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Biomedical subjects

J Kolberg

Publications and source records attributed to J Kolberg.

At least 73 records · Page 4Linked to original sources
PubMed1991

Use of a signature nucleotide sequence of hepatitis C virus for detection of viral RNA in human serum and plasma.

The nucleic acid sequence of the putative 5'-untranslated (5PUT) region of hepatitis C virus (HCV), determined for samples obtained from a variety of geographic origins, was found to be over 98% conserved among all isolates. On the basis of this signature sequence for HCV, a viral RNA assay was developed by using cDNA synthesis with reverse transcriptase, followed by polymerase chain reaction (PCR). The new assay was compared with the Ortho-Chiron C100-3 HCV enzyme-linked immunosorbent assay to research radioimmunoassays for antibodies to the C33c and C22 HCV antigens and to the first reported set of HCV PCR primers designed from the NS3 domain. Plasma samples from 16 Japanese patients with non-A, non-B hepatitis (NANBH) and 16 immunoassay-positive blood donors from the United States were investigated. The 5PUT PCR primers were found to be superior to the NS3 primers in sensitivity and specificity (15 of 25 versus 3 of 25 of the C100 enzyme-linked immunosorbent assay-positive samples, respectively). Samples from two C100-negative patients with acute NANBH were found to react with the 5PUT primers but not with the NS3 primers. Also, two of three patients with chronic NANBH converted from reverse transcriptase PCR positive to negative after interferon treatment. Although the clinical significance of the presence or absence of HCV RNA in samples from patients is not fully understood, the use of probes and primers from the 5PUT region (as opposed to primers from other segments) should not lead to false-negative results due to nucleic acid sequence variations in viral isolates.

Base Sequence↗
PubMed1991

Production and characterization of a mouse monoclonal antibody specific for lentil lectin.

A murine IgA monoclonal antibody (MoAb) was produced against the widely used glucose/mannose-specific two-chain mitogen from Lens culinaris (lentil) belonging to the Vicieae tribe of the Leguminosae family. The MoAb designated, 98, F-10, was found to be specific for lentil lectin when tested in dot blotting against 22 different native lectins. The antigenic specificity was also tested against subunits of 13 completely sequenced legume lectins separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and electrotransferred to nitrocellulose filters. The MoAb showed a strong reaction only against the lentil heavy subunit. Comparison of the amino-acid sequences revealed 13 amino-acid residues which might be involved in the epitope reactive with this antibody. The MoAb did not react with synthetic peptides from the heavy subunit of lentil.

Amino Acid Sequence↗
PubMed1989

Further characterization of monoclonal antibody 6,F-8 reacting with Lathyrus, Lens and Pisum lectins.

The murine monoclonal antibody (MoAB) 6,F-8 made against the glucose/mannose-specific Lathyrus odoratus mitogen has previously been shown to react with Lens culinaris and Pisum sativum lectins, but not with the lectin from Vicia faba [(1988) Biol. Chem. Hoppe-Seyler 369, 365-370]. The reactivity against seven other completely sequenced Lathyrus lectins has now been tested after separation of the subunits by SDS-polyacrylamide gel electrophoresis and electroblotting to nitrocellulose filters. Two of these lectins reacted with the antibody. Comparison of the amino acid sequences of the examined lectins and the predicted hydrophilic, flexible and accessible regions of Pisum sativum suggest that valine-147 is involved in antibody binding.

Amino Acid Sequence↗
PubMed1989

Application of a rapid non-radioisotopic nucleic acid analysis system to the detection of sexually transmitted disease-causing organisms and their associated antimicrobial resistances.

We devised a versatile method for detecting nucleic acids in crude lysates of biological samples. A controlled network of nucleic acid hybrids composed of the target fragment, several oligonucleotide probes, branched DNA amplifiers, and labeled oligonucleotides is produced on a solid phase to ultimately incorporate 60 to 300 molecules of alkaline phosphatase, which are detected with a chemiluminescent substrate. The visible light output can be recorded on a luminometer or on instant black-and-white film. Assays have been developed for the detection of Chlamydia trachomatis, Neisseria gonorrhoeae, and for genes conferring penicillin and tetracycline resistance. Conducted much like ELISAS, the assays are performed in about 4 h (for 96 samples) in microliter dishes. The molecular detection limit of approximately 50,000 molecules of double-stranded DNA has permitted us to detect 1 to 10 x 10(3) of C. trachomatis and N. gonorrhoeae with specific probe sequences. Both plasmid and genomic target sequences can be detected by the same procedure. All of the assay components, except for a set of unmodified oligonucleotide probes, are universally applicable for all targets.

