16 results
Search Results
2. Immobilizing affinity proteins to nitrocellulose: a toolbox for paper-based assay developers.
- Author
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Holstein CA, Chevalier A, Bennett S, Anderson CE, Keniston K, Olsen C, Li B, Bales B, Moore DR, Fu E, Baker D, and Yager P
- Subjects
- Antibodies chemistry, Antibodies metabolism, Bacterial Proteins chemistry, Bacterial Proteins metabolism, Carrier Proteins chemistry, Collodion chemistry, Immobilized Proteins chemistry, Recombinant Proteins chemistry, Streptavidin chemistry, Carrier Proteins metabolism, Chromatography, Affinity methods, Collodion metabolism, Immobilized Proteins metabolism, Paper, Recombinant Proteins metabolism, Streptavidin metabolism
- Abstract
To enable enhanced paper-based diagnostics with improved detection capabilities, new methods are needed to immobilize affinity reagents to porous substrates, especially for capture molecules other than IgG. To this end, we have developed and characterized three novel methods for immobilizing protein-based affinity reagents to nitrocellulose membranes. We have demonstrated these methods using recombinant affinity proteins for the influenza surface protein hemagglutinin, leveraging the customizability of these recombinant "flu binders" for the design of features for immobilization. The three approaches shown are: (1) covalent attachment of thiolated affinity protein to an epoxide-functionalized nitrocellulose membrane, (2) attachment of biotinylated affinity protein through a nitrocellulose-binding streptavidin anchor protein, and (3) fusion of affinity protein to a novel nitrocellulose-binding anchor protein for direct coupling and immobilization. We also characterized the use of direct adsorption for the flu binders, as a point of comparison and motivation for these novel methods. Finally, we demonstrated that these novel methods can provide improved performance to an influenza hemagglutinin assay, compared to a traditional antibody-based capture system. Taken together, this work advances the toolkit available for the development of next-generation paper-based diagnostics.
- Published
- 2016
- Full Text
- View/download PDF
3. Identification of a neuroregulated phosphoprotein in skeletal muscle as the regulatory subunit of cyclic AMP-dependent protein kinase II.
- Author
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Squinto SP, McLane JA, and Held IR
- Subjects
- Animals, Carrier Proteins physiology, Collodion, Cyclic AMP metabolism, Electrophoresis, Polyacrylamide Gel, Male, Molecular Weight, Muscles metabolism, Paper, Phosphoproteins physiology, Rats, Rats, Inbred Strains, Subcellular Fractions metabolism, Trypsin, Carrier Proteins metabolism, Intracellular Signaling Peptides and Proteins, Muscles innervation, Neurons metabolism, Phosphoproteins metabolism, Protein Kinases metabolism
- Abstract
When soluble proteins in cytosolic fractions of rat soleus muscles are 32P-phosphorylated in vitro by an ATP:protein phosphotransferase reaction, the major substrate is a 56-kilodalton (56K) protein. As we have also reported previously, the onset and development of increased 32P-phosphorylation of this 56K protein, which are observed after the soleus is denervated, temporally correlate with the denervation period and length of the distal nerve stump [Held et al, 1983]. Conclusive evidence which identifies this neuroregulated muscle protein as the regulatory subunit of cyclic AMP-dependent protein kinase type II (R-II) is presented in this paper. The 56K soleus protein and purified bovine heart R-II were 32P-phosphorylated and subjected to limited proteolysis with bovine pancreas trypsin. After resolution of the generated 32P-phosphopeptides by SDS slab PAGE and visualization by autoradiography, no tryptic products were observed from the 56K soleus protein which were not also produced by proteolysis of the purified R-II. These tryptic phosphopeptides included 39, 16.5, and 12K fragments which retained the autophosphorylation site of R-II. After denervation, the 32P-phosphorylation of the 56K soleus protein and of the 39K tryptic peptide product were comparably increased. The identification of the neuroregulated 56K soleus protein as R-II was also confirmed by Western blotting with a specific anti-R-II sera. Taken together, our results demonstrate that the previously observed neuroregulation of the 32P-phosphorylation of the 56K soleus protein is identifiable with some alteration which affects the intramolecular 32P-autophosphorylation of R-II.
