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O J Bjerrum

Publications and source records attributed to O J Bjerrum.

At least 19 recordsLinked to original sources
PubMed2003

Two polyclonal antisera detect different AGE epitopes in human plasma samples.

Advanced glycation end products (AGEs), involved in the pathogenesis of diabetic complications, comprise a series of related chemical structures which might possess dissimilar immunogenic characteristics. In this study the levels of AGE in plasma samples from normal subjects (N=41) and diabetic patients (N=44) were measured by ELISA using two polyclonal antisera (named CF5 and CF199, respectively, and immunologically characterized) raised using two different immunogens and immunization techniques. Age levels were significantly higher in diabetic than in normal plasma samples (P<0.0001) with both antisera. However, CF199 detected higher AGE levels than CF5 both in normal (P<0.0001) and diabetic (P<0.005) samples. Pre-incubation with AGE-bovine serum albumin (BSA) caused the loss of most the reactivity of both antisera. Pre-incubation with carboxy-methyl-lysine-BSA (an oxidation-derived AGE) induced the loss of nearly all CF5 reactivity while CF199 retained a significant amount of activity against AGE antigens. Moreover, CF5 lost over 90% of its reactivity against BSA incubated with high glucose under non-oxidative conditions, suggesting its recognition of mainly oxidation-derived AGE epitopes. The different AGE levels measured by the two antisera suggests, therefore, that one single antiserum is unable to recognize all the various AGE epitopes which might be present, at any time, in tissues and body fluids in health and disease.

Diabetes Mellitus↗
PubMed2000

New safe medicines faster: A proposal for a key action within the European union's 6th framework programme.

The global competitiveness of the European pharmaceutical industry is under threat. Technology currently available for the development of new medicines is unable to match the pace of drug discovery and design; and the ever-growing demand for safety, efficacy and quality documentation has increased the cost and time involved in getting new medicines on the market. Although the pharmaceutical industry is one of the strongest in Europe in terms of research, innovation, exports and employment, there are severe restrictions on its ability to create wealth and launch safe drugs for the treatment of common and rare afflictions. The present situation should not be allowed to continue. For this reason, the European Federation for Pharmaceutical Sciences (EUFEPS) has proposed a key action under the title "New safe medicines faster" for the forthcoming EU 6th RTD framework programme. The key action has three main objectives: to seek new technologies capable of more effective selection of potential drug candidates for innovative medicines while accommodating safety demands; to use such technologies to speed up the pharmaceutical development process and eliminate bottlenecks created by initial exploratory drug research; and to cultivate a pan-European interdisciplinary network that bridges the gap between industry, academia and regulatory authorities. By involving regulatory authorities early on and fuelling research and innovation with EU money it should be possible to create a new set of recognised European standards whereby new safe medicines can be brought onto the market faster and cheaper.

Drug Approval↗
PubMed1999

A novel inhibitor of advanced glycation end-product formation inhibits mesenteric vascular hypertrophy in experimental diabetes.

AIMS/HYPOTHESIS: Previous studies in our laboratory have shown that the vascular changes in diabetes include hypertrophy of the mesenteric vasculature and that this process can be attenuated by the inhibition of advanced glycation with aminoguanidine. Since aminoguanidine can also act as an inhibitor of nitric oxide synthase, the effect of a novel inhibitor of advanced glycation end-products, formation that does not inhibit nitric oxide synthase, known as 2,3 diaminophenazine (2,3 DAP) was evaluated. METHODS: Initially, in vitro assessment of the ability of 2,3 diaminophenazine to inhibit formation of advanced glycation products was performed. Subsequently, in vivo studies evaluating 2,3 diaminophenazine and aminoguanidine were carried out. Animals were followed for 3 weeks after induction of diabetes and randomised to no treatment, aminoguanidine or 2,3 diaminophenazine. Mesenteric vessels were weighed and advanced glycation end-products were measured by radioimmunoassay in vessel and kidney homogenates. In addition, these products were assessed in mesenteric vessels by immunohistochemistry. RESULTS: When compared with control animals, diabetes was associated with an increase in mesenteric vascular weight. Treatment of diabetic rats with aminoguanidine or 2,3 diaminophenazine resulted in attenuation of vascular hypertrophy. Both aminoguanidine and 2,3 diaminophenazine reduced the formation of advanced glycation end-products as measured by radioimmunoassay and as assessed immunohistochemically in these vessels. This reduction was also observed in the kidney. CONCLUSION/INTERPRETATION: These data support the concept that the effects of aminoguanidine in reducing diabetes associated vascular hypertrophy are via inhibition of advanced glycation end-products dependent pathways.

