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Biomedical subjects

N H Heegaard

Publications and source records attributed to N H Heegaard.

At least 19 recordsLinked to original sources
PubMed2001

Conformational intermediate of the amyloidogenic protein beta 2-microglobulin at neutral pH.

Aggregation and fibrillation of beta(2)-microglobulin are hallmarks of dialysis-related amyloidosis. We characterize perturbations of the native conformation of beta(2)-microglobulin that may precede fibril formation. For a beta(2)-microglobulin variant cleaved at lysine 58, we show using capillary electrophoresis that two conformers spontaneously exist in aqueous buffers at neutral pH. Upon treatment of wild-type beta(2)-microglobulin with acetonitrile or trifluoroethanol, two conformations were also observed. These conformations were in equilibrium dependent on the sample temperature and the percentage of organic solvent present. Circular dichroism showed a loss of beta-structures and gain of alpha-helices. Reversal to the native conformation occurred when removing the organics. Affinity capillary electrophoresis experiments showed increased specific interactions of the nonnative beta(2)-microglobulin conformation with the dyes 8-anilino-1-naphthalene sulfonic acid and Congo red. The observations may relate to early folding events prior to amyloid fibrillation and facilitate the development of methods to detect and inhibit pro-amyloid protein and peptide conformations.

Antibodies, Monoclonal↗
PubMed2001

Smoking, but not lipids, lipoprotein(a) and antibodies against oxidised LDL, is correlated to the expansion of abdominal aortic aneurysms.

OBJECTIVES: to study the role of smoking, lipids, lipoprotein (a), and autoantibodies against oxidised low density lipoprotein (Ab-oxLDL) in the expansion of small abdominal aortic aneurysms (AAA). To study the role of Ab-oxLDL and lp(a) in the progression of lower limb atherosclerosis. METHODS AND MATERIALS: one hundred and thirty-eight male patients with AAA were interviewed, examined, and their serum lipids and S-Ab-oxLDL determined. Of these, 117 were followed annually with ultrasound and underwent control scans and blood pressure measurements for a mean of 2.5 (range 1-5) years. RESULTS: initial AAA size, smoking and level of triglycerides were positively correlated to increased aneurysmal expansion, while beta-blocker medication was associated with decreased expansion. Besides initial AAA size, only smoking had persisting significance after adjustment of the other significant variables. Initial ankle brachial pressure index (ABI) and Lp(A) but not ab-oxLDL were significantly correlated to ABI change. CONCLUSION: smoking cessation may inhibit aneurysmal expansion. Lipids seem to play a minor role in the progression of AAA.

Aged↗
PubMed2001

Association of beta2-glycoprotein I IgG and IgM antibodies with thrombosis and thrombocytopenia.

Antiphospholipid antibodies (APA) have been known for decades. Their relation to clinical manifestations, primarily thromboses and thrombocytopenia, was recognised in the 1980s. In this clinical study two cohorts of patients, a population-based (84 patients with systemic lupus erythematosus (SLE)) and a hospital-based (87 patients with SLE and 53 with other connective tissue diseases) were investigated for APA and associated clinical manifestations. Anticardiolipin antibodies (ACA) of IgG and IgM classes were found in 13 and 38% of the population-based patients and in 29 and 58% of the hospital-based patients, respectively. The corresponding figures for antibodies against beta2-glycoprotein I (anti-beta2GPI) were 15 and 10% in the population-based patients and 14 and 8% in the hospital-based cohort. Anti-beta2GPI antibodies were always found in association with the corresponding immunoglobulin class of ACA. In both cohorts anti-beta2GPI of the IgG class were associated with arterial/venous occlusion, a result concordant with other studies. A novel finding in both cohorts, however, was an association between thrombocytopenia and IgM anti-beta2GPI.

Adolescent↗
PubMed2000

Congophilicity (Congo red affinity) of different beta2-microglobulin conformations characterized by dye affinity capillary electrophoresis.

