaminant antimicrosome antibodies were removed from immune sera by immunoadsorption onto the purified antigen, and the F(ab')2 fragments of the pure antireductase antibody thus obtained were found to inhibit the NADH-cytochrome c reductase activity equally well in the four membrane fractions investigated, with similar dose-response relationships. Moreover, the purified water-soluble fragment of microsomal reductase, which by itself is very inefficient in reducing cytochrome c, competed for antibody binding with the membrane-bound enzymes, and therefore prevented the inhibition of their activity not only in microsomes but also in the other fractions. The reductases isolated from detergent-solubilized microsomes, mitochondria, GF3, and GF1+2 by immunoadsorption had identical mobilities in SDS polyacrylamide gels. The corresponding bands were eluted from gels, fragmented with pepsin or CNBr treatment, and the two families of peptides thus obtained were analyzed by two-dimensional mapping and SDS polyacrylamide gel electrophoresis, respectively. Both analyses failed to reveal differences among reductases of the four fractions. These findings support the hypothesis that NADH-cytochrome b5 reductase in its various subcellular locations is molecularly identical.

Location of post-translational cleavage events within F and HN glycoproteins of Newcastle disease virus.

The biologically active form of the fusion glycoprotein F from Newcastle disease virus (NDV) comprises two polypeptides, F1 and F2 (derived from a precursor polypeptide F0 by a post translational cleavage event), which are covalently linked together (F1,2) by disulphide bonds. This feature was exploited in a two-dimensional SDS-polyacrylamide gel electrophoretic analysis to orientate the position of the cleavage event within F0. Separation of proteins from NDV-infected CEF in the first dimension in the absence of reducing agent resolved F1,2 protein from all NDV-induced proteins other than F0. Reduction of the first dimension gel with 2-mercaptoethanol, followed by electrophoresis in the second dimension, resolved F1 (55K), F2 (12.5K) and F0 (64K) proteins. The only polypeptides other than F1 and F2 which fell below the diagonal, indicating the positions of the polypeptides from infected cells, were two minor glycoproteins designated HN1 (51.5K) and HN2 (27.5K) which took up positions vertically beneath the major haemagglutinin-neuraminidase glycoprotein HN (75K). Dual isotope labelling experiments with NDV-infected chick embryo fibroblasts, which had previously received a salt shock to effect synchronization of polypeptide initiation upon release of salt shock, revealed the following orientations within the parent molecules: NH2-F2-F1-COOH and NH2-HN1-HN2-COOH. The existence of intermolecular disulphide bonds, orientation and relative lengths of the two NDV HN fragments is analogous to the HA1 and HA2 proteins of influenza virus haemagglutinin.

NGF stimulates incorporation of fucose or glucosamine into an external glycoprotein in cultured rat PC12 pheochromocytoma cells

Rat PC12 pheochromocytoma cells respond to Nerve Growth Factor (NGF) by ceasing to undergo mitosis and acquiring neuronal characteristics, including outgrowth of neurites and electrical excitability. We have found that NGF treatment results in no consistent qualitative and only a few minor quantitative changes in the two-dimensional electrophoresis pattern of 14C-amino acid-labeled proteins synthesized by the cells. On the other hand, NGF stimulates the incorporation of radiolabeled fucose or glucosamine into several components, including one of apparent molecular weight 230,000 daltons on one- and two-dimensional SDS-polyacrylamide gels. This component is removed by mild trypsinization of intact cells and therefore appears to be a glycoprotein (named here NILE) at least partially exposed on the cell surface. Stimulation of NILE glycoprotein labeling can first be detected after 2 days of exposure to NGF and increases progressively with time of treatment. This change is not solely a consequence of the cessation of cell division caused by NGF, since it does not occur in nondividing PC12 populations prepared by cytosine arabinoside treatment. NGF stimulates labeling of NILE glycoprotein even when attachment and process outgrowth are prevented by growing the cells in spinner suspension cultures. The relative rate of labeling attained under these conditions is less, however, than in NGF-treated, substrate-attached cells. Stimulation of NILE glycoprotein labeling by NGF is selectively blocked (as is neurite outgrowth) by camptothecin, an RNA synthesis inhibitor, and thus may require transcription. Despite their similarities in apparent size and exposure on cell surfaces, the NILE and LETS glycoproteins are shown to be immunologically distinct.

Mobilities of envelope proteins of Escherichia coli in two-dimensional SDS-polyacrylamide gel electrophoresis with different buffer systems.

Mobilities of envelope proteins of Escherichia coli in two-dimensional SDS-polyacrylamide gel electrophoresis with different buffer systems.

A simple and rapid method for the differentiation of the members of the genus Klebsiella.

Nitric oxide (NO) is an endogenous, diffusible, transcellular messenger shown to affect regulatory and signaling pathways with impact on cell survival. Exposure to NO can impart direct post-translational modifications on target proteins such as nitration and/or nitrosylation. As an alternative, after interaction with oxygen, superoxide, glutathione, or certain metals, NO can lead to <i>S</i>-glutathionylation, a post-translational modification potentially critical to signaling pathways. A novel glutathione <i>S</i>-transferase π (GSTπ)-activated pro-drug, <i>O</i>²-{2,4-dinitro-5-[4-(<i>N</i>-methylamino)benzoyloxy]phenyl}1-(<i>N</i>,<i>N</i>-dimethylamino)diazen-1-ium-1,2-diolate (PABA/NO), liberates NO and elicits toxicity in vitro and in vivo. We now show that PABA/NO induces nitrosative stress, resulting in undetectable nitrosylation, limited nitration, and high levels of <i>S</i>-glutathionylation. After a single pharmacologically relevant dose of PABA/NO, <i>S</i>-glutathionylation occurs rapidly (&lt;5 min) and is sustained for ∼7 h, implying a half-life for the deglutathionylation process of approximately 3 h. Two-dimensional SDS-polyacrylamide gel electrophoresis and immunoblotting with a monoclonal antibody to <i>S</i>-glutathionylated residues indicated that numerous proteins were <i>S</i>-glutathionylated. Subsequent matrix-assisted laser desorption ionization/time of flight analysis identified 10 proteins, including β-lactate dehydrogenase, Rho GDP dissociation inhibitor β, ATP synthase, elongation factor 2, protein disulfide isomerase, nucleophosmin-1, chaperonin, actin, protein tyrosine phosphatase 1B (PTP1B), and glucosidase II. In addition, we showed that sustained <i>S</i>-glutathionylation was temporally concurrent with drug-induced activation of the stress kinases, known to be linked with cell death pathways. This is consistent with the fact that PABA/NO induces <i>S</i>-glutathionylation and inactivation of PTP1B, one phosphatase that can participate in deactivation of kinases. These effects were consistent with the presence of intracellular PABA/NO or metabolites, because cells overexpressing MRP1 were less sensitive to the drug and had reduced levels of <i>S</i>-glutathionylated proteins.