Animals↗
PubMed1999

Structural heterogeneity of the binding sites of HSA for phenyl-groups and medium-chain fatty acids. Demonstration of equilibrium between different binding conformations.

A new facet of the very heterogeneous albumin molecule is described. Chromatography at pH 6-9 of human serum albumin (HSA) on a phenyl-sepharose column separates it into two nonconvertible conformations that are, in turn, in equilibrium with its binding and nonbinding forms. The hydrophobic interaction of HSA with phenyl-sepharose depends on ionic strength, pH, and time of contact with the immobilized ligand. Binding as a function of pH shows a minimum at pH 6.5, and the binding profile at pH 7-9 fits the titration of a weak monoprotic acid with a pKa of 7.3. There was no observable difference in the CD spectra or the masses of the two forms. The equilibrium between the albumin forms was examined under defined conditions and cannot be explained by a simple two-state model. Thus rechromatography of the nonbinding fraction derived from a sample in which 50% of the protein was originally retained resulted only in 10-20% bound protein. Correspondingly only 70-80% of the binding form was retained. A model explaining the observations can be derived if two species, I and II, exist in the solution, both being in an equilibrium with a binding and a nonbinding form, but in which I is not in equilibrium with II. The rate of conversion between the binding and nonbinding conformations was determined to be faster than 15 s at room temperature.

Binding Sites↗
PubMed1997

A simple method for the preparation and purification of C1 complement cleaved beta 2-microglobulin from human serum.

A simple method is described for the preparation of proteolytically processed forms of beta 2-microglobulin suitable for structural and biological studies. PEG 6000 was added to the serum of healthy individuals to precipitate the C1 complement complex from C1 esterase inhibitor (C1-inh). After dissolving the precipitate containing the C1 complement in Tris-HCl buffer, pH 7.6, efficient conversion of added beta 2-microglobulin to desLys58 beta 2-microglobulin was observed. Addition of a specific carboxypeptidase B inhibitor (Plummers inhibitor) could partly prevent the deletion of Lys-58 from cleaved beta 2-microglobulin, whereby Lys58-cleaved beta 2-microglobulin was obtained. The proteolytically processed forms were subsequently purified by G-75 Sephadex gel filtration followed by chromatofocusing. A yield of 10-40% of proteolytically processed beta 2-microglobulin was obtained. Only one component was seen by SDS-PAGE stained with Coomassie Brilliant Blue.

Chromatography, Gel↗
PubMed1997

Relative contributions of advanced glycation and nitric oxide synthase inhibition to aminoguanidine-mediated renoprotection in diabetic rats.

Advanced glycation end products (AGEs) have previously been shown to be increased in the diabetic kidney. Aminoguanidine, an inhibitor of advanced glycation, has been shown to attenuate the development of AGEs as well as the progression of renal disease in experimental diabetes. However, the precise mechanisms through which aminoguanidine acts remain to be elucidated since it is also able to act as an inhibitor of nitric oxide synthase (NOS). This study has therefore compared the effects of aminoguanidine with the effects of two other inhibitors of NOS, L-NAME and methylguanidine, on the development of experimental diabetic nephropathy. Diabetic rats were randomised to receive no treatment, aminoguanidine (1 g/l in drinking water), L-NAME (5 mg/l in drinking water) or methylguanidine (1 g/l in drinking water). Diabetic rats had increased levels of albuminuria and urinary nitrite/nitrate excretion when compared to control rats. Renal AGEs measured by fluorescence as well as by a carboxymethyllysine reactive radioimmunoassay, were elevated in diabetic rats. No changes in inducible NOS (iNOS) protein expression were detected in experimental diabetes nor did aminoguanidine affect iNOS expression. Aminoguanidine did not affect blood glucose or HbA1c but it did prevent increases in albuminuria, urinary nitrites/nitrates and renal AGE levels as measured by fluorescence and radioimmunoassay. L-NAME and methylguanidine did not retard the development of albuminuria, nor did they prevent increases in renal AGE levels, as assessed by fluorescence. However, these treatments did prevent increases in AGEs, as measured by radioimmunoassay. This study indicates that the renoprotective effect of aminoguanidine in experimental diabetes cannot be reproduced by L-NAME or methylguanidine. It is likely that the effect of aminoguanidine is mediated predominantly by decreased AGE formation rather than via NOS inhibition. It also raises the possibility that inhibition of fluorescent AGE formation may be more renoprotective than inhibition of the formation of carboxymethyllysine-containing AGEs.

