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Biomedical subjects

H Towbin

Publications and source records attributed to H Towbin.

54 records · Page 3Linked to original sources
PubMed1988

Serological activity against galactosyl-alpha(1-3)galactose in sera from patients with several kinetoplastida infections.

Using rabbit erythrocyte-derived neutral glycosphingolipids enriched for a defined ceramide pentasaccharide as antigens, we have detected elevated anti-galactosyl-alpha(1-3)galactose (anti-G alpha G) antibody values in patients with American cutaneous leishmaniasis (ACL), chronic Chagas' disease, and Trypanosoma rangeli infections compared with normal subjects or with patients suffering from any of 15 other infectious diseases. The specificity of the G alpha G antibodies was determined by inhibition enzyme-linked immunosorbent assays, which revealed that several alpha-galactosyl- but not beta-galactosyl-bearing sugars blocked absorption of G alpha G antibodies to the specific antigen used. G alpha G antibodies were mainly distributed between immunoglobulin classes G and M in three Kinetoplastida infections studied, with a lower increase in reactivity detected in immunoglobulin A. Absorption of highly reactive G alpha G antibodies with purified murine laminin and nidogen, two basement membrane proteins, almost abolished G alpha G reactivity, suggesting the identity of anti-G alpha G with laminin and nidogen antibodies previously reported as elevated in Kinetoplastida infections. In ACL, G alpha G antibodies were detected in 71% of patients having skin lesions with a clinical evolution time of 0.5 month. This percentage increased with the time of evolution of skin lesions, reaching 93% in lesions older than 3 months, and tended to decrease inversely to the induration diameter in the skin leishmanin test. It is proposed that similar epitopes may exist on kinetoplast protozoa and that the determination of G alpha G antibodies may be a highly sensitive assay for the detection of humoral responses to Kinetoplastida infections.

ABO Blood-Group System↗
PubMed1987

Circulating antibodies to mouse laminin in Chagas disease, American cutaneous leishmaniasis, and normal individuals recognize terminal galactosyl(alpha 1-3)-galactose epitopes.

Sera from patients with American cutaneous leishmaniasis and Chagas disease and from monkeys infected with either Trypanosoma cruzi or Trypanosoma rhodesiense show, in RIAs, strong binding to mouse laminin. A distinct although weaker binding activity is also detected in normal human sera. The antibodies recognize a common carbohydrate epitope present on mouse laminin, which was assigned to a terminal galactosyl(alpha 1-3)-galactose group. Distinct crossreactions were observed with some other basement membrane proteins, rabbit glycosphingolipids, defucosylated human B blood group substance and components produced by some human tumor cells. Only little activity was, however, found on laminin obtained from human placenta. The data indicate that the antibodies arising in infectious diseases are stimulated by similar carbohydrate epitopes present on the surface of parasites. Tissue-specific occurrence of such epitopes may exist and explain the involvement of distinct tissues in autoimmune disorders.

Animals↗
PubMed1985

Chromogenic labeling of monosaccharides using 4'-N,N-dimethylamino-4-aminoazobenzene.

Twenty-three monosaccharides, e.g., D- or L-pentoses, D- or L-hexoses, heptose, 2- or 6-deoxyhexoses, 2-deoxy-2-aminohexoses, hexuronic acids, and N-acetylmuramic acid, were coupled to the azo dye 4'-N,N-dimethylamino-4-aminoazobenzene by reductive amination using sodium cyanoborohydride as reducing agent and in the presence of pentaerythritol. The structure of the colored glycamines was established by mass spectrometry. The average yield of the reaction was more than 80%. The sugar derivatives were separated either by silica-gel thin-layer chromatography or by high-performance liquid chromatography. Spectrophotometric quantitation was performed in the visible range at the picomole level. The method was applied to the determination of the sugar composition of the glycosphingolipid globotetraosyl ceramide and the human milk oligosaccharide lacto-N-fucopentaose I.

Amination↗
PubMed1984

Glycosphingolipid-blotting: an immunological detection procedure after separation by thin layer chromatography.

A method for detecting glycosphingolipids (GSL) in situ after thin layer chromatography is described. The separated GSL are transferred by diffusion to nitrocellulose. The replica is incubated with poly- or monoclonal antibodies and bound antibodies are detected with second antibodies coupled to peroxidase. Advantages of the procedure are its speed, the non-radioactive detection method, and its suitability for screening applications. In addition, small scale affinity purification of antibodies from the replicas is possible. The presence of Forssman antigen in mouse tissues and the reaction of monoclonal antibodies with human GSL is demonstrated.

