scholarly journals Mapping of the properdin-binding site in the third component of complement

1984 ◽  
Vol 217 (1) ◽  
pp. 323-326 ◽  
Author(s):  
J D Lambris ◽  
J Alsenz ◽  
T F Schulz ◽  
M P Dierich
Keyword(s):  
Binding Site ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Enzyme Linked Immunosorbent Assay ◽  
Chain Fragment ◽  
Alpha Chain ◽  
Factor I ◽  
Assay Procedure ◽  
Complement Component C3 ◽  
Third Component Of Complement

The properdin-binding site in the human third complement component (C3) was mapped by using isolated C3b, C3c, alpha- and beta-chains of C3 and C3 polypeptide fragments and an enzyme-linked-immunosorbent-assay procedure. The C3 chains and the polypeptide fragments were purified to homogeneity by preparative sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The alpha-chain polypeptides included a 68 kDa and a 43 kDa polypeptide, which were generated by cleavage of C3b with factors I and H, and a 40 kDa, 33 kDa (C3d) and 27 kDa polypeptide, which were generated by cleavage of C3b with porcine elastase. It was shown that properdin binds to C3b, C3c, alpha-chain, and to the 43 kDa (factor-I + H-derived), as well as to 40 kDa (elastase-derived) alpha-chain fragment, but not to the beta-chain 68 kDa, 33 kDa (C3d) and 27 kDa alpha-chain fragments. Thus the binding site for properdin resides on the 40-43 kDa C-terminal alpha-chain fragment of C3.

scholarly journals Structural and functional analysis of the complement component factor H with the use of different enzymes and monoclonal antibodies to factor H

1985 ◽  
Vol 232 (3) ◽  
pp. 841-850 ◽  
Author(s):  
J Alsenz ◽  
T F Schulz ◽  
J D Lambris ◽  
R B Sim ◽  
M P Dierich
Keyword(s):  
Monoclonal Antibodies ◽  
Binding Sites ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Factor H ◽  
Enzyme Linked Immunosorbent Assay ◽  
Terminal Sequence ◽  
Tryptic Fragment ◽  
Cofactor Activity ◽  
Third Component Of Complement

The action of six different enzymes on the function and structure of Factor H was investigated by use of sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, haemagglutination, two enzyme-linked immunosorbent assay systems and an assay for Factor I cofactor activity. Six monoclonal antibodies directed against the 38 kDa tryptic fragment of Factor H [which contains the binding site for C3b (a 180 kDa fragment of the third component of complement) and the cofactor activity] were also used to detect cleavage products derived from the same fragment. Elastase, chymotrypsin A4 or trypsin first cleaved Factor H to 36-38 kDa fragments carrying all six monoclonal anti-(Factor H)-binding sites. In parallel, the interaction of Factor H with surface-bound C3b was lost, whereas the cofactor function was preserved. Further cleavage of the 36-38 kDa fragments into two 13-19 kDa fragments (one carrying the MAH4 and MRC OX 24 epitopes, the other the MAH1, MAH2, MAH3 and MRC OX 23 epitopes) destroyed cofactor activity. Pepsin, bromelain or papain rapidly split off a 13-15 kDa fragment of Factor H carrying the MAH1, MAH2, MAH3 and MRC OX 23 epitopes and destroyed all tested functions of Factor H. Ficin cleaved Factor H into disulphide-linked fragments smaller than 25 kDa, but did not affect the functions of the Factor H molecule. The 38 kDa tryptic fragment of Factor H is the N-terminal end of the Factor H molecule, as determined by N-terminal sequence analysis. A model is presented of the substructure of Factor H.