Administration, Oral↗
PubMed1995

Electrophoretic and chromatographic differentiation of two forms of albumin in equilibrium at neutral pH: new screening techniques for determination of ligand binding to albumin.

Analysis of normal human serum by crossed hydrophobic interaction immunoelectrophoresis with Phenyl-Sepharose revealed a biphasic appearance of the albumin peak. The molecular mechanism behind this apparent albumin heterogeneity was investigated. Analysis of defatted purified albumin showed that a major fraction bound to the Phenyl-Sepharose and that addition of ligands (e.g. long-chain fatty acids, bilirubin, sulfonamides and warfarin) before electrophoresis blocked this binding to different degrees. A quantitative relation between ligand binding and the amount of nonbinding albumin was found. Thus the technique might be suitable for screening of ligand binding to albumin. Analysis of serum samples from newborns with hyperbilirubinemia revealed a positive correlation between the fraction of the nonretarded albumin and the bilirubin concentration. By chromatography on Phenyl-Sepharose, defatted albumin was separated into a binding and a nonbinding form and this technique was subsequently used to determine the kinetics of the intramolecular conversion. After rechromatography, each of the fractions could again be separated into two fractions, indicating the presence of an equilibrium. By varying the passage time for albumin on the column or varying the period between the first and the second separation it was possible to calculate the conversion rates. The half-life for the conversion was found to be as long as 1 1/4 h. It is the first time that a conformational change for albumin with such a long conversion time has been described experimentally.

Bilirubin↗
PubMed1993

Peptide dot immunoassay and immunoblotting: electroblotting from aluminum thin-layer chromatography plates and isoelectric focusing gels to activated nitrocellulose.

Nitrocellulose membrane was preactivated with divinyl sulfone, and a spacer of 1,6-diaminohexane was coupled to the membrane which was functionalized by glutaraldehyde, leaving a reactive carbonyl group. The peptides were coupled to the carbonyl by the side chain and terminal amino groups. The octapeptide angiotensin II (sequence: DRVYIHPF) and peptide analogs containing 6-10 amino acid residues were dotted directly onto the matrix at 45 degrees C for 15 min and detected by specific antisera, which were raised in rabbits against angiotensin I and II, respectively. They were visualized by peroxidase-coupled anti-rabbit IgG antibodies. The detection limit for synthetic angiotensin II was 500 fg per cm2 (= 500 amol per cm2) and for the decapeptide angiotensin I (sequence: DRVYIHPFHL) it was 500 pg per cm2 (= 400 fmol per cm2). Separation of synthetic angiotensin analogs by high performance thin-layer chromatography on silica coated aluminum plates was followed by electroblotting onto activated nitrocellulose and detection with specific antibodies, showing a sensitivity of 100 fg and 1 pg for angiotensin II and angiotensin I, respectively. Isoelectric focusing in agarose using Ampholine carrier ampholytes and immunoblotting with specific antisera displayed a lower sensitivity for angiotensin II and angiotensin I of 2 ng and 20 ng, respectively. The isoelectric focusing and immunoblotting technique was applied for separation of angiotensin I and II and related peptides in serum, where synthetic angiotensin I was degraded in the presence of 1 mM phenylmethylsulfonyl fluoride and 10 mM ethylenediaminetetraacetic acid.(ABSTRACT TRUNCATED AT 250 WORDS)

Acquired Immunodeficiency Syndrome↗
PubMed1993

Glycogen synthase and phosphofructokinase protein and mRNA levels in skeletal muscle from insulin-resistant patients with non-insulin-dependent diabetes mellitus.

