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Biomedical subjects

U Nilsson

Publications and source records attributed to U Nilsson.

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

Analysis of an invariant cofactor-protein interaction in thiamin diphosphate-dependent enzymes by site-directed mutagenesis. Glutamic acid 418 in transketolase is essential for catalysis.

A homologous expression system and a purification protocol for pure, highly active recombinant yeast transketolase have been developed. The invariant transketolase residue Glu418, which forms a hydrogen bond to the N-1' nitrogen atom of the pyrimidine ring of the cofactor thiamin diphosphate has been replaced by glutamine and alanine. Crystallographic analyses of the mutants show that these amino acid substitutions do not induce structural changes beyond the site of mutation. In both cases, the cofactor binds in a manner identical to the wild-type enzyme. Significant differences in the CD spectra of the mutant transketolases compared with the spectrum of wild-type enzyme indicate differences in the electron distribution of the aminopyrimidine ring of the cofactor. The E418Q mutant shows 2% and the E418A mutant shows about 0.1% of the catalytic activity of wild-type enzyme. The affinities of the mutant enzymes for thiamin diphosphate are comparable with wild-type transketolase. The hydrogen bond between the coenzyme and the side chain of Glu418 is thus not required for coenzyme binding but essential for catalytic activity. The results demonstrate the functional importance of this interaction and support the molecular model for cofactor deprotonation, the first step in enzymatic thiamin catalysis.

Animals↗
PubMed1994

Oligosaccharide-receptor interaction of the Gal alpha 1-4Gal binding adhesin of Streptococcus suis. Combining site architecture and characterization of two variant adhesin specificities.

The sugar binding specificities of two groups of Streptococcus suis, a pig pathogen that causes meningitis also in man, were determined. Both the group represented by a recently characterized strain inhibitable by galactose and N-acetylgalactosamine (type PN) and the group inhibitable by galactose (type PO) were found by hemagglutination and solid-phase binding inhibition experiments to recognize the disaccharide Gal alpha 1-4Gal of the P1 and Pk blood group antigens. Both types preferred the disaccharide in terminal position. PN showed some, whereas PO showed almost no, binding to the globoside oligosaccharide containing an additional GalNAc beta 1-3 residue. The complete hydrogen bonding patterns were determined by using deoxy and other synthetic derivatives of the receptor disaccharide, and the constructed models of the interactions were compared with that of Escherichia coli PapG396 adhesin. The essential hydroxyls for binding were the HO-4', HO-6', HO-2, and HO-3 hydroxyls on the beta' alpha-side of the Gal alpha 1-4Gal molecule. Type PO adhesin also formed weak interactions with the hydroxyls HO-6 and HO-3'. The mechanism differed from that of E. coli, which binds to a cluster of five hydroxyls (HO-6, HO-2', HO-3', HO-4', and HO-6') and thus to a different part of the receptor disaccharide. These results represent the first example of the comparison of the saccharide receptor hydrogen bonding patterns of two bacterial organisms of different origin and show that the same saccharide may be recognized by two different binding mechanisms.

Adhesins, Bacterial↗
PubMed1994

Nuclear magnetic resonance and conformational investigations of the pentasaccharide of the Forssman antigen and overlapping di-, tri-, and tetra-saccharide sequences.

The 1H and 13C NMR parameters, i.e., chemical shifts and coupling constants, for the pentasaccharide of the Forssman antigen and overlapping di-, tri-, and tetra-saccharide sequences thereof have been measured and assigned completely using 1D and 2D techniques, and the oligosaccharide structures have thereby been confirmed. Nuclear Overhauser effect (NOE) experiments have been carried out at three different temperatures to assess the preferred conformations of the pentasaccharide and the component oligosaccharides. The conformational preferences of the compounds mentioned above have subsequently been investigated by theoretical calculations. The flexibility and dynamics of the molecules have been studied by Metropolis Monte Carlo simulations using a modified HSEA force field, and ensemble average data have been generated and compared to data obtained experimentally.