- Published
- 1985
- Full Text
- View/download PDF
4. Molecular heterogeneity of the bullous pemphigoid antigens as detected by immunoblotting.
- Author
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Labib RS, Anhalt GJ, Patel HP, Mutasim DF, and Diaz LA
- Subjects
- Antigen-Antibody Reactions, Autoantibodies immunology, Autoantigens immunology, Collodion, Dystonin, Electrophoresis, Polyacrylamide Gel, Epidermis analysis, Epitopes analysis, Epitopes immunology, Fluorescent Antibody Technique, Humans, Molecular Weight, Paper, Tissue Extracts analysis, Collagen Type XVII, Antigens analysis, Autoantigens analysis, Carrier Proteins, Collagen, Cytoskeletal Proteins, Nerve Tissue Proteins, Non-Fibrillar Collagens, Pemphigoid, Bullous immunology, Skin Diseases, Vesiculobullous immunology
- Abstract
Sera from 28 patients with bullous pemphigoid (BP), four patients with cicatricial pemphigoid (CP), and 24 controls (normal volunteers and patients with pemphigus, systemic lupus erythematosus, or other skin diseases) were tested against extracts of human epidermis by immunoblotting techniques. The extraction buffer included 1% SDS, 5% beta-mercaptoethanol, and six protease inhibitors with various specificities. BP sera from individual patients showed different patterns of reactivity with the same epidermal extract, and each pattern consisted of one or more bands. A total of five bands of 240 kD, 200 kD, 180 kD, 97 kD, and 77 kD reacted with BP sera; the 240-kD band reacted with one CP sera, and none of these bands was detected by the control sera. The 240-kD and 180-kD bands reacted very strongly with some sera and were most frequently observed (43% and 29%, respectively). The 200-kD, 97-kD, and 77-kD bands were less frequently observed (25%, 7%, and 7%, respectively), but when present, their reactions were usually strong. Eleven percent of the BP sera did not react with any bands. Contrary to previous reports, this study shows that BP autoantibodies react with several protein bands, as detected by immunoblotting. We have recently shown by immunoelectron microscopy that BP autoantibodies bind to the basal cell hemidesmosomes. It remains to be determined which of these protein bands represent specific hemidesmosomal proteins and which antibody-antigen interactions are relevant to the pathogenesis of this disease.
- Published
- 1986
5. Functional comparison of paper-based immunoassays based on antibodies and engineered binding proteins
- Author
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Hadley D. Sikes, Quinlan R. Johns, Ki-Joo Sung, Yara Jabbour Al Maalouf, and Eric A. Miller
- Subjects
Paper ,02 engineering and technology ,Viral Nonstructural Proteins ,Biochemistry ,DNA-binding protein ,Antibodies ,Analytical Chemistry ,Antigen-Antibody Reactions ,03 medical and health sciences ,chemistry.chemical_compound ,Limit of Detection ,Electrochemistry ,Humans ,Environmental Chemistry ,Cellulose ,Spectroscopy ,030304 developmental biology ,Immunoassay ,0303 health sciences ,biology ,Chemistry ,Binding protein ,Diagnostic test ,Zika Virus ,Paper based ,021001 nanoscience & nanotechnology ,3. Good health ,biology.protein ,Antibody ,Carrier Proteins ,0210 nano-technology - Abstract
Binding protein scaffolds, such as rcSso7d, have been investigated for use in diagnostic tests; however, the functional performance of rcSso7d has not yet been studied in comparison to antibodies. Here, we assessed the analyte-binding capabilities of rcSso7d and antibodies on cellulose with samples in buffer and 100% human serum.