In patients with non-insulin-dependent diabetes mellitus (NIDDM) and matched control subjects we examined the interrelationships between in vivo nonoxidative glucose metabolism and glucose oxidation and the muscle activities, as well as the immunoreactive protein and mRNA levels of the rate-limiting enzymes in glycogen synthesis and glycolysis, glycogen synthase (GS) and phosphofructokinase (PFK), respectively. Analysis of biopsies of quadriceps muscle from 19 NIDDM patients and 19 control subjects showed in the basal state a 30% decrease (P < 0.005) in total GS activity and a 38% decrease (P < 0.001) in GS mRNA/microgram DNA in NIDDM patients, whereas the GS protein level was normal. The enzymatic activity and protein and mRNA levels of PFK were all normal in diabetic patients. In subgroups of NIDDM patients and control subjects an insulin-glucose clamp in combination with indirect calorimetry was performed. The rate of insulin-stimulated nonoxidative glucose metabolism was decreased by 47% (P < 0.005) in NIDDM patients, whereas the glucose oxidation rate was normal. The PFK activity, protein level, and mRNA/microgram DNA remained unchanged. The relative activation of GS by glucose-6-phosphate was 33% lower (P < 0.02), whereas GS mRNA/micrograms DNA was 37% lower (P < 0.05) in the diabetic patients after 4 h of hyperinsulinemia. Total GS immunoreactive mass remained normal. In conclusion, qualitative but not quantitative posttranslational abnormalities of the GS protein in muscle determine the reduced insulin-stimulated nonoxidative glucose metabolism in NIDDM.

Adult↗
PubMed1991

Affinity electrophoresis used for determination of binding constants for antibody-antigen reactions.

The use of affinity electrophoresis in agarose gels for determination of binding constants for the interaction of antigens with monoclonal antibodies is exemplified for monoclonal anti-human serum albumin and anti-alpha 1-fetoprotein antibodies. The calculated binding constants are verified by independent binding assays. The electrophoretic separation of antigen-antibody complexes of different stoichiometry is also demonstrated. Thus, affinity electrophoresis represents an alternative method for both qualitative and quantitative assessment of antigen-antibody interactions.

Antibodies, Monoclonal↗
PubMed1989

Has immunoblotting replaced electroimmunoprecipitation? Examples from the analysis of autoantigens and transglutaminase-induced polymers of the human erythrocyte membrane.

The virtues and drawbacks of immunoblotting and electroimmunoprecipitation in the characterization of macromolecules in crude mixtures are presented. Interactions between autoantibodies and human erythrocyte membrane proteins were studied by means of crossed-affinoimmunoelectrophoresis with autologous immunoglobulins incorporated into the first dimension gel and by immunoblotting of sodium dodecyl sulphate-polyacrylamide gel electrophoresis separated erythrocyte membrane proteins with autologous immunoglobulins as primary antibodies. Substrates for transglutaminase in calcium-activated human erythrocyte membranes were examined by immunoelectrophoretic and immunoblotting methods. The experiments concerning autoantibodies complemented each other and showed that epitopes on Band 3 protein, spectrin and ankyrin are recognized by circulating immunoglobulin autoantibodies in normal individuals. The polymer experiments showed the presence of spectrin, ankyrin, Band 3, Band 4.1, glucose transporter, actin and haemoglobin epitopes in the polymer (Mr 3.10(6)-5.10(6]. It is concluded that the two techniques complement each other. The most evident advantage of immunoblotting is its sensitivity and applicability while electroimmunoprecipitation in some instances allows an easier identification of distinct protein species and still has a role for quantification and certain monitoring purposes.

Autoantibodies↗
PubMed1987

Electroimmunochemical characterization of endothelial cell proteins: antigenic relationship with platelet and erythrocyte membrane proteins.

Human endothelial cells isolated from umbilical cords and cultured in primary cultures were solubilized in Triton X-100 and examined by crossed immunoelectrophoresis using rabbit antiserum against endothelial cells. Endogeneous labelling of the endothelial cell proteins with 35S-methionine or 14C-mannose followed by crossed immunoelectrophoresis and autoradiography revealed about 30 or 8 immunoprecipitates, respectively. Antigenic relationship between endothelial cell proteins and proteins in human platelets or erythrocyte membranes was demonstrated by use of the corresponding antisera and by antigen addition experiments. One of the endothelial cell proteins cross-reacted with antiserum against erythrocyte membranes and showed a partial antigenic identity reaction with the band 3 protein complex of erythrocyte membranes. The same protein showed antigenic relationship also with a platelet protein. In addition, endothelial cells contain at least 7 proteins antigenically related to platelet proteins, of which at least 5 were labelled with 14C-mannose and thus were glycoproteins. Three of these glycoproteins were antigenically related to proteins from isolated platelet membranes and three were related to the release products obtained after thrombin treatment of platelets. The present study demonstrated numerous platelet and endothelial cell proteins that were antigenically related, more than previously anticipated.

Antigens↗
PubMed1987

Immunochemical determination of acetylcholinesterase in amniotic fluid--an evaluation of eleven monoclonal antibodies.