Animals↗
PubMed1982

Monoclonal antibodies in analysis of cathepsin G-digested proteolytic fragments of human plasma fibronectin.

Proteolytic fragments of fibronectin, obtained by digestion with cathepsin G, were transferred electrophoretically from sodium dodecyl sulphate (NaDodSO4) polyacrylamide gels to nitrocellulose sheets and used as antigens for monoclonal antibodies. All 9 monoclonal antibodies tested reacted with undenatured intact fibronectin or its fragments applied directly to nitrocellulose sheets. Two of the clones did not react with the NaDodSO4-treated transferred material suggesting reactivity with conformational determinants. Distinct fragments of fibronectin could be detected by several of the antibodies. None of the monoclonal or the polyclonal antibodies used reacted with the Mr = 40,000 or Mr = 30,000 gelatin-binding fragments of fibronectin. However, one of the monoclonal antibodies reacted specifically with their precursor Mr = 64,000 fragment, but apparently with its gelatin-nonbinding segment. The apparent non-immunogenicity of the gelatin-binding domain is conspicuous, suggesting that it may be highly conserved in evolution. The present method, combination of controlled proteolytic cleavage with electrophoretic transfer, provides an effective means for characterization of monoclonal antibodies raised against proteins.

Animals↗
PubMed1982

Monoclonal antibodies against eucaryotic ribosomes. Use to characterize a ribosomal protein not previously identified and antigenically related to the acidic phosphoproteins P1/P2.

Mice were immunized against chick ribosomes with the use of various protocols and immunogen preparations. Hybridomas were prepared, clones screened, and specific antibodies identified by reversible protein staining followed by immunoperoxidase staining on nitrocellulose blots. Clones were obtained which secreted specific antibodies against ribosomal proteins S6, L7, L18a, P1/P2, and also against ribosomal RNA. Antibodies were typed by means of a dot-binding assay with typing antibodies immobilized on a solid support of nitrocellulose, and also characterized by their species cross-reactivities. The common determinant on proteins P1 and P2 cross-reacted with proteins of similar molecular weight in all eucaryotes tested, and with a determinant in a previously uncharacterized 38,000-dalton protein of the large ribosomal subunit. We designate this protein P0. The determinant of P0 was also present in a protein of similar molecular weight in all eucaryotes tested. Unlike P1 and P2, P0 was not removable from ribosomes by an ethanol-NH4Cl washing procedure. No evidence for a precursor-product relationship between P0 and P1/P2 was found. P0, P1, and P2 were found in active polysomes and in the nucleolus. The molecular weights of the nucleolar forms were not identical with those of the cytoplasmic forms, suggesting some processing during ribosomal assembly and/or transport.

Animals↗
PubMed1982

The phosphorylation of ribosomal protein S6 in rat tissues following cycloheximide injection, in diabetes, and after denervation of diaphragm. A simple immunological determination of the extent of S6 phosphorylation on protein blots.

The extent of phosphorylation of S6 was measured in a variety of cells, organs, and tissues using a simplified procedure. Ribosomal proteins were separated by electrophoresis on a one-dimensional urea-sodium dodecyl sulfate gel. They were transferred electrophoretically to nitrocellulose, and S6 was specifically stained by means of its reaction with a monoclonal antibody. The mobility of the stained protein was shown to be a measure of the extent of its phosphorylation. Using this method, we found that cycloheximide injection of rats gives rise to the phosphorylation of S6 in a variety of organs and tissues: diaphragm, heart and skeletal muscle liver and kidney, but not brain. Diabetes induced with alloxan did not result in the phosphorylation of S6 in any of the tissues examined, and subsequent insulin injection resulted in a slight increase in the phosphorylation of S6 in liver. Insulin injection in normal rats also caused the phosphorylation of liver S6. Unilateral denervation of rat diaphragm resulted in phosphorylation of S6 in the denervated portion but not in the innervated portion of the diaphragm.

Animals↗
PubMed1982

Ribosomal protein S6 from Xenopus laevis ovaries. Isolation, phosphorylation in vivo and cross-reaction with heterologous anti-S6 antibodies.

Ribosomal protein S6 from Xenopus laevis ovaries was prepared by ion-exchange chromatography on phosphocellulose and gel filtration on Sephadex G-75. The protein was identified as S6 from its position on two-dimensional polyacrylamide gels and from its immunological cross-reaction with monoclonal antibody raised against chicken liver S6, and from the fact that it is the major phosphorylated protein of the small subunit. When oocytes were incubated with [32P]orthophosphate in the presence of progesterone, 32P incorporation of 40-S ribosomal proteins was stimulated about 10-fold over controls without hormone. The bulk of the 32P radioactivity was incorporated into protein S6.