Chlamydia Infections↗
PubMed1988

Reactivity with legume lectins of three monoclonal antibodies made against the Lathyrus odoratus lectin.

Three murine IgG1 monoclonal antibodies (MoAb) were produced against the glucose/mannose-specific two-chain mitogen from seeds of Lathyrus odoratus (sweet pea) belonging to the Vicieae tribe of the Leguminosae family. Their antigenic specificities were tested against subunits of two-chain and one-chain legume lectins separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and electrotransferred to nitrocellulose filters. Different binding to native and detergent-treated lectins in dot-blots indicated that two of the antibodies bound to conformational epitopes which by immunoblotting were shown to be expressed on the heavy subunits from all the five tested two-chain lectins and on the six one-chain lectins, including PHA (Phaseolus vulgaris) and Con A (Canavalia ensiformis). However, ELISA cross-inhibition studies showed that these two antibodies bound to different epitopes on the lectin molecules. The third MoAb reacted with a continuous epitope not revealed by classical taxonomy since only three of five heavy subunits of the two-chain lectins stained with the antibody. Comparison of the known amino-acid sequences of the chains indicates eight positions as possible binding sites for this antibody.

Antibodies, Monoclonal↗
PubMed1987

Immunoblotting detection of lectins in gluten and white rice flour.

The gluten lectin was isolated by affinity chromatography, separated by sodium dodecyl sulphate-gel electrophoresis together with purified wheat germ agglutinin (WGA) and electrotransferred to nitrocellulose filters. The binding pattern of anti-WGA to the blotted filters confirmed the presence of WGA in gluten. A lectin from rice bran and white rice flour, respectively, was isolated by affinity chromatography. Both lectins reacted with anti-WA in immunoblotting. As patients with coeliac disease are known to tolerate rice flour, the finding of a WGA-like lectin questioned the suggestion that WGA in gluten is involved in the pathogenesis of coeliac disease. A second lectin was also isolated from rice flour which reacted only with antibodies against soybean lectin on immunoblots. This may indicate a contamination of soybean proteins in rice flour.

Antibodies↗
PubMed1986

Serum antibodies to wheat germ agglutinin and gluten in patients with dermatitis herpetiformis.

It has been speculated that gluten may play a role in the pathogenesis of dermatitis herpetiformis (DH) because it can act as a lectin. The lectin activity of gluten preparations was recently identified as wheat germ agglutinin (WGA). IgG and IgA serum antibodies to WGA and gluten were therefore measured in patients with DH and coeliac disease (CD) by an enzyme-linked immunosorbent assay (ELISA). Compared with healthy controls, both patients categories had increased IgG and IgA activities to WGA and gluten, the CD group showing the highest antibody levels. DH patients with subtotal villous atrophy tended to have higher activities than those with no villous changes or only minor changes. No significant difference in the gluten-to-WGA ratio of IgA or IgG antibodies was found when DH patients were compared with CD patients. If WGA plays a pathogenetic role in DH, then DH patients must have dermal characteristics, as yet undefined, that explain the initiation of their skin disease.

Adult↗
PubMed1986

Antibodies to wheat germ agglutinin in coeliac disease.

Serum IgG and IgA antibodies to wheat germ agglutinin (WGA) were measured by an enzyme-linked immunosorbent assay (ELISA) with N-acetyl-D-glucosamine in all incubation steps to inhibit sugar-specific binding. Patients with coeliac disease (CD) had significantly higher antibody levels to WGA than patients with other intestinal disorders or healthy controls. Similar results were obtained for antibodies to the gluten fraction glyc-gli. The WGA antibodies did apparently not cross-react with gluten antigens, but commercial gluten powder contained traces of WGA or a similar lectin. Our findings support the proposal that WGA may be involved in the pathogenesis of CD.

Adult↗
PubMed1985

Lectin activity of gluten identified as wheat germ agglutinin.