Eleven monoclonal antibodies and a polyclonal rabbit antiserum were evaluated with respect to reactivity with acetylcholinesterase (AChE, EC 3.1.1.7) from erythrocytes and brain. Employing our enzyme antigen immunoassay for AChE five selected antibodies were evaluated with regard to their clinical usefulness in the prenatal diagnosis of neural tube defects (NTD). Of these, one antibody preferentially bound the enzyme from human brain, and discerned better than the others pathological samples (anencephaly, spina bifida and encephalocele) from normal ones. With this antibody no false positive values were obtained even if amniotic fluid samples were blood contaminated.

Acetylcholinesterase↗
PubMed1987

Beta 2-microglobulin in neutrophils: an intragranular protein.

The subcellular localization of beta2-microglobulin (beta 2m) in human neutrophils was determined by an enzyme-linked immunosorbent assay on subcellular fractions obtained by Percoll density gradient centrifugation of neutrophils disrupted by nitrogen cavitation. The neutrophils were found to contain 160 ng beta 2m/mg protein. Approximately two-thirds co-located with the markers for specific granules and was released from intact cells during degranulation, whereas one-third of the beta 2m was located together with markers of the plasma membrane. This fraction was not further enriched during degranulation. These results indicate that beta 2m cannot be universally used as a plasma membrane marker as hitherto assumed, but beta 2m may serve as an indicator of neutrophil degranulation.

Cell Compartmentation↗
PubMed1987

Autoantibodies against spermatozoal antigens detected by immunoblotting and agglutination. A longitudinal study of vasectomized males.

Sera taken pre- and post-operatively at regular intervals within a year from 16 men undergoing vasectomy were analysed for autoantibodies against spermatozoal proteins by immunoblotting. The reaction patterns were compared with the results of sperm agglutination tests. Immunoblotting revealed the presence of autoantibodies against various spermatozoal polypeptides in all sera taken pre-operatively and post-operatively. On average, seven polypeptides showed reaction. During the post-operative period two patients developed spermatozoal agglutinins in moderate titers (greater than 16) but in immunoblotting no change in band reactivity was observed for these two patients. However, scanning of the immunoblotting results revealed that one of the patients, although without sperm agglutinins, during the post-operative period showed an increasing band colouring of a polypeptide of Mr 31,500, reflecting an increased level of the corresponding antibodies.

Adult↗
PubMed1987

Modification of beta-2-microglobulin in sera from patients with small cell lung cancer: evidence for involvement of a serine protease.

Modification of beta-2-microglobulin has been shown to occur in vitro in serum of patients suffering of small cell lung cancer, where it clearly correlated to the clinical course of disease. The cause of serum beta-2-microglobulin modification occurring in these patients is investigated in the present study. This revealed that modification of beta-2-microglobulin is due to cleavage by a serine protease, which is dependent on divalent cations and has a trypsin-like specificity. The process showed the following characteristics: pH optimum at 8.5, heat inactivation by incubation at 56 degrees C for 20 min and temperature optimum at 20 degrees C.

Carcinoma, Small Cell↗
PubMed1985

Auto- and iso-antigens of human spermatozoa detected by immunoblotting with human sera after SDS-PAGE.

A sodium dodecylsulphate-polyacrylamide gel electrophoretic system for analysis of the proteins of human spermatozoa was established. Subsequent immunoblotting of the gels with human sera gave a reproducible immunolabelling of distinctive polypeptide bands. To identify auto- and isoantigens, 28 well-characterized sera from the WHO Reference Bank for Reproductive Immunology (1977, Acta Pathol. Microbiol. Scand. Sect. C, Suppl. 252) containing agglutinating and complement fixating antibodies (9 female (F) and 19 male (M) and 30 normal sera (14 F and 16 M) were analysed with reference to binding of IgG. Three spermatozoal antigens with Mr values in reduced state of 120,000 (6), 41,000 (6) and 32,000 (15) were found to be specifically correlated to the agglutinating activity of the reactive sea. (The number of these are given in parentheses). Furthermore, IgG from 2 and 3 of the normal sera and 14 and 17 of the agglutinating sera reacted with 78 and 64 kDA polypeptide, respectively. Identical binding patterns of IgG to spermatozoal polypeptides were obtained with IgG from male and female sera. The IgG-binding could not be correlated to the modes of spermatozoal agglutination. In a similar analysis of the IgM binding antigens of 21 agglutinating and 12 normal sera no differences in binding between the sera were found, except for a specific reaction to a 78 kDa antigen for 5 of the agglutinating sera.

Antigens↗