Screening of Single-Chain Variable Fragments against TSP50 from a Phage Display Antibody Library and Their Expression as Soluble Proteins

2006 ◽  
Vol 11 (5) ◽  
pp. 546-552 ◽  
Author(s):  
Jingyan Wei ◽  
Yang Liu ◽  
Songchuan Yang ◽  
Junjie Xu ◽  
Hangtian Kong ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Phage Display ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sandwich Elisa ◽  
Sodium Dodecyl ◽  
Enzyme Linked Immunosorbent Assay ◽  
Antibody Library ◽  
Single Chain ◽  
Single Chain Variable Fragments ◽  
Phage Display Antibody

A novel gene, testes-specific protease 50 ( TSP50), is abnormally activated and differentially expressed in most patients with breast cancer, suggesting it as a novel biomarker for this disease. The possibility that TSP50 may be an oncogene is presently under investigation. In this study, the single-chain variable fragments (scFvs) against TSP50 were panned from a phage display antibody library using TSP50-specific peptide, pep-50, as a target antigen. After 4 rounds of panning, 3 clones (A1, A11, and C8) from the library were verified to show strong binding affinities for TSP50 by enzyme-linked immunosorbent assay (ELISA) and to contain the variable region genes of the light and heavy chains of scFv antibodies but different complementary determining regions by sequencing. The genes of scFv-A1 and scFv-A11 were cloned into expression vector pPELB and successfully expressed as a soluble protein in Escherichia coli Rosetta. The yields of expressions were about 4.0 to 5.0 mg of protein from 1 L of culture. The expressed proteins were purified by a 2-step procedure consisting of ion-exchange chromatography, followed by immobilized metal affinity chromatography. The purified proteins were shown a single band at the position of 31 KDa on sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Sandwich ELISA demonstrated that the expressed scFv proteins were able to specifically react with pep-50, laying a foundation for the investigation of the function of TSP50 in the development and treatment of breast cancer.

scholarly journals Allamanda Mosaic Caused by Cucumber mosaic virus in Taiwan

Plant Disease ◽  
2005 ◽  
Vol 89 (5) ◽  
pp. 529-529 ◽  
Author(s):  
Y. K. Chen ◽  
C. C. Yang ◽  
H. T. Hsu
Keyword(s):  
Cucumber Mosaic Virus ◽  
Mosaic Virus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Chenopodium Quinoa ◽  
Rabbit Antiserum ◽  
Nucleotide Sequence Analysis ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rt Pcr ◽  
Indicator Plants

Allamanda (Allamanda cathartica L., family Apocynaceae) is native to Brazil and is a popular perennial shrub or vine ornamental in Taiwan. Plants showing severe mosaic, rugosity, and leaf distortion symptoms on leaves are common in commercial nurseries and private gardens. Examination of crude sap prepared from symptomatic leaves using an electron microscope revealed the presence of spherical virus particles with a diameter of approximately 28 nm. The virus was mechanically transmitted to indicator plants and induced symptoms similar to those incited by Cucumber mosaic virus (CMV). The virus caused local lesions on inoculated leaves of Chenopodium quinoa and C. amaranticolor and systemic mosaic in Cucumis sativus, Lycopersicon esculentum, Nicotiana benthamiana, N. glutinosa, N. rustica, and N. tabacum. On N. tabacum, necrotic ringspots developed on inoculated leaves followed by systemic mosaic. Tests of leaf sap extracted from naturally infected allamanda and inoculated indicator plants using enzyme-linked immunosorbent assay were positive to rabbit antiserum prepared to CMV. Viral coat protein on transblots of sodium dodecyl sulfate-polyacrylamide gel electrophoresis reacted with CMV subgroup I specific monoclonal antibodies (2). With primers specific to the 3′-half of RNA 3 (1), amplicons of an expected size (1,115 bp) were obtained in reverse transcription-polymerase chain reaction (RT-PCR) using total RNA extracted from infected allamanda and N. benthamiana. The amplified fragment (EMBL Accession No. AJ871492) was cloned and sequenced. It encompasses the 3′ part of the intergenic region of RNA 3 (158 nt), CP ORF (657 nt), and 3′ NTR (300 nt) showing 91.8–98.9% and 71.4–72.8% identities to those of CMV in subgroups I and II, respectively. Results of MspI-digested restriction fragment length polymorphism patterns of the RT-PCR fragment and the nucleotide sequence analysis indicate that the CMV isolate from allamanda belongs to subgroup IB, which is predominant on the island. To our knowledge, CMV is the only reported virus that infects allamanda and was first detected in Brazil (3), and this is the first report of CMV infection in allamanda plants occurring in Taiwan. References: (1) Y. K. Chen et al. Arch. Virol. 146:1631, 2001. (2) H. T. Hsu et al. Phytopathology 90:615, 2000. (3) E. W. Kitajima. Acta. Hortic. 234:451, 1988.