The amyloidogenic protein beta-microglobulin was characterized by affinity capillary electrophoresis (CE). CE could separate conformational variants of beta2-microglobulin and with the amyloid-specific dye Congo red as a buffer additive it was possible to measure different Congo red-affinities of native and abnormally folded beta2-microglobulin. We find that native beta2-microglobulin has an intermediate affinity for Congo red at pH 7.3 and that binding involves electrostatic interactions. The conformational variant of beta2-microglobulin that appears in acetonitrile solutions binds Congo red more strongly. Affinity CE using Congo red as a buffer additive is a new, simple, fast, and quantitative micromethod for the characterization of soluble conformational intermediates of amyloidogenic proteins.

Coloring Agents↗
PubMed2000

Heterogeneous nuclear ribonucleoproteins C1/C2 identified as autoantigens by biochemical and mass spectrometric methods.

The antigenic specificity of an unusual antinuclear antibody pattern in three patient sera was identified after separating HeLa-cell nuclear extracts by two-dimensional (2D) gel electrophoresis and localizing the antigens by immunoblotting with patient serum. Protein spots were excised from the 2D gel and their contents were analyzed by matrix-assisted laser desorption-ionization (MALDI) or nanoelectrospray ionization time-of-flight (TOF) tandem mass spectrometry (MS) after in-gel digestion with trypsin. A database search identified the proteins as the C1 and C2 heterogeneous nuclear ribonucleoproteins. The clinical spectrum of patients with these autoantibodies includes arthritis, psoriasis, myositis, and scleroderma. None of 59 patients with rheumatoid arthritis, 19 with polymyositis, 33 with scleroderma, and 10 with psoriatic arthritis had similar antibodies. High-resolution protein-separation methods and mass-spectrometric peptide mapping in combination with database searches are powerful tools in the identification of novel autoantigen specificities.

Adult↗
PubMed2000

Polypeptide binding properties of the chaperone calreticulin.

Calreticulin is a highly conserved eukaryotic ubiquitious protein located mainly in the endoplasmic reticulum. Two major characteristics of calreticulin are its chaperone activity and its lectin properties, but its precise function in intracellular protein and peptide processing remains to be elucidated. We have investigated the interactions of human calreticulin with denatured ovalbumin, proteolytic digests of ovalbumin, and different available peptides by solid phase assays, size-exclusion chromatography, capillary electrophoresis, and MS. The results show that calreticulin interacts better with unfolded ovalbumin than with native ovalbumin, that calreticulin strongly binds components in proteolytic digests of denatured ovalbumin, and that calreticulin interacts strongly with certain synthetic peptides.

Amino Acid Sequence↗
PubMed2000

Established and emerging biological activity markers of inflammatory bowel disease.

Assessment of disease activity in inflammatory bowel disease (IBD), i.e., ulcerative colitis (UC) and Crohn's disease (CD), is done using clinical parameters and various biological disease markers. Ideally, a disease marker must: be able to identify individuals at risk of a given disorder, be disease specific, mirror the disease activity and, finally, be easily applicable for routine clinical purposes. However, no such disease markers have yet been identified for IBD. In this article, classical disease markers including erythrocyte sedimentation rate, acute phase proteins (especially orosomucoid and CRP), leukocyte and platelet counts, albumin, neopterin, and beta2-microglobulin will be reviewed together with emerging disease markers such as antibodies of the ANCA/ASCA type, cytokines (e.g., IL-1, IL-2Ralpha, IL-6, IL-8, TNF-alpha, and TNF-alpha receptors) and with various adhesion molecules. It is concluded that none of the pertinent laboratory surrogate markers of disease activity in IBD are specific or sensitive enough to replace basic clinical observation such as the number of daily bowel movements, general well-being, and other parameters in parallel. Further studies are highly warranted to identify and assess the clinical importance and applicability of new laboratory markers for the diagnosis or the disease activity of IBD.

Acute-Phase Proteins↗
PubMed1999

Microscale characterization of the structure-activity relationship of a heparin-binding glycopeptide using affinity capillary electrophoresis and immobilized enzymes.