- Published
- 2020
6. Zinc sequestration by human calprotectin facilitates manganese binding to the bacterial solute-binding proteins PsaA and MntC
- Author
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Tomer Rosen, Rose C Hadley, Aaron T Bozzi, Daniel Ocampo, Jason Shearer, and Elizabeth M Nolan
- Subjects
Paper ,Manganese ,Bacteria ,Metals and Alloys ,Biophysics ,Biochemistry ,Biomaterials ,Zinc ,Streptococcus pneumoniae ,Bacterial Proteins ,Chemistry (miscellaneous) ,Humans ,Carrier Proteins ,Leukocyte L1 Antigen Complex - Abstract
Zinc is an essential transition metal nutrient for bacterial survival and growth but may become toxic when present at elevated levels. The Gram-positive bacterial pathogen Streptococcus pneumoniae is sensitive to zinc poisoning, which results in growth inhibition and lower resistance to oxidative stress. Streptococcus pneumoniae has a relatively high manganese requirement, and zinc toxicity in this pathogen has been attributed to the coordination of Zn(II) at the Mn(II) site of the solute-binding protein (SBP) PsaA, which prevents Mn(II) uptake by the PsaABC transport system. In this work, we investigate the Zn(II)-binding properties of pneumococcal PsaA and staphylococcal MntC, a related SBP expressed by another Gram-positive bacterial pathogen, Staphylococcus aureus, which contributes to Mn(II) uptake. X-ray absorption spectroscopic studies demonstrate that both SBPs harbor Zn(II) sites best described as five-coordinate, and metal-binding studies in solution show that both SBPs bind Zn(II) reversibly with sub-nanomolar affinities. Moreover, both SBPs exhibit a strong thermodynamic preference for Zn(II) ions, which readily displace bound Mn(II) ions from these proteins. We also evaluate the Zn(II) competition between these SBPs and the human S100 protein calprotectin (CP, S100A8/S100A9 oligomer), an abundant host-defense protein that is involved in the metal-withholding innate immune response. CP can sequester Zn(II) from PsaA and MntC, which facilitates Mn(II) binding to the SBPs. These results demonstrate that CP can inhibit Zn(II) poisoning of the SBPs and provide molecular insight into how S100 proteins may inadvertently benefit bacterial pathogens rather than the host.
- Published
- 2022
7. MTM1 displays a new function in the regulation of nickel resistance in Saccharomyces cerevisiae
- Author
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Naifeng Xu, Yuan Xu, Nathan Smith, Huizhu Chen, Ziguo Guo, Jaekwon Lee, and Xiaobin Wu
- Subjects
Paper ,Saccharomyces cerevisiae Proteins ,Superoxide Dismutase ,Metals and Alloys ,Biophysics ,Saccharomyces cerevisiae ,Biochemistry ,Trace Elements ,Mitochondrial Proteins ,Biomaterials ,Nickel ,Chemistry (miscellaneous) ,Metalloproteins ,RNA, Messenger ,Carrier Proteins ,Reactive Oxygen Species - Abstract
Nickel (Ni) is an essential yet toxic trace element. Although a cofactor for many metalloenzymes, nickel function and metabolism is not fully explored in eukaryotes. Molecular biology and metallomic methods were utilized to explore the new physiological functions of nickel in Saccharomyces cerevisiae. Here we showed that MTM1 knockout cells displayed much stronger nickel tolerance than wild-type cells and mitochondrial accumulations of Ni and Fe of mtm1Δ cells dramatically decreased compared to wild-type cells when exposed to excess nickel. Superoxide dismutase 2 (Sod2p) activity in mtm1Δ cells was severely attenuated and restored through Ni supplementation in media or total protein. SOD2 mRNA level of mtm1Δ cells was significantly higher than that in the wild-type strain but was decreased by Ni supplementation. MTM1 knockout afforded resistance to excess nickel mediated through reactive oxygen species levels. Meanwhile, additional Ni showed no significant effect on the localization of Mtm1p. Our study reveals the MTM1 gene plays an important role in nickel homeostasis and identifies a novel function of nickel in promoting Sod2p activity in yeast cells.