TNF-alpha and cyclooxygenase metabolites do not modulate C. albicans septic shock with disseminated candidiasis

1993 ◽  
Vol 74 (5) ◽  
pp. 2432-2442 ◽  
Author(s):  
G. M. Matuschak ◽  
C. A. Klein ◽  
T. L. Tredway ◽  
D. R. Schilly ◽  
A. J. Lechner
Keyword(s):  
Septic Shock ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Passive Immunization ◽  
Organ Injury ◽  
Tnf Alpha ◽  
Enzyme Linked Immunosorbent Assay ◽  
Necrosis Factor Alpha ◽  
Disseminated Candidiasis ◽  
Alpha Production

We analyzed differences in host regulation of tumor necrosis factor-alpha (TNF-alpha) production and pathophysiological responses in conscious rats after infection with two strains of pathogenic Candida albicans spp. (CA-1 and CA-2) compared with Escherichia coli serotype 055:B5 (EC). The hypothesis was tested that, in contrast to EC, hypotension, organ injury, and mortality after candidemia are not obligatorily dependent on TNF-alpha or TNF-alpha-induced cyclooxygenase pathway metabolites. Dose, viability, and strain-specific dependencies were established after intravenous 10(6) or 10(9) viable CA, as well as heat-killed (HK) or Formalin-inactivated (FI) CA blastospores, compared with live EC at the 24-h LD25 [10(9) colony-forming units (CFU)] and LD100 (10(10) CFU). Shock without endotoxemia developed 4–8 h after 10(9) live CA-1 or CA-2 (LD100 at 24 h) with disseminated yeast-mycelial transformation and increased microvascular permeability in multiple organs but not after HK or FI CA-1. Peak serum TNF-alpha after an LD100 of CA-1 or CA-2 was < 3% of LD25 EC values and was < 1% of peak values during lethal bacteremia. Similar pathogen-specific differences were found in liver- and lung-associated TNF. Production of functionally inactive TNF-alpha during candidemia was excluded by enzyme-linked immunosorbent assay and sodium dodecyl sulfate-polyacrylamide gel electrophoresis with Western blotting. Passive immunization against TNF-alpha 2 h before microbial challenge was not protective against CA but prevented otherwise lethal EC sepsis. Cyclooxygenase inhibition also failed to attenuate candidemic shock. We conclude that the magnitude and kinetics of TNF-alpha production and TNF-alpha-dependent immunophysiological responses are differentially regulated after lethal fungal vs. gram-negative bacterial infection. Thus TNF-alpha is not a pivotal mediator of the acute Candida septic shock syndrome with disseminated candidiasis.

scholarly journals A 13-mer peptide straddling the leucine33/proline33 polymorphism in glycoprotein IIIa does not define the PLA1 epitope

Blood ◽  
1991 ◽  
Vol 77 (9) ◽  
pp. 1964-1969 ◽  
Author(s):  
F Flug ◽  
R Espinola ◽  
LX Liu ◽  
C SinQuee ◽  
R DaRosso ◽  
...  
Keyword(s):  
Amino Acid ◽  
Polyclonal Antibodies ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Adenosine Diphosphate ◽  
Enzyme Linked Immunosorbent Assay ◽  
Amino Acid Polymorphism ◽  
Glycoprotein Iiia ◽  
Polymerase Chain ◽  
Induced Aggregation