A heparin-binding glycopeptide (T3) from human serum amyloid P component was characterized by taking advantage of two important features of capillary electrophoresis: the low sample consumption and the possibility of doing on-line binding studies. Incubations with neuraminidase and proteolytic enzymes were carried out with enzymes immobilized on paramagnetic microbeads. Affinity capillary electrophoresis subsequently was used to characterize T3 and its fragments with respect to heparin binding. We find that an intact glycan moiety makes the C-terminal part of T3 relatively resistant to chymotryptic clevage. This protection is lost upon desialylation. Also, the C-terminus of T3 is involved in heparin binding while the N-terminal part of the molecule has no appreciable binding activity. The micromethods presented here make it feasible to perform structure-function studies even on the small amounts of analytes that are typically available when working with glycopeptides from natural sources.

Amino Acid Sequence↗
PubMed1999

Monitoring nitrotyrosinylation of a synthetic peptide by capillary zone electrophoresis.

Proteins may be nitrated on tyrosyl residues (nitrotyrosinylated) by the action of reactive nitrogen species in inflamed tissues. Capillary electrophoresis was used to monitor this reaction in a model system with tetranitromethane as the nitrotyrosinylating reagent and a synthetic pentapeptide containing one tyrosine as the target molecule. The reaction was readily followed by capillary electrophoresis performed at pH 8 and, using an absorption wavelength of 436 nm, the signature spectral characteristics of the nitrotyrosinylated peptide were verified on-line. The peak appearance time for the nitrotyrosinylated peptide was more than 1 min longer than that of the starting material and a single main product was observed in contrast to the case when peroxynitrite was used as the nitrotyrosinylating reagent. Capillary electrophoresis appears to be a convenient method for the optimization of nitrotyrosinylation, examination of reaction inhibitors, and for studies of the consequences of nitrotyrosinylation, e.g., for antibody binding and for the function of the target protein or peptide.

Electrophoresis, Capillary↗
PubMed1999

Identification, quantitation, and characterization of biomolecules by capillary electrophoretic analysis of binding interactions.

The high resolving power of capillary electrophoresis combined with the specificity of binding interactions may be used with advantage to characterize the structure-function relationship of biomolecules, to quantitate specific analytes in complex sample matrices, and to determine the purity of pharmaceutical and other molecules. We here review recent and innovative methodologies and applications of high resolution affinity electrophoresis within the fields of binding constant determination, structure-activity studies, quantitative microassays, analysis of drug purity and protein conformation, and immobilized affinity ligands. Despite the virtues of these approaches with respect to applicability, resolving power, speed, and low sample consumption, problems remain with respect to analyte identification and low concentration limits of detection. The ongoing development of new detector technologies for capillary electrophoresis such as mass spectrometry, and possibly nuclear magnetic resonance and other spectroscopic methods, is therefore very promising for the continued increased use of affinity capillary electrophoresis.

Binding, Competitive↗
PubMed1999

Autoantibodies to molecular targets in neutrophils in patients with ulcerative colitis.

Autoantibodies against neutrophil granulocytes are frequently observed in patients with ulcerative colitis, but a precise description of the autoantigens involved is lacking. Therefore, sera from 75 patients with ulcerative colitis were studied for antibody specificities by means of indirect immunofluorescence microscopy, enzyme-linked immunosorbent assays, and immunoblotting of extracts of neutrophils, neutrophil granules and lymphocytes. Fifty-six percent of the sera reacted in indirect immunofluorescence with ethanol-fixed neutrophils. On formalin-fixed cells most sera shifted fluorescence pattern, indicating a cytoplasmic origin of antigen(s). Only a few sera reacted with specific, known antigens. Immunoblots showed reactions with a broad panel of antigens with preferences for proteins around 55-65 kDa, which were present in azurophilic granules and in cytosol, an 80-kDa protein found in the specific granules, and a 110-kDa unknown cytosol component. In conclusion, neutrophil-specific IgG autoantibodies from ulcerative colitis patients react with several different antigens, most of which are of nonnuclear origin.