- Published
- 2022
8. Pharmacogenomics: marshalling the human genome to individualise drug therapy
- Author
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W E Evans
- Subjects
Paper ,Drug ,Polymorphism, Genetic ,Genome, Human ,Receptors, Drug ,media_common.quotation_subject ,Gastroenterology ,Biology ,Pharmacology ,Molecular diagnostics ,Bioinformatics ,Pharmacotherapy ,Cytochrome P-450 Enzyme System ,Drug Therapy ,Pharmacogenetics ,Drug metabolising enzymes ,Pharmacogenomics ,Drug response ,Humans ,Human genome ,Carrier Proteins ,Glutathione Transferase ,media_common - Abstract
Pharmacogenomics aims to identify the inherited basis for interindividual differences in drug response, and translate this to molecular diagnostics that can be used to individualise drug therapy. This review uses a number of published examples of inherited differences in drug metabolising enzymes, drug transporters, and drug targets (for example, receptors) to illustrate the potential importance of inheritance in determining the efficacy and toxicity of medications in humans. It seems that this field is at the early stages of developing a powerful set of molecular diagnostics that will have profound utility in optimising drug therapy for individual patients.
- Published
- 2003
9. The molecular basis of allergenicity: comparative analysis of the three dimensional structures of diverse allergens reveals a common structural motif
- Author
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R. Furmonaviciene and Farouk Shakib
- Subjects
Paper ,UoA 12 Allied Health Professions and Studies ,Protein Data Bank (RCSB PDB) ,Beta sheet ,Biology ,Antiparallel (biochemistry) ,Pathology and Forensic Medicine ,immune system diseases ,Sequence Analysis, Protein ,Animals ,Senecio ,Antigens, Dermatophagoides ,Cysteine ,Binding site ,Structural motif ,Glycoproteins ,Binding Sites ,RAE 2008 ,Allergens ,Cysteine protease ,respiratory tract diseases ,Cysteine Endopeptidases ,Plants, Toxic ,Biochemistry ,Carrier Proteins ,Sequence motif ,Sequence Alignment ,Software ,Lipocalin 1 - Abstract
Background—Although a large number of allergens have been characterised, the structural, functional, and biochemical features that these molecules have in common, and that could explain their ability to elicit powerful IgE antibody responses, are still uncertain. Recently, there has been considerable interest in the role of the cysteine protease activity of the house dust mite allergen Der p 1 in biasing the immune response in favour of IgE production. Aims—To search for remote homologues of Der p 1 with sequences similar to the 30 conserved amino acids surrounding the catalytic cysteine residue (Cys34). Methods—Potential homologues were analysed by examining their three dimensional structures and multiple sequence alignments using the programs PROPSEARCH, ClustalW, GeneDoc, and Swiss Pdb Viewer. Results—Diverse allergens (for example, the plant cysteine protease papain, the transport protein lipocalin Mus m 1, and the ragweed allergen Amb a 5) have a similar structural motif; namely, a groove resembling the substrate binding groove of Der p 1. The groove is located inside an α–β motif, between an α helix on one side and an antiparallel β sheet on the other side. A similar common motif (a cysteine stabilised α–β fold) can also be found in some toxins and defensins. Conclusion—Allergens of diverse sources have a common structural motif, namely a groove located inside an α–β motif, which could potentially serve as a ligand binding site.
- Published
- 2001
10. Sudomotor function in familial dysautonomia
- Author
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Bickel, A., Axelrod, F. B., Marthol, H., Martin Schmelz, and Hilz, M. J.