Abstract We confirm the recent report (J Clin Invest 83:1778, 1989) of a polymorphism at amino acid 33 of platelet GPIIIa associated with the PLA1/PLA2 phenotype by using the polymerase chain reaction on cDNA derived from platelet RNA, using the base-pair primers 105–129 and 452- 428. Platelet cDNA from three PLA2-homozygous individuals, when digested with Nci I, gave two bands of 256 bp and 91 bp, whereas eight PLA1 cDNAs gave a single band of 347 bp. Two 13-mer amino acid peptides straddling the amino acid polymorphism: SDEALP (L/P) GSPRCD were synthesized for epitope studies. Two mouse polyclonal antibodies were raised: one against the PLA1-associated peptide, the other against the PLA2 peptide. Both antibodies react with either peptide, as well as with both PLA1 and PLA2 platelets. The PLA1 peptide did not block the binding of two different human anti-PLA1 antibodies to the 100-Kd GPIIIa band on immunoblot of platelet extracts; neither did it block the binding of the same antibodies to PLA1-platelet extracts in an enzyme-linked immunosorbent assay. Further studies were performed on the PLA1 epitope following subtilisin digestion of purified GPIIIa. A 55-Kd fragment was obtained that retained the PLA1 epitope as well as the first 13 N-terminal amino acids of GPIIIa. Reduction of the 55-Kd fragment resulted in loss of the PLA1 epitope with production of a 67- Kd, 21-Kd, and 10-Kd band on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The 55-Kd band does not react with LK-2, a monoclonal antibody versus GPIIIa that inhibits adenosine diphosphate, collagen, epinephrine, and thrombin-induced aggregation. Thus, the PLA1 epitope is conformation-induced, resides on an N-terminal 55-Kd fragment composed of two or more peptides held together by -SH bonds, and is not required for platelet aggregation.

scholarly journals Plasma thrombomodulin in health and diseases

Blood ◽  
1990 ◽  
Vol 76 (10) ◽  
pp. 2024-2029 ◽  
Author(s):  
S Takano ◽  
S Kimura ◽  
S Ohdama ◽  
N Aoki
Keyword(s):  
Distress Syndrome ◽  
Pulmonary Thromboembolism ◽  
Polyacrylamide Gel Electrophoresis ◽  
Plasma Concentrations ◽  
Sodium Dodecyl ◽  
Acute Hepatic Failure ◽  
Immunoblot Analysis ◽  
Enzyme Linked Immunosorbent Assay ◽  
Molecular Weights ◽  
Disease States

Abstract Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblot analysis of plasma thrombomodulin concentrate revealed that four degraded forms of thrombomodulin with different molecular weights are present in plasma. Plasma concentrations of thrombomodulin in patients with various diseases were measured by two methods of enzyme- linked immunosorbent assay using monoclonal antibodies. One method measures intact thrombomodulin and degraded forms of thrombomodulin; the other does not detect the two smaller degraded forms of thrombomodulin present in plasma. The results indicated that thrombomodulin was increased in the circulating blood of patients with disseminated intravascular coagulation syndrome, pulmonary thromboembolism, adult respiratory distress syndrome, chronic renal failure, or acute hepatic failure. The different values obtained by the two methods indicate that the increase of plasma thrombomodulin found in these patients was mainly due to an increase of the smaller fragments of degraded forms, suggesting that the release of thrombomodulin from endothelial cells was accelerated in various disease states by proteolytic activity generated on the surface of the endothelium and may be removed from the circulation mostly by the kidneys and liver.

scholarly journals Comparison of the Proteosomes and Antigenicities of Secreted and Cellular Proteins Produced by Mycobacterium paratuberculosis

2006 ◽  
Vol 13 (10) ◽  
pp. 1155-1161 ◽  
Author(s):  
Donghee Cho ◽  
Michael T. Collins
Keyword(s):  
Solid Phase ◽  
Expression Profiles ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Size Exclusion ◽  
Enzyme Linked Immunosorbent Assay ◽  
Infected Cattle ◽  
Mycobacterium Paratuberculosis ◽  
Serologic Tests ◽  
Sds Page