Calorimetry↗
PubMed1994

Synthesis of the globotetraose tetrasaccharide and terminal tri- and di-saccharide fragments.

The 2-(trimethylsilyl)ethyl (TMSEt) beta-glycosides of globotetraose [beta-D-GalNAc-(1-->3)-alpha-D-Gal-(1-->4)-beta-D-Gal-(1-->4)-D-Glc] and the terminal trisaccharide, as well as the methyl alpha-glycoside 1 of the terminal disaccharide, were synthesised by silver trifluoromethanesulfonate-promoted beta-glycosylation of suitably protected galactoside, galabioside, and globotrioside alcohols with 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-beta-D-galactopyranosyl chloride, followed by removal of protecting groups. Removal of the anomeric TMSEt group of the globotetraoside and of the terminal trisaccharide, using trifluoroacetic acid-dichloromethane, gave the corresponding hemiacetal sugars 8 and 3. The TMSEt glycoside of the terminal trisaccharide was converted, via the 1-acetate, into the corresponding isobutyl (4) and 3-butylsulfonyl-2-[(butylsulfonyl)methyl]propyl (5) glycosides and into the TMSEt thioglycoside 6 via the glycosyl bromide.

Antigens↗
PubMed1994

Synthesis of the Forssman pentasaccharide and terminal tetra-, tri-, and di-saccharide fragments.

The 2-(trimethylsilyl)ethyl (TMSEt) beta-glycosides of the Forssman pentasaccharide [alpha-D-GalNAc-(1-->3)-beta-D-GalNAc-(1-->3)-alpha-D-Gal- (1-->4)-beta-D-Gal-(1-->4)-D-Glc] and the terminal tetrasaccharide, as well as the methyl glycosides 1 and 2 of the terminal di- and tri-saccharides, were synthesised by silver trifluoromethanesulfonate-promoted alpha-glycosylation of suitably protected mono-, di-, tri-, and tetrasaccharide alcohols with 3,4,6-tri-O-acetyl-2-azido-2-deoxy-alpha-D-galactopyranosyl bromide, followed by removal of protecting groups. The anomeric TMSEt group of the Forssman pentasaccharide and terminal tetrasaccharide was removed with trifluoroacetic acid-dichloromethane, to give the corresponding hemiacetal sugars 4 and 6.

Carbohydrate Sequence↗
PubMed1994

Hydroperoxides in oxidized d-limonene identified as potent contact allergens.

Hydroperoxides of d-limonene were shown to be potent contact allergens when studied in guinea-pigs. Limonene-2-hydroperoxide (2-hydroperoxy-p-mentha-6,8-diene, a mixture of trans and cis isomers) was synthesized for the first time. The ratio between the trans and cis forms was 3:1. These two hydroperoxides were identified as the major hydroperoxides in autoxidized d-limonene. In photo-oxidized d-limonene, they constituted a minor part of the hydroperoxide fraction. Hydroperoxides may bind to proteins of the skin to make antigens either via a radical mechanism or after reactions to give epoxides. The cross-reactivity between the epoxide limonene-1,2-oxide, a potent contact allergen, and the hydroperoxides was therefore studied. No significant pattern of cross-reactivity was found. Further studies to identify and test the allergenicity of single hydroperoxides are needed to elucidate the mechanism of the allergenicity.

Allergens↗
PubMed1994

Influence of an anti-oxidant on the formation of allergenic compounds during auto-oxidation of d-limonene.

Butylated hydroxytoluene (BHT), a common anti-oxidant, was added to different samples of d-limonene. The decrease in concentration of d-limonene and the formation of oxidation products were compared between the samples and with samples without anti-oxidant using gas chromatography. The aim of the study was to investigate how long d-limonene to which BHT was added could be handled when air-exposed at room temperature, without formation of oxidation products which according to previous studies increase the risk of skin sensitization. In experiments trying to mimic the handling of limonene products at workplaces the addition of BHT prevented auto-oxidation for periods depending on the purity of the products and on the room temperature. Cold and dark storage of d-limonene in closed vessels prevented auