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Biomedical subjects

M Mäder

Publications and source records attributed to M Mäder.

At least 55 records · Page 3Linked to original sources
PubMed1994

Identification of beta-trace as prostaglandin D synthase.

Human cerebrospinal fluid (CSF) is the richest source of prostaglandin (PG) D synthase, a key enzyme in sleep regulation, having a specific activity of 20-130 nmol/min/mg protein that is almost two orders of magnitude higher than that of crude rat brain homogenate (2-7 nmol/min/mg protein). PGD synthase was purified from human CSF approximately 8-fold to apparent homogeneity in a yield of 6% by ammonium sulfate fractionation followed by column chromatographies on phenyl-Sepharose and DEAE-cellulose. The purified enzyme displayed enzymatic properties almost identical to those of rat brain PGD synthase in terms of Mr, specific activity, optimum pH, Km value, and SH requirement. The purified PGD synthase cross-reacted with an antibody against rat brain PGD synthase and also with that against human beta-trace, a major protein in the CSF. Furthermore, beta-trace cross-reacted with rat brain PGD synthase antibody. The N-terminal sequences of human PGD synthase, beta-trace, and purified PGD synthase from human CSF were essentially identical. These results clearly show that beta-trace is structurally and enzymologically identical to PGD synthase.

Amino Acid Sequence↗
PubMed1994

Human intestinal alkaline phosphatase-binding IgG in patients with severe bacterial infections.

Patterns of alkaline phosphatase (AP)-binding proteins were observed in the alkaline pH range of 6.5-9.5 upon isoelectric focusing and blotting of serum from patients with inflammatory diseases. After isolation using affinity chromatography on protein A or immunoaffinity chromatography on AP coupled to cyanogen bromide (CNBr)-activated Sepharose, the AP-binding protein was identified as IgG on Western blots and in ELISA using human IgG-specific antibodies. It was shown that this IgG binds to AP from both calf (bovine) and human intestine. However, it binds neither to the human liver-bone-kidney (LBK) isoform nor to bacterial AP. Moderate reaction was observed with human placental AP. Comparing patients with various diagnoses (n = 284), AP-binding antibodies were mainly found in severe bacterial infections. They were not detected in serum from healthy blood donors (n = 300). The presence of AP-binding IgG was independent of the infected organ and the bacterial species causing infection. This antibody may be useful for discriminating bacterial from viral infection and for indicating severe bacterial inflammation.

Alkaline Phosphatase↗
PubMed1993

Purification and N-terminal sequence of beta-trace, a protein abundant in human cerebrospinal fluid.

beta-Trace, a protein that represents a major constituent of human cerebrospinal fluid with unknown function has been purified to apparent homogeneity by gel chromatography and electrophoresis. After sodium dodecylsulfate electrophoresis on polyacrylamide gels (SDS-PAGE) and Western blotting, the N-terminal sequence (28 amino acids) has been determined. The high degree of identity with the corresponding sequences of prostaglandin D synthetase from rat (68%) and human (93%) suggests a strong relationship if not identity with this enzyme. Thus, 30 years after its discovery beta-trace might unravel as a well-known protein of the prostaglandin metabolism.

Amino Acid Sequence↗
PubMed1993

An efficient sandwich-ELISA for the determination of choline acetyltransferase.

A specific, sensitive, and reliable sandwich-ELISA (enzyme-linked immunosorbent assay) has been established for the determination of choline acetyltransferase (CHAT) from porcine brain. The detection limit of the assay was 30 micrograms/l and the assay was linear up to 300 micrograms/l. The within-day and day-to-day coefficients of variation were found to be 3.3% and 4.7% respectively for low CHAT concentrations (30 micrograms/l) and 3.1% and 3.4% respectively for high levels of CHAT (300 micrograms/l). The immunoassay was more sensitive than the radiometric assay of Fonnum which is widely used for the measurement of enzyme activity. In the assay monoclonal antibody was adsorbed to the polystyrene surface of the immunoplate as the capture reagent. Using a standard peroxidase protocol the immobilized antigen was detected with a highly specific anti-CHAT antiserum raised in rabbits. Two monoclonal antibodies were available for antigen binding. One of the two--A10.29B4--reacted preferentially with the active enzyme the other one--B3.9B3--reacted only with a degraded form. The polyclonal antiserum recognized both native and denatured enzyme. The effectiveness of employing the two monoclonal antibodies separately or in combination was demonstrated by measurement of porcine CHAT diluted in human cerebrospinal fluid and serum. After some minor modifications the sandwich-ELISA could be used for the determination of CHAT from the central nervous system of the rat.

Animals↗
PubMed1993

Cationic glycoproteins in sera of patients with acute infections identified as kappa light chain glycosylated IgG.

In half of the sera from patients with acute bacterial infections and 15% of the sera from patients with acute viral infections glycoproteins were found that form a scalariform pattern in the cationic range upon isoelectric focusing. The cationic glycoproteins appeared with the clinical illness. After subsidence of the symptoms they disappeared within 4 to 6 weeks. The proteins were identified as immunoglobulin G (IgG) by determination of the NH2-terminal amino acid sequence. Remarkably, these IgG only contained light chains of the kappa type with high proportions of carbohydrates. Both, N-glycosidic- and O-glycosidic-bound glycans were present. The glycosylated light chains may render the cationic IgG multireactive. Thus, it may be part of an early nonspecific immune defense mechanism.

Acute Disease↗
PubMed1993

Reactivity of locally produced CSF antibodies in patients with neurosyphilis against antigens of Treponema pallidum.

The reactivity and specificity of locally produced cerebrospinal fluid (CSF) antibodies against antigens of Treponema pallidum were assessed by Western blotting in patients with clinical signs of parenchymal or meningovascular neurosyphilis. All nine patients showed local production of treponeme-specific antibodies in the central nervous system (CNS). In most of the patients serum and CSF antibodies were bound to the same antigens: the common treponemal 48/45 kDa protein and the putative specific T. pallidum protein in the range of 12-14 kDa. In some patients the intensity of staining obtained by CSF antibodies was higher than that derived from serum, indicating locally produced antibodies. In contrast to other more acute inflammatory CNS diseases, no expanded or different antigen binding of the CSF antibodies compared with serum antibodies was found in neurosyphilic patients. The results presented are discussed with regard to the role of the blood-brain barrier in antibody concentrations of CSF and serum.

Adult↗
PubMed1993

Antibodies raised against synthetic peptides react with choline acetyltransferase in various immunoassays and in immunohistochemistry.

Antisera were raised in rabbits against five synthetic peptides. These peptides have been identified as potentially antigenic epitopes from the sequence of porcine choline acetyltransferase (ChAT) using primary and secondary structure analysis. All five antisera recognized immunoaffinity-purified antigen from porcine brain in an ELISA and on western blots. Four antisera recognized ChAT on dot blots, and another four antisera reacted with native and degraded enzyme in a sandwich ELISA using monoclonal antibodies as the capture antibody. One peptide antiserum was of similar avidity in this sandwich ELISA as a polyclonal antibody raised against immunoaffinity-purified ChAT. The same antiserum reacted with the enzyme from human placenta in an ELISA and on western and dot blots and recognized ChAT in rat, primate, and human neurons. Thus, a single peptide (amino acids 168-189) provides the means for easy, reliable, and reproducible generation of antibodies against ChAT suitable for replacing conventional polyclonal and monoclonal antibodies.

Animals↗
PubMed1993

Identification of cholinergic neurons in enteric nervous system by antibodies against choline acetyltransferase.

Several different monoclonal and polyclonal antibodies to choline acetyltransferase (ChAT) were screened to identify effective antibodies for immunocytochemical marking of cholinergic neurons in the enteric nervous system. Excellent immunohistochemical results were obtained with two of the antibodies in the myenteric plexus of the guinea pig stomach and small intestine. One was a mouse monoclonal antibody designated B3.9B3, and the second was a rabbit polyclonal antibody referred to as Peptide 3. Both antibodies clearly stained neuronal cell bodies as well as nerve fibers to the muscle layers and fibers encircling stained and unstained cell bodies. Cell counts indicated that approximately 64% (21.0 +/- 8.6 cells/ganglion) of gastric myenteric neurons are ChAT positive. Pelvic ganglia and the inferior mesenteric ganglia were examined as controls. Strong labeling of the majority of neurons was found in the pelvic ganglia, whereas few immunoreactive cells were apparent in the predominantly noradrenergic inferior mesenteric ganglion. Lack of effective antibodies to enteric neuronal ChAT has hampered progress in the study of the neurophysiology of cholinergic neurons in the digestive tract. Application of the B3.9B3 and Peptide 3 antibodies now promises to facilitate investigation of this important subset of enteric neurons.

Animals↗
PubMed1992

Characterization of mono- and polyclonal antibodies against highly purified choline acetyltransferase: a monoclonal antibody shows reactivity in human brain.

Choline acetyltransferase (ChAT) from porcine brain was purified by immunoaffinity chromatography, and the highly purified enzyme was subsequently used for immunization of mice and rabbits. After fusion of mouse spleen cells, 32 cultures producing monoclonal antibodies directed against ChAT were detected by an enzyme-linked immunosorbent assay (ELISA) with immunoaffinity-purified ChAT. Of these original 32, the most active 11 cultures were cloned and used for ascites production. The 11 clones generated monoclonal antibodies of the immunoglobulin (Ig) M class (three), the IgG1 subclass (seven), and the IgG2b subclass (one). The isoelectric points of the antibodies of the IgG class were different in each case. The monoclonal antibodies exhibited different binding characteristics in the above ELISA and on western blots. Two monoclonal antibodies demonstrated excellent immunohistological results with neurons of rat brain and spinal cord. One of them reacted well immunohistochemically with neurons of human brain and also recognized partially purified human placenta ChAT in the ELISA.

Animals↗
PubMed1991

Neuronal acetylcholinesterase levels in cerebrospinal fluid and serum determined by a specific and sensitive immunoassay.

Acetylcholinesterase levels were determined in cerebrospinal fluid and serum of 272 patients with various neurological disorders. The patients were ordered in 13 diagnostic groups. The assay employed was an antigen capture assay based on an immobilized monoclonal antibody selective for neuronal acetylcholinesterase (Rasmussen et al., Clin. Chim. Acta 166 (1987) 17-25 (1)). In 100% of the cases the acetylcholinesterase levels in cerebrospinal fluid were 500-10,000 fold higher than expected for a regular serum-derived protein (0.1-0.5%) at the intact blood-brain barrier. In both compartments the test values showed wide variations, which were much greater in serum than in cerebrospinal fluid. No specific relationship was discernible between the values obtained and any of the diagnostic groups, although decreased levels of acetylcholinesterase were found in bacterial and viral meningitis, and elevated levels were found in groups with more chronic diseases. A possible transfer of acetylcholinesterase from cerebrospinal fluid to blood is discussed.

Acetylcholinesterase↗
PubMed1991

A cationic glycoprotein pattern in human serum and cerebrospinal fluid with pathological implications.

Patterns of highly glycosylated proteins with mainly cationic isoelectric points, pH 6.5-9.5, were observed in serum and cerebrospinal fluid of patients with various disorders. Detection was performed after isoelectric focusing, using an immunoassay specific for digoxigenylated carbohydrate moieties of glycoconjugates. To our knowledge, these glycoproteins have not hitherto been described as regular serum proteins. The patterns were found among 7% of the patients studied (n = 400). Similar bands were not detectable in a reference group of 100 persons without clinical symptoms. The glycoprotein pattern was specific for each individual. The pathophysiological meaning of these glycoproteins as well as the basic biochemistry has not yet been evaluated. The glycoproteins, however, were shown to differ from immunoglobulin G, oligoclonal immunoglobulin G, paraprotein or from regular cationic serum protein. By comparison with standard glycoproteins a carbohydrate content of 30 +/- 10% was roughly suggested. The oligosaccharides contain probably sialic acid as evidenced by lectin binding. Although the diagnoses varied, 90% of the patients with this glycoprotein pattern had inflammatory processes.

Blotting, Western↗
PubMed1990

Large-scale purification of choline acetyltransferase and production of highly specific antisera.

Choline acetyltransferase (ChAT) was purified by immunoaffinity chromatography using a covalently immobilized monoclonal antibody. In a two-step procedure, 10 kg porcine brain yielded 750 micrograms active enzyme of apparent homogeneity. This amount of ChAT was purified routinely. The purification factor was 18,000 and the yield of activity 4.3%. The affinity resin was stable under the experimental conditions applied and was used many times. The highly purified enzyme was subsequently employed to obtain a specific anti-ChAT antiserum of high titer.