Animals↗
PubMed1979

Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.

A method has been devised for the electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets. The method results in quantitative transfer of ribosomal proteins from gels containing urea. For sodium dodecyl sulfate gels, the original band pattern was obtained with no loss of resolution, but the transfer was not quantitative. The method allows detection of proteins by autoradiography and is simpler than conventional procedures. The immobilized proteins were detectable by immunological procedures. All additional binding capacity on the nitrocellulose was blocked with excess protein; then a specific antibody was bound and, finally, a second antibody directed against the first antibody. The second antibody was either radioactively labeled or conjugated to fluorescein or to peroxidase. The specific protein was then detected by either autoradiography, under UV light, or by the peroxidase reaction product, respectively. In the latter case, as little as 100 pg of protein was clearly detectable. It is anticipated that the procedure will be applicable to analysis of a wide variety of proteins with specific reactions or ligands.

Animals↗
PubMed1978

A photoaffinity labelling study of the messenger RNA-binding region of Escherichia coli ribosomes.

A photoaffinity labelling study of the messenger RNA-binding region of E. coli ribosomes has been made, using oligoadenylic acids as mRNA analogs. The oligonucleotides, of chain length 6 to 8 and thus several nucleotides longer than oligonucleotides previously employed for this purpose, carried a radioactive photolabile aromatic azide reagent bound covalently to the 3'-terminal ribose moiety. The synthesis of the reagent, p-azidobenzoyl-(3H)-glycylhydrazide, is described. The derivatized oligonucleotides were shown to be functional messengers. They stimulated the binding of the cognate aminoacyl-tRNA, lysyl-tRNA: their binding was reciprocally stimulated by lysyl-tRNA; and they competed with underivatized oligoadenylates for ribosomal binding sites. When the 70 S ribosomal binding complex was irradiated, the photolabile reagent reacted covalently with both RNA and proteins of the 30 S subunit and with tRNA, but not with the 50 S subunit. The 16 S RNA appeared to be labelled at more than one site. Of the proteins, S3 and S5 reacted with the reagent with high specificity; and the possibility was not eliminated that S4 may have been labelled to a minor degree. Functional studies in other laboratories have implicated S3 and S5 in the decoding process, but these proteins were not labelled by any of the previously reported mRNA affinity labelling analogs. The results reported here therefore indicate that S3 and S5 not only affect the decoding process, but are located in the mRNA-binding region of the ribosome, presumably to the 3' side of the decoding site.

Affinity Labels↗
PubMed1976

The use of a cleavable crosslinking reagent to identify neighboring proteins in the 30-S ribosomal subunit of Escherichia coli.

A cleavable bifunctional reagent, dimethyl 3,3'-dithiobispropionimidate, has been used to crosslink proteins that occupy neighboring positions in the 30-S ribosomal subunit of Escherichia coli. The crosslinked proteins were identified, fully or partly, by their positions in two two-dimensional gel electrophoretic systems, one diagonal and the other quasi-diagonal, in which the complexes were cleaved after the first-dimensional run. It was found to be necessary to block the protein sulfhydryl groups in order to prevent artifactual disulfide crosslinking after extraction of the protein from ribosome. Eleven crosslinked complexes were detected. Four were fully identified: the triplet S4-S5-S8, and the pairs S2-S3, S4-S5, and S5-S8. In five others one component was identified unambiguously. No additional complexes were seen when the longer homologous butyro and capro reagents were used.

Binding Sites↗
PubMedDate not supplied

Direct chemiluminescence immunoassay (CLIA) for muramyl tripeptide phosphatidyl-ethanolamine in plasma.

A competitive chemiluminescent immunoassay for quantitation of muramyl tripeptide phosphatidyl-ethanolamine (MTP-PE) in plasma has been developed. The assay is based on the use of an acridinium ester-labelled analogue of muramyl tripeptide and a rabbit antiserum. It includes an overnight incubation and a separation with a second antibody covalently coupled to paramagnetic particles. The sensitivity of detection is 0.012 nmol/l, the assay working range is 0.1-5 nmol/l, and the inter-assay CVs are less than or equal to 10%. Using up to 6000-fold sample dilutions, a wide working range (0.1-30,000 nmol/l) is obtained. Rat plasma samples were collected during and one day after intravenous infusion of MTP-PE. Following infusion, the concentrations in plasma declined multiphasically. Half-life time was 0.37 h +/- 0.03 (mean +/- SD, alpha phase) and 1.76 h +/- 0.08 (mean +/- SD, beta phase), clearance and volume of distribution were 0.09 +/- 0.02 l/h x kg (mean +/- SD) and 0.06 +/- 0.01 l/kg (mean +/- SD) respectively. The use of an acridinium ester as a chemiluminescent (CL) label overcomes the problems associated with reagents of limited shelf-life.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMedDate not supplied

Acridinium ester labelled cytokines: receptor binding studies with human interleukin-1 alpha, interleukin-1 beta and interferon-gamma.