A lectin in gluten was detected by agglutination of papain-treated human erythrocytes. A partially purified lectin preparation was obtained by chromatography on immobilized ovalbumin. This fraction showed the same sugar specificity as wheat germ agglutinin (WGA). There was no indication of lectins with carbohydrate specificities different from WGA in the various gluten fractions examined. Small amounts of the gluten lectin was then isolated by using an affinity column with specificity for WGA. Analyses of this gluten lectin by sodium dodecyl sulphate-gel electrophoresis showed bands with the same mobility as that of purified WGA. Our results indicate that the lectin properties of gluten are due to traces of WGA. This finding is relevant for theories about the pathogenesis of coeliac disease.

Carbohydrates↗
PubMed1984

Antigenic similarities both inside and outside the carbohydrate-binding sites of two-chain and one-chain leguminous lectins.

Antibodies were made against the two-chain lectin Lath-O from the seeds of Lathyrus odoratus as well as its isolated light (alpha) and heavy (beta) chains. These antibodies were used to antigenically compare the Lath-O with other two-chain lectins like Lath-S, lentil and Vicia cracca glc specific and with one-chain lectins like Con A, PHA, peanut and soybean. Sensitive ELISA tests showed that there was a distinct antigenic cross-reaction between the one-chain and two-chain lectins both by using antibodies against Lath-O whole molecules and its isolated beta-chains, while the anti-Lath-O alpha-chain antibodies barely reacted at all even with Lath-O alpha-chains. The antibodies against Lath-O whole molecules also strongly inhibited the mitogenic responses induced by two-chain lectins. Antibodies reacting inside the carbohydrate-binding site of the lectins were isolated by eluting them with glucose from lectin-Sepharose columns and antibodies reacting outside the carbohydrate-binding site were subsequently eluted with guanidine or buffer with low pH. The two antibody fractions were separately used to antigenically compare one-chain and two-chain lectins, demonstrating that both kinds of antibodies showed cross-reaction between one-chain and two-chain lectins. Both the antibodies presumably reacting inside the carbohydrate-binding site of the lectins and those reacting outside the carbohydrate-binding site inhibited haemagglutination activity of two-chain lectins, while haemagglutination by one-chain lectins was unaffected. The carbohydrate-binding site specificity of the glucose-eluted antibodies was assessed by glucose-inhibition in ELISA tests. Furthermore, galactose neither inhibited in these tests nor eluted antibodies from lectin-Sepharose columns. Control experiments were also performed with normal rabbit IgG.

Animals↗
PubMed1983

Interactions of purified human ceruloplasmin with Lathyrus odoratus, Lens culinaris and Canavalia ensiformis lectins.

Human ceruloplasmin was isolated from normal serum by fractional polyethylene glycol precipitation and subsequent ion exchange chromatography. The molecular mass determined by ultracentrifugation was 132 kDa and an optical density ratio (A610/A280) of 0.046 was observed in the preparation. Immunoelectrophoresis, agarose-gel electrophoresis and N-terminal amino-acid sequence analyses showed that the ceruloplasmin preparation was highly purified, while dodecyl sulphate polyacrylamide-gel electrophoresis indicated some degradation of the protein. Affinity chromatography showed that only a fraction of ceruloplasmin was bound to Lens culinaris or Lathyrus odoratus lectin-Sepharose, whereas nearly all the protein was bound to Canavalia ensiformis lectin-Sepharose. The carbohydrate composition of the ceruloplasmin fractions was analysed. It is suggested that fucose might be a determinant for the microheterogeneity in the carbohydrate chains of ceruloplasmin.

Amino Acid Sequence↗
PubMed1983

Properties of a lectin purified from the seeds of Cicer arietinum.