Autoantibodies↗
PubMed1999

Distinct differences in autoantigen specificity of anti-neutrophil cytoplasm antibodies in systemic vasculitides and other inflammatory diseases.

BACKGROUND: Anti-neutrophil cytoplasm antibodies in necrotizing vasculitides need to be distinguished from ANCAs in other inflammatory conditions to avoid clinical misinterpretation. OBJECTIVES: To help clinicians and laboratory scientists recognize and utilize vasculitis-related ANCAs as an aid in diagnostic workup and patient follow-up, and be aware that ANCAs with different characteristics are commonly found in other chronic inflammatory conditions that persistently engage neutrophils in the inflammatory process. METHODS: Indirect immunofluorescence and enzyme immunoassay methods were used to detect ANCAs with both known and unknown neutrophil autoantigenic targets. RESULTS: Primary necrotizing small vessel vasculitides such as Wegener's granulomatosis, Churg-Strauss syndrome, microscopic polyangiitis, and renal-limited rapidly progressive necrotizing glomerulonephritis target either the serine protease proteinase 3 or myeloperoxidase in azurophilic granules. In ulcerative colitis and rheumatoid arthritis, we found multiple ANCA targets contained in azurophilic and specific granules, the cytosol and the nucleus, whereas PR3 and MPO were not, or only weakly, recognized. CONCLUSIONS: ANCAs typically found in active SVV are demonstrable both by indirect immunofluorescence and antigen-specific enzyme immunoassay, and strong reactivity to either PR3 or MPO is characteristic. Strong ANCA with MPO reactivity is also found in some patients with drug-induced syndromes (lupus, vasculitis). Intermediate to strong perinuclear ANCAs are found in a substantial proportion of patients with UC (40-60%) and RA (30-70%), but in these conditions the ANCAs have many antigen targets that are only weakly recognized.

Antibodies, Antineutrophil Cytoplasmic↗
PubMed1998

Affinity capillary electrophoresis: important application areas and some recent developments.

Affinity capillary electrophoresis (ACE) is a broad term referring to the separation by capillary electrophoresis of substances that participate in specific or non-specific affinity interactions during electrophoresis. The interacting molecules can be found free in solution or can be immobilized to a solid support. Every ACE mode has advantages and disadvantages. Each can be used for a wide variety of applications. This paper focuses on applications that include purification and concentration of analytes present in diluted solutions or complex matrices, quantitation of analytes based on calibration curves, and estimation of binding constants from direct and derived binding curves based on quantitation of analytes or on analyte migration shifts. A more recent chemicoaffinity strategy in capillary electrophoresis/capillary electrochromatography (CE/CEC) termed molecular imprinting ('plastic antibodies') is discussed as well. Although most ACE studies are aimed at characterizing small-molecular mass analytes such as drugs, hormones, and peptides, some efforts have been pursued to characterize larger biopolymers including proteins, such as immunoglobulins. Examples of affinity interactions that have been studied are antigen-antibody, hapten-antibody, lectin-sugar, drug-protein, and enzyme-substrate complexes using ultraviolet, laser-induced fluorescence, and mass spectrometer detectors. This paper also addresses the critical issue of background electrolyte selection and quantitation of analytes. Specific examples of bioaffinity applications are presented, and the future of ACE in the biomedical field is discussed.

Electrophoresis, Capillary↗
PubMed1998

Capillary electrophoresis for the study of affinity interactions.

Molecular recognition may be characterized both qualitatively and quantitatively by electrophoretic methods if complexed molecules differ in electrophoretic mobility from unbound ones. The use of capillary zone electrophoresis (CE) for the characterization of affinity interactions is advantageous because of the high resolution, reproducibility and wide applicability of the technique and because of the mild conditions, i.e., physiological buffers without additions of organics or detergents, that are often sufficient for highly efficient separations. CE gives the ability to characterize binding between small amounts of unlabelled reactants in solution, has few requirements for special characteristics of the interacting molecules and is also applicable to the study of interactions of individual components in mixtures, as detection of binding and analytical separation are achieved in one step. This is unique compared with other techniques for the study of non-covalent interactions. The advantages and disadvantages of using CE to demonstrate molecular interactions, to screen for specific ligand binding in complex mixtures and to calculate binding constants will be discussed.