- Subjects
Paper ,Adult ,Male ,Nerve Fibers, Unmyelinated ,integumentary system ,Adolescent ,Injections, Intradermal ,Microdialysis ,Plasmapheresis ,Middle Aged ,Acetylcholine ,Axons ,Sweat Glands ,Reflex ,Dysautonomia, Familial ,Humans ,Hyperhidrosis ,Female ,Transcriptional Elongation Factors ,Carrier Proteins - Abstract
Patients with familial dysautonomia (FD) manifest episodic hyperhidrosis despite the reduction of sudomotor fibres and sweat glands associated with this autonomic neuropathy. We assessed peripheral sudomotor nerve fibre and sweat gland function to determine if this symptom was due to peripheral denervation hypersensitivity.In 14 FD patients and 11 healthy controls, direct and axon reflex mediated sweat responses were determined by measuring transepidermal water loss (TEWL) after application of acetylcholine via a microdialysis membrane, a novel method to evaluate sudomotor function in neuropathy patients. Results were compared with data from conventional quantitative sudomotor axon reflex testing (QSART). Using microdialysis, interstitial fluid was analysed for plasma proteins to evaluate protein extravasation induced by acetylcholine as an additional parameter of C-fibre function.Although reduced axon reflex sweating was expected in FD patients, neither direct or axon reflex mediated sweat responses, nor acetylcholine induced protein extravasation differed between control and patient groups. However, the baseline resting sweat rate was higher in FD patients than controls (p0.05). TEWL and QSART test results correlated (r = 0.64, p = 0.01), proving the reliability of TEWL methodology in evaluating sudomotor function.The finding of normal direct and axon reflex mediated sweat output in FD patients supports our hypothesis that, in a disorder with severe sympathetic nerve fibre reduction, sudomotor fibres, but not the sweat gland itself, exhibit chemical hypersensitivity. This might explain excessive episodic hyperhidrosis in situations with increased central sympathetic outflow.
- Published
- 2004
11. CSF hypocretin-1 levels in narcolepsy, Kleine-Levin syndrome, and other hypersomnias and neurological conditions
- Author
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Claudio L. Bassetti, T Blatter, Michel Lecendreux, Yves Dauvilliers, Matthias Bischof, Michel Billiard, A Besset, Mehdi Tafti, Bertrand Carlander, Friedrich E. Maly, Christian R. Baumann, and Jacques Touchon
- Subjects
Paper ,Adult ,Male ,medicine.medical_specialty ,Cataplexy ,Adolescent ,Neurological disorder ,macromolecular substances ,Disorders of Excessive Somnolence ,Asymptomatic ,Gastroenterology ,REM Sleep Parasomnias ,Normal pressure hydrocephalus ,Internal medicine ,mental disorders ,medicine ,Humans ,Sleep disorder ,Orexins ,Neuropeptides ,Intracellular Signaling Peptides and Proteins ,Feeding Behavior ,Middle Aged ,medicine.disease ,nervous system diseases ,Orexin ,Psychiatry and Mental health ,Endocrinology ,Phenotype ,Kleine–Levin syndrome ,nervous system ,Surgery ,Female ,Neurology (clinical) ,medicine.symptom ,Nervous System Diseases ,Psychology ,Carrier Proteins ,psychological phenomena and processes ,Narcolepsy - Abstract
Objective: To determine the role of CSF hypocretin-1 in narcolepsy with and without cataplexy, Kleine-Levin syndrome (KLS), idiopathic and other hypersomnias, and several neurological conditions. Patients: 26 narcoleptic patients with cataplexy, 9 narcoleptic patients without cataplexy, 2 patients with abnormal REM-sleep-associated hypersomnia, 7 patients with idiopathic hypersomnia, 2 patients with post-traumatic hypersomnia, 4 patients with KLS, and 88 patients with other neurological disorders. Results: 23 patients with narcolepsy-cataplexy had low CSF hypocretin-1 levels, while one patient had a normal hypocretin level (HLA-DQB1*0602 negative) and the other two had intermediate levels (familial forms). One narcoleptic patient without cataplexy had a low hypocretin level. One patient affected with post-traumatic hypersomnia had intermediate hypocretin levels. The KLS patients had normal hypocretin levels while asymptomatic, but one KLS patient (also affected with Prader-Willi syndrome) showed a twofold decrease in hypocretin levels during a symptomatic episode. Among the patients without hypersomnia, two patients with normal pressure hydrocephalus and one with unclear central vertigo had intermediate levels. Conclusion: Low CSF hypocretin-1 is highly specific (99.1%) and sensitive (88.5%) for narcolepsy with cataplexy. Hypocretin ligand deficiency appears not to be the major cause for other hypersomnias, with a possible continuum in the pathophysiology of narcolepsy without cataplexy and idiopathic hypersomnia. However, partial hypocretin lesions without low CSF hypocretin-1 consequences cannot be definitely excluded in those disorders. The existence of normal hypocretin levels in narcoleptic patients and intermediate levels in other rare aetiologies needs further investigation, especially for KLS, to establish the functional significance of hypocretin neurotransmission alterations.