ABSTRACT The protein expression profiles and antigenicities of both culture filtrates (CF) and cellular extracts (CE) of Mycobacterium paratuberculosis were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), one-dimensional electrophoresis (1-DE) and 2-DE immunoblotting, and enzyme-linked immunosorbent assay (ELISA). The CF proteins were harvested from supernatants of stationary-phase liquid cultures and concentrated by size exclusion filtration. The CE proteins were extracted by mechanical disruption of cells using glass beads and a high-speed agitator. Analysis of SDS-PAGE gels showed that the majority of CF proteins had low molecular masses (<50 kDa), whereas CE protein mass ranged more evenly over a broader range up to 100 kDa. By 2-DE, CF proteins had a narrow array of pI values, with most being between pH 4.0 and 5.5; CE proteins spanned pI values from pH 4.0 to 7.0. The antigenicities of CF and CE proteins were first determined by 1-DE and 2-DE immunoblotting with serum from a cow naturally infected with M. paratuberculosis. The serum reacted strongly to more proteins in the CF than the CE. Sera from 444 infected and 412 uninfected cattle were tested by ELISA with CF and CE as solid-phase antigens. Receiver-operator characteristic curve analysis of the ELISA results showed a significantly greater area under the curve for CF compared to CE (P < 0.05). A high degree of variability in protein binding patterns was shown with 1-DE immunoblot analysis with 31 sera from M. paratuberculosis-infected cattle. Collectively, these results indicate that serologic tests for bovine paratuberculosis may be improved by using proteins derived from CF instead of CE. To maximize the diagnostic sensitivity of serologic tests, multiple proteins will be required. Even so, a CF ELISA may not be able to detect all M. paratuberculosis-infected cattle, in particular those in the early stages of infection that have yet to mount an antibody response.

scholarly journals Antivinculin antibodies in sera of patients with immune thrombocytopenia and in sera of normal subjects

Blood ◽  
1992 ◽  
Vol 79 (1) ◽  
pp. 161-168 ◽  
Author(s):  
Y Tomiyama ◽  
R Kekomaki ◽  
J McFarland ◽  
TJ Kunicki
Keyword(s):  
Immune Thrombocytopenia ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Surface Protein ◽  
Normal Subjects ◽  
Enzyme Linked Immunosorbent Assay ◽  
Two Dimensional ◽  
Platelet Protein ◽  
Sds Page ◽  
Significant Difference

Abstract We have characterized a 120-Kd antigen that frequently reacts with serum antibodies from patients with immune thrombocytopenia or normal subjects. Immunoblots made after two-dimensional nonreduced/reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or two-dimensional isoelectric focusing/SDS-PAGE demonstrated that this 120-Kd protein has the same molecular weight under nonreduced or reduced conditions, is not a surface protein, and has an isoelectric point (pl) of 6.4 to 6.5. From these data, one likely candidate is the intracellular platelet protein, vinculin. Monoclonal antivinculin antibody reacts with this 120-Kd protein, and purified human platelet vinculin is bound by antibodies that recognize the 120-Kd protein. Therefore, we conclude that this 120-Kd protein is identical to vinculin. Data obtained from a sensitive enzyme-linked immunosorbent assay demonstrate the presence of naturally occurring antivinculin antibodies in many normal sera. However, the incidence of antivinculin antibodies in patient sera (67%; 55 of 82 sera) is significantly (P less than .01) higher than that in normal sera (40%; 32 of 80 sera), and there is a significant difference (P less than .05) between the mean levels of antivinculin antibodies in patient and normal sera. Whereas the levels of these antibodies in patient and normal sera overlap, 2 of 82 sera from patients with thrombocytopenia express unusually high levels of such antibodies. The pathologic significance of these antibodies remains to be determined.