Affinity Labels↗
PubMed1998

A heparin-binding peptide from human serum amyloid P component characterized by affinity capillary electrophoresis.

Affinity capillary electrophoresis (CE) was used for a detailed characterization of the binding between heparin and a peptide isolated from the heparin-binding serum protein amyloid P component (SAP). The peptide corresponds to a tryptic fragment (T3) comprising amino acids 14-38 of SAP. By including ligands in the electrophoresis buffer various glycosaminoglycans could be screened for binding of T3 using one sample aliquot. The binding was found to be highly specific for heparin and heparin fragments down to tetramers and appeared strongest at a slightly alkaline pH while no binding could be demonstrated with heparan sulfate, chondroitin sulfate, desulfated heparin, mannose 6-phosphate and phosphotyrosine. The T3-heparin complexes were sufficiently stable to perform quantitative measurements of the binding using preequilibration of samples prior to a CE-mediated separation of bound and free T3-peptide. Plots based on quantitation of analyte peaks corresponding to free and complexed T3 yielded a dissociation constant of 1.5 microM for the interaction with heparin. The results indicate that a specific subfraction of the heparin molecules is active in binding interactions with the peptide. The affinity CE approach proved to be useful for these studies because of its sensitivity to complex formation involving charged ligands and the possibility of achieving separations under native conditions. Also advantageous is the low sample consumption and the ability to analyze unlabeled reactants in solution.

Amino Acid Sequence↗
PubMed1998

Lower leg electrical impedance after distal bypass surgery.

Electrical impedance was determined in 13 patients following distal bypass surgery to evaluate lower leg oedema as reflected by its circumference. Tissue injury was assessed by the plasma concentration of muscle enzymes. After surgery, the volume of the control lower leg increased from 1250 (816-2373) to 1384 (874-2345) ml (median and range; P < 0.05), where the impedance did not change significantly from 140 (92-181) ohms. The volume of the operated leg increased more [from 1129 (824-2373) to 1600 (1090-2837) ml], and the decrease in electrical impedance was also pronounced [137 (125-169) to 83 (69-104) ohms (P < 0.001)]. Tissue injury after surgery was indicated by an increase in total creatine kinase (n = 17) and MB isoenzyme of creatine kinase (n = 8) (P < 0.05). Myoglobin (n = 8) had increased already during surgery (P < 0.05), whereas there was no significant change in the plasma concentration of troponin I (n = 8). In conclusion, tissue injury was reflected by increases in muscle enzymes in plasma. We found an inverse correlation between lower leg electrical impedance and volume, but the deviation in electrical impedance was approximately twice that of the leg volume. Electrical impedance appears to be a useful method for the evaluation of lower leg oedema after distal bypass surgery.

Adult↗
PubMed1997

Interactions of the human class II major histocompatibility complex protein HLA-DR4 with a peptide ligand demonstrated by affinity capillary electrophoresis.

The interactions of empty recombinant major histocompatibility complex (MHC) class II molecules (DRA1*0101/DRB1*0401) with a known peptide ligand [the HA(307-319) fragment of influenza virus hemagglutinin] were studied by capillary electrophoresis. Using an alkaline buffer system with the addition of non-ionic or zwitterionic detergent and high sensitivity laser-induced fluorescence detection, both slowly and rapidly equilibrating binding could be demonstrated. This was accomplished using a pre-equilibration approach as well as migration shift experiments where receptor molecules were added to the electrophoresis buffer. This system may be useful for the study of both peptide binding to MHC molecules and screening for inhibition or amplification of binding by other ligands as well as for the study of the interactions of T-cell receptors with MHC-peptide complexes.

Amino Acid Sequence↗