A lectin was isolated from seed extracts of Cicer arietinum by (NH4)2SO4 precipitation and subsequent ion exchange chromatography and gel filtration. Affinity chromatography on desialylated human IgM coupled to AH-Sepharose was also performed, but the amount bound was very low. The lectin has a molecular mass of about 44000 Da, as determined by ultracentrifugation and gel filtration. Dodecyl sulphate polyacrylamide-gel electrophoresis showed one band corresponding to a molecular mass of 26000 Da. N-Terminal amino acid sequence analyses indicate only one type of chain, suggesting that the lectin is probably dimeric. The amino acid composition is given. Papainized human erythrocytes of the different ABO groups were agglutinated equally well by the Cicer lectin, whereas untreated cells reacted weakly and only in the presence of bovine serum albumin. Simple sugars did not inhibit the agglutination, but some glycoproteins did inhibit. The lectin is probably nonmitogenic against human lymphocytes. Antigenic analyses in an enzyme-linked immunosorbent assay (ELISA) showed only a weak cross-reaction between Cicer and the lectins in the Vicieae tribe. Thus, our physicochemical and antigenic studies of the Cicer lectin support the botanical reasons recently given for removing the genus Cicer from the Vicieae tribe.

Amino Acid Sequence↗
PubMed1983

The primary structure of the alpha chain of a mitogenic lectin from the seeds of Lathyrus sativus.

The complete primary structure of the alpha chain of the glucose- and mannose-specific lectin from Lathyrus sativus seeds has been determined. The structure was elucidated by Edman degradation on the whole chain as well as on fragments derived from cleaving the chain with BNPS-skatole and of peptides derived after tryptic digestion of the citraconylated chain. The analyses revealed a heterogeneity in the chain length (53 or 55 residues) and a microheterogeneity in positions 47 (aspartic acid and glycine). The sequence was highly homologous to the alpha chain of the lectin from Lathyrus odoratus.

Amino Acid Sequence↗
PubMed1982

Purification and properties of a mitogenic lectin from Lathyrus sativus seeds.

A mitogenic lectin has been isolated from saline extract of Lathyrus sativus by (NH4)2SO4 precipitation, chromatography on DEAE-Sepharose and subsequent affinity chromatography on Sephadex G-100. The lectin has a molecular weight of 49,000, as determined by ultracentrifugation, and consists of heavy (Mr 19,000) and light subunits (Mr about 4,400). The amino acid composition and N-terminal sequence of both subunits are given. The lectin agglutinated human erythrocytes of different ABO groups equally well, and the agglutination is inhibited best by D-mannose and D-glucose and their alpha-methyl-glucosides. High concentrations of the lectin were needed for optimal stimulation of human lymphocytes.

Amino Acids↗
PubMed1979

Mitogenic stimulation of human lymphocyte subpopulations by Lathyrus odoratus lectin.

Investigation was made of the mitogenic activation by Lathyrus odoratus (LATH) lectin and phytohaemagglutinin (PHA) of human T-enriched and non-T lympohcytes isolated by nylon wool filtration and sheep erythrocyte rosette depletion, respectively. LATH stimulated only weakly the T-enriched and non-T fractions as compared to PHA which strongly stimulated the T-enriched population and weakly stimulated the non-T cells. Removal of phagocytic cells from the unfractionated lymphocytes caused only a slight reduction in the response to LATH. The responses of mixtures of T-enriched and non-T lymphocytes were similar to that of unfractionated lymphocytes. The LATH lectin seems, therefore, to be neither a pure T nor a pure non-T cell mitogen, since both populations must be present for optimal response. When alpha-methyl-D-mannoside was added to unfractionated lymphocytes together with LATH, there was a strong inhibition of thymidine incorporation. D-glucose and N-acetyl-D-glucosamine diminished the stimulation effect to a lesser degree.

Cells, Cultured↗
PubMed1978

Isolation and partial characterization of a mitogenic lectin from Lathyrus odoratus seeds.

A mitogenic lectin has been isolated from saline extract of Lathyrus odoratus seeds by (NH4)2SO4 precipitation and subsequent chromatography on DEAE-Sepharose and CM-Sepharose. The isolated lectin was almost homogeneous by SDS gel electrophoresis and gel filtration, but multiple bands were obtained on isoelectric focusing. The molecular weight, determined by gel filtration, was 37,000. Gel electrophoresis in the presence of SDS gave a molecular weight of 19,000, thus suggesting that the lectin is a dimer. It agglutinated human erythrocytes of the different ABO groups equally well. Human lymphocytes were exposed to the lectin or PHA, and the incorporation of thymidine was measured. Compared with PHA, which stimulated maximally in the concentration range of 0.25--1microgram/ml, the L. odoratus lectin had optimal activity at concentrations of 35 microgram/ml and higher.

Carbohydrates↗