Animals↗
PubMed1990

Selection of brain choline acetyltransferase synthetic peptides.

Choline acetyltransferase (ChAT) of pig brain is a 640 amino acid enzyme. The sequence has recently been published (Berrard et al., 1988). We identified potentially antigenic epitopes on porcine ChAT by primary and secondary structure analysis. We used 12 structural parameters derived from the amino acid sequence, especially surface antigenicity, internal repetitions and protease cleavage site clusters and superposed gliding averages for the complete ChAT protein. Four peptides from 13 to 21 amino acids in length were defined for antibody production. In detail, we describe a nine-step selection process and discuss costs of synthesis and purification.

Amino Acid Sequence↗
PubMed1988

A reevaluation of choline acetyltransferase activity in human cerebrospinal fluid and serum.

Choline acetyltransferase (EC 2.3.1.6) was studied in human cerebrospinal fluid and blood. Sensitivity, precision and reproducibility of the radioactive assay were evaluated with a homogenate from pig brain and with partially purified enzymes from bovine brain and human placenta. Contrary to other reports, with the assay used in the present study, choline acetyltransferase activity was not detectable in the cerebrospinal fluid or blood of 50 patients with various neurological disorders. Only a very slight but significant non-enzymatic formation of a radioactive product by concentrated cerebrospinal fluid was observed. Nonenzymatic but enzyme-like formation of acetylcholine by thiol reagents and other compounds is discussed.

Animals↗
PubMed1982

Role of peroxidase in lignification of tobacco cells : I. Oxidation of nicotinamide adenine dinucleotide and formation of hydrogen peroxide by cell wall peroxidases.

The two peroxidase isoenzyme groups (G(I) and G(III)) localized in the cell walls of tobacco (Nicotiana tabacum L.) tissues were compared with respect to their capacity for NADH-dependent H(2)O(2) formation. Peroxidases of the G(III) group are slightly more active than those of the G(I) group when both are assayed under optimal conditions. This difference is probably not of major regulatory importance. NADH-dependent formation of H(2)O(2) required the presence of Mn(2+) and a phenol as cofactors. The addition of H(2)O(2) to the reaction mixture accelerated subsequent NADH-dependent H(2)O(2) formation. In the presence of both cofactors or Mn(2+) alone, catalase oxidized NADH. However, if the cofactors were absent or if only dichlorophenol was present, catalase inhibited NADH oxidation. No H(2)O(2) accumulation occurred in the presence of catalase. Superoxide dismutase inhibited NADH oxidation quite significantly indicating the involvement of the superoxide radical in the peroxidase reaction. These results are interpreted to mean that the reactions whereby tobacco cell wall peroxidases catalyze NADH-dependent H(2)O(2) formation are similar to those proposed for horseradish peroxidase (Halliwell 1978 Planta 140: 81-88).

Journal Article↗
PubMed1982

Role of Peroxidase in Lignification of Tobacco Cells : II. Regulation by Phenolic Compounds.

Coniferyl alcohol is the primary substrate for peroxidase-mediated lignification, a process which depends on the generation of H(2)O(2) by NADH oxidation. We measured the concentrations of various phenols (synthetic and natural) at which maximal enhancement of NADH oxidation occurs. Coniferyl alcohol was found to stimulate NADH oxidation at a much lower concentration (0.01 mm) than other natural or synthetic phenols (1-100 mm). In addition, coniferyl alcohol prevented the conversion of active peroxidase into the inactive intermediate compound III-which is usually formed in the presence of NADH-at equally low concentrations. This conversion was found to be a prerequisite for stimulation of NADH-oxidation, but it was not necessarily connected to stimulation.The oxidation of NADH and coniferyl alcohol (or guaiacol) can occur simultaneously, but there is a strong competitive interaction between these two substrates. At pH 5, the presence of NADH at concentrations 30 to 60 times lower than the phenols completely prevents their oxidation. The results are discussed in relation to the role of cell wall peroxidases in conversion of coniferyl alcohol to lignin and in formation of H(2)O(2).

Journal Article↗