- Published
- 2003
12. Bi-directional transport of GABA in human embryonic kidney (HEK-293) cells stably expressing the rat GABA transporter GAT-1
- Author
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Harald H, Sitte, Ernst A, Singer, and Petra, Scholze
- Subjects
Paper ,GABA Plasma Membrane Transport Proteins ,Nipecotic Acids ,Biological Transport, Active ,Gene Expression ,Organic Anion Transporters ,Nerve Tissue Proteins ,Kidney ,Tritium ,Cell Line ,GABA Antagonists ,Receptors, GABA ,Animals ,Humans ,Drug Interactions ,Neurotransmitter Uptake Inhibitors ,Ouabain ,Tiagabine ,Cells, Cultured ,gamma-Aminobutyric Acid ,Dose-Response Relationship, Drug ,Aminobutyrates ,Membrane Proteins ,Membrane Transport Proteins ,Rats ,Carrier Proteins - Abstract
1. Bi-directional GABA-transport was studied by performing uptake and superfusion experiments in human embryonic kidney 293 cells stably expressing the rat GABA transporter rGAT-1. 2. K(M) and V(max) values for [(3)H]-GABA uptake were 11.7+/-1.8 microM and 403+/-55 pmol min(-1) 10(-6) cells (n=9), respectively. 3. Kinetic analysis of outward transport was performed by pre-labelling the cells with increasing concentrations of [(3)H]-GABA and triggering outward transport with 333 microM GABA. Approximate apparent K(M) and V(max) values were 12 mM and 50 pmol min(-1) 10(-6) cells, respectively. 4. GABA re-uptake inhibitors (RI; e.g. tiagabine), as well as, substrates of the rGAT-1 (e.g. GABA, nipecotic acid) concentration dependently decreased [(3)H]-GABA uptake and increased efflux of [(3)H]-GABA from pre-labelled cells. The IC(50) values for inhibiting uptake and the EC(50) values for increasing efflux were significantly correlated (r(2)=0.99). 5. On superfusion, RI antagonized the efflux-enhancing effect of the substrates. The effect of the latter was markedly augmented in the presence of ouabain (100 microM), whereas the effect of RI remained unchanged. The most likely explanation for the release enhancing effect of RI is interruption of ongoing re-uptake. 6. The structural GABA-analogue 2,4-diamino-n-butyric acid (DABA) exhibited a bell-shaped concentration response curve on [(3)H]-GABA efflux with the maximum at 1 mM, and displayed a deviation from the sigmoidal inhibition curve in uptake experiments in the same concentration range. At concentrations below 1 mM, DABA inhibited [(3)H]-GABA uptake non-competitively, while at 1 mM and above the inhibition of uptake followed a competitive manner. 7. The results provide information of GABA inward and outward transport, and document a complex interaction of the rGAT-1 with its substrate DABA.