As a consequence of environmental protection and legal restrictions, increasing efforts are made to avoid radioactivity. One alternative is the labelling of ligands with chemiluminescent acridinium esters such as 2,6-dimethyl-4-(N-succinimidyloxy-carbonyl)phenyl 10-methylacridinium-9-carboxylate methosulphate (DMAE-NHS). When exposed to hydrogen peroxide in a basic solution, the DMAE-moiety decays with emission of a short-lasting chemiluminescent flash. With the goal of replacing the radioactive label in protein ligands with a DMAE label, and of increasing the efficiency by using microtitre plate technology for DMAE detection, we compared the receptor binding properties of iodinated interleukin-1 alpha (125I-IL-1 alpha), interleukin-1 beta (125I-IL-1 beta) and interferon-gamma (125I-IFN-gamma) with the corresponding DMAE-labelled ligands. The luminescence signal was assessed in a single-tube luminometer and in the prototype of a chemiluminescent microtitre plate reader. Derivatization of the three proteins with DMAE-N-hydroxy-succinimide resulted in photon yields of up to 100,000 counts per femtomole. As shown by Scatchard analysis, no significant loss of receptor binding affinity was observed, which might have been expected as a consequence of the chemical modification of the proteins. The use of DMAE labelling of proteins has the following advantages as compared to iodination: (i) the coupling reaction and binding assay can be performed in a normal laboratory, (ii) since there is no radiolysis, the DMAE-labelled proteins remain stable, (iii) the detection sensitivity may be improved as a consequence of higher specific activity of the DMAE label.(ABSTRACT TRUNCATED AT 250 WORDS)

Acridines↗
PubMedDate not supplied

Antibodies directed against ribosomal protein determinants in the sera of patients with connective tissue diseases.

A collection of sera from patients with systemic lupus erythematosus, rheumatoid arthritis, mixed connective tissue disease, Sjögren's syndrome, progressive systemic sclerosis, and the reflex sympathetic dystrophy syndrome was screened for the presence of antibodies recognizing determinants on the protein moiety of human ribosomes after electrophoretic separation in polyacrylamide and blotting on nitrocellulose. In addition to the antibodies already described against the RNA moiety or against the whole ribosome, some autoimmune sera contain antibodies against the protein moiety. The results obtained by this methodology were compared with those obtained by a microtiter procedure.

Antibodies↗
PubMedDate not supplied

Neutralization of interleukin-1 beta activity in vivo with a monoclonal antibody alleviates collagen-induced arthritis in DBA/1 mice and prevents the associated acute-phase response.

Interleukin-1 (IL-1) has been implicated in the development and progression of a variety of acute and chronic inflammatory diseases. Due to its pro-inflammatory and tissue-degrading activities, IL-1 is regarded as a major mediator of chronic inflammatory joint diseases, including rheumatoid arthritis in man, adjuvant arthritis in rats and collagen-induced arthritis in mice. However, conclusive experimental evidence for the crucial role of IL-1 in the development of joint destruction has not been presented as yet. In the present study, we investigated the effect of a neutralizing monoclonal mouse antibody against mouse IL-1 beta (IgG1 isotype) on the development and progression of collagen-induced arthritis in DBA/1 mice. The antibody was injected intraperitoneally 3 times a week, either from day 3 or from day 21 after primary immunization, to day 60. In the positive control group an arthritis incidence of 80% was observed after 60 days. The injection of a control antibody of the same isotype did not influence the incidence of arthritis, whereas injection of anti-IL-1 beta from day 21 reduced the arthritis incidence to about 30%. Injection of anti-IL-1 beta starting at day 3 totally prevented both the development of arthritis and the associated increase of the acute phase protein serum amyloid P (SAP). Anti-collagen antibody titers, which increased significantly after immunization, were not influenced by the injection of anti-IL-1 beta antibodies, in spite of the suppressive effect on arthritis development.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute-Phase Reaction↗