scholarly journals High-molecular weight kininogen is present in cultured human endothelial cells: localization, isolation, and characterization

Blood ◽  
1988 ◽  
Vol 71 (5) ◽  
pp. 1268-1276 ◽  
Author(s):  
F van Iwaarden ◽  
PG de Groot ◽  
JJ Sixma ◽  
M Berrettini ◽  
BN Bouma
Keyword(s):  
Molecular Weight ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
High Molecular Weight ◽  
Light Chain ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Enzyme Linked Immunosorbent Assay ◽  
Human Endothelial Cells ◽  
Isolation And Characterization

Abstract The presence of high-molecular weight (mol wt) kininogen was demonstrated in cultured human endothelial cells derived from the umbilical cord by immunofluorescence techniques. Cultured human endothelial cells contain 58 +/- 11 ng (n = 16) high-mol wt kininogen/10(6) cells as determined by an enzyme-linked immunosorbent assay (ELISA) specific for high-mol wt kininogen. High-mol wt kininogen was isolated from cultured human endothelial cells by immunoaffinity chromatography. Nonreduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that endothelial cell high-mol wt kininogen consisted of five protein bands with mol wts of 95,000, 85,000, 65,000, 46,000, and 30,000 daltons. Immunoblotting of the endothelial cell high-mol wt kininogen by using specific antisera against the heavy and light chain indicated that the 95,000-, 85,000-, and 65,000-dalton bands consisted of the heavy and light chain whereas the 46,000- and 30,000-dalton bands reacted only with the anti-light chain antiserum. Immunoprecipitation studies performed with lysed, metabolically labeled endothelial cells and monospecific antisera directed against high-mol wt kininogen suggested that high-mol wt kininogen is not synthesized by the endothelial cells. Endothelial cells cultured in high-mol wt kininogen-free medium did not contain high-mol wt kininogen. These studies indicate that endothelial cell high-mol wt kininogen was proteolytically cleaved in the culture medium and subsequently internalized by the endothelial cells. Binding and internalization studies performed with 125I-labeled, proteolytically cleaved, high-mol wt kininogen showed that endothelial cells can indeed bind and internalize proteolytically cleaved high-mol wt kininogen in a specific and saturable way.

Characterization of galanin receptor in canine small intestinal circular muscle synaptosomes

1994 ◽  
Vol 266 (1) ◽  
pp. G106-G112 ◽  
Author(s):  
C. K. Chen ◽  
T. J. McDonald ◽  
E. E. Daniel
Keyword(s):  
Small Intestine ◽  
Binding Site ◽  
G Protein ◽  
Binding Capacity ◽  
Specific Binding ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Circular Muscle ◽  
Galanin Receptor ◽  
High Affinity

We used 125I-galanin (porcine) as ligand to study the galanin receptors in circular muscle and deep muscular plexus from canine small intestine. Specific binding sites were found in both nerve and muscle membranes. On synaptosomal membranes, the equilibrium binding study showed a high-affinity (dissociation constant, Kd = 1.1 +/- 0.13 nM; maximum binding capacity, Bmax = 244 +/- 2.1 fmol/mg) binding site. The specific binding of 125I-galanin to nerve membrane was inhibited by galanin or NH2-terminal galanin fragments but not by the COOH-terminal fragment. Computer analysis suggested a two-site model (inhibitor constants, Ki1 = 0.02 +/- 0.005 nM and Ki2 = 1.05 +/- 0.3 nM) for competition by galanin-(1-29). Kinetic and competition studies using guanosine 5'-O-(3-thiotriphosphate) or pertussis toxin (PTX) suggested that the high-affinity binding site involved a PTX-sensitive G protein which acted to slow dissociation of bound galanin from the receptor. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the galanin receptor complex revealed a radioactive band at 50 kDa. We conclude that, in canine small intestine, galanin may act as an inhibitory neuromodulator by a PTX-sensitive G protein-coupled interaction of galanin and its specific receptor on enteric nerve synaptosomes

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