- Published
- 2002
13. Identification of a neuroregulated phosphoprotein in skeletal muscle as the regulatory subunit of cyclic AMP-dependent protein kinase II
- Author
-
Irene R. Held, Jerry A. McLane, and S. P. Squinto
- Subjects
Male ,Paper ,Neuroregulation ,Protein subunit ,Proteolysis ,Biology ,Cellular and Molecular Neuroscience ,Cyclic AMP ,medicine ,Animals ,Trypsin ,Protein phosphorylation ,Protein kinase A ,Neurons ,medicine.diagnostic_test ,Muscles ,Autophosphorylation ,Intracellular Signaling Peptides and Proteins ,Collodion ,Rats, Inbred Strains ,Phosphoproteins ,Molecular biology ,Rats ,Molecular Weight ,Biochemistry ,Phosphoprotein ,Electrophoresis, Polyacrylamide Gel ,Carrier Proteins ,Protein Kinases ,Subcellular Fractions ,medicine.drug - Abstract
When soluble proteins in cytosolic fractions of rat soleus muscles are 32P-phosphorylated in vitro by an ATP:protein phosphotransferase reaction, the major substrate is a 56-kilodalton (56K) protein. As we have also reported previously, the onset and development of increased 32P-phosphorylation of this 56K protein, which are observed after the soleus is denervated, temporally correlate with the denervation period and length of the distal nerve stump [Held et al, 1983]. Conclusive evidence which identifies this neuroregulated muscle protein as the regulatory subunit of cyclic AMP-dependent protein kinase type II (R-II) is presented in this paper. The 56K soleus protein and purified bovine heart R-II were 32P-phosphorylated and subjected to limited proteolysis with bovine pancreas trypsin. After resolution of the generated 32P-phosphopeptides by SDS slab PAGE and visualization by autoradiography, no tryptic products were observed from the 56K soleus protein which were not also produced by proteolysis of the purified R-II. These tryptic phosphopeptides included 39, 16.5, and 12K fragments which retained the autophosphorylation site of R-II. After denervation, the 32P-phosphorylation of the 56K soleus protein and of the 39K tryptic peptide product were comparably increased. The identification of the neuroregulated 56K soleus protein as R-II was also confirmed by Western blotting with a specific anti-R-II sera. Taken together, our results demonstrate that the previously observed neuroregulation of the 32P-phosphorylation of the 56K soleus protein is identifiable with some alteration which affects the intramolecular 32P-autophosphorylation of R-II.
- Published
- 1985
14. A monovalent cation-sensitive actin-binding factor in a myeloid leukemia cell line
- Author
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Takashi Hashida, Kuniaki Takagi, Yasuo Ichikawa, and Kazuhiro Nagata
- Subjects
Paper ,Antigenicity ,Physiology ,Cell ,Peptide ,macromolecular substances ,Chromatography, DEAE-Cellulose ,Cell Line ,Potassium Chloride ,Mice ,medicine ,Animals ,Molecular Biology ,Gelsolin ,Actin ,chemistry.chemical_classification ,biology ,Binding protein ,Microfilament Proteins ,Collodion ,Skeletal muscle ,Cell Biology ,General Medicine ,biology.organism_classification ,Actina ,Actins ,Molecular Weight ,Microscopy, Electron ,medicine.anatomical_structure ,chemistry ,Leukemia, Myeloid ,Cell culture ,Immunology ,Biophysics ,Electrophoresis, Polyacrylamide Gel ,Carrier Proteins - Abstract
Cell extracts of a murine leukemia cell line, M1, apparently contain three kinds of actin-gelation factors; a filamin-like protein, and 38K-dimer and 105K-dimer proteins. Unlike gelation by the filamin-like protein, gelation by the latter two proteins is inhibited by low concentrations of KCl. Our study of the 38K protein has been reported elsewhere (Takagi, K. et al., J. Biochem. Tokyo 97, 605-616, 1985). We here describe the purification and characterization of the 105K protein. The 105K protein differs from the alpha-actinin group of proteins in its antigenicity, peptide components and Ca2+-insensitivity. The saturated binding ratio of the protein to purified skeletal muscle actin is 1:8; when this ratio exceeds 1:20, gelation takes place. This gelation is inhibited completely by the presence of 25 mM KCl. Electron microscopy revealed that, in the absence of KCl, the 105K protein/actin mixture forms short actin bundles that are accompanied by a meshwork of short single filaments. The presence of 25 mM KCl did not prevent actin-bundling, but the bundles became longer and the meshwork of short filaments was no longer present.
- Published
- 1985
15. Molecular heterogeneity of the bullous pemphigoid antigens as detected by immunoblotting
- Author
-
R S, Labib, G J, Anhalt, H P, Patel, D F, Mutasim, and L A, Diaz
- Subjects
Paper ,Skin Diseases, Vesiculobullous ,Dystonin ,Tissue Extracts ,Collodion ,Fluorescent Antibody Technique ,Nerve Tissue Proteins ,Non-Fibrillar Collagens ,Autoantigens ,Antigen-Antibody Reactions ,Molecular Weight ,Cytoskeletal Proteins ,Epitopes ,Pemphigoid, Bullous ,Humans ,Electrophoresis, Polyacrylamide Gel ,Collagen ,Antigens ,Epidermis ,Carrier Proteins ,Autoantibodies - Abstract
Sera from 28 patients with bullous pemphigoid (BP), four patients with cicatricial pemphigoid (CP), and 24 controls (normal volunteers and patients with pemphigus, systemic lupus erythematosus, or other skin diseases) were tested against extracts of human epidermis by immunoblotting techniques. The extraction buffer included 1% SDS, 5% beta-mercaptoethanol, and six protease inhibitors with various specificities. BP sera from individual patients showed different patterns of reactivity with the same epidermal extract, and each pattern consisted of one or more bands. A total of five bands of 240 kD, 200 kD, 180 kD, 97 kD, and 77 kD reacted with BP sera; the 240-kD band reacted with one CP sera, and none of these bands was detected by the control sera. The 240-kD and 180-kD bands reacted very strongly with some sera and were most frequently observed (43% and 29%, respectively). The 200-kD, 97-kD, and 77-kD bands were less frequently observed (25%, 7%, and 7%, respectively), but when present, their reactions were usually strong. Eleven percent of the BP sera did not react with any bands. Contrary to previous reports, this study shows that BP autoantibodies react with several protein bands, as detected by immunoblotting. We have recently shown by immunoelectron microscopy that BP autoantibodies bind to the basal cell hemidesmosomes. It remains to be determined which of these protein bands represent specific hemidesmosomal proteins and which antibody-antigen interactions are relevant to the pathogenesis of this disease.
- Published
- 1986
16. The earliest form of C-protein expressed during striated muscle development is immunologically the same as cardiac-type C-protein
- Author
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M, Kawashima, S, Kitani, T, Tanaka, and T, Obinata
- Subjects
Paper ,Muscles ,Myocardium ,Antibodies, Monoclonal ,Collodion ,Fluorescent Antibody Technique ,Muscle Proteins ,Heart ,Chick Embryo ,Muscle Development ,Pectoralis Muscles ,Antibody Specificity ,Animals ,Electrophoresis, Polyacrylamide Gel ,Carrier Proteins ,Cells, Cultured - Abstract
A monoclonal antibody (C-315) specific for cardiac-type C-protein was prepared and, in combination with other antibodies specific for fast and slow skeletal muscle C-proteins, it was used to investigate the expression of C-protein isoforms in developing striated muscle cells in vivo and in vitro. During embryonic development of skeletal muscles, a C-protein recognized by C-315 appeared first but only transiently, it being replaced subsequently by two other isoforms recognized by the antibodies to slow and fast skeletal muscle C-proteins in a fiber-type specific manner as previously demonstrated (Obinata et al. (1984) Develop. Biol. 101, 116-124). In contrast, only cardiac-type C-protein was detected in cardiac muscle throughout the developmental stages. When myogenesis in vitro was monitored using the same antibodies, C-315 binding appeared first in multinucleated myotubes as in vivo which was followed by the sequential expression of two other C-protein variants. The reactivity of C-315 as well as that of anti-slow and anti-fast skeletal C-protein antibodies persisted during muscle development in culture. Thus, this study demonstrates that the earliest form of C-protein expressed in striated muscles may either be a cardiac-type isoform or a unique embryonic protein containing an epitope in common with the adult cardiac-type protein, and that transitions of C-protein isoform expression characteristic of each fiber-type occur during muscle development in vivo but not in vitro.
- Published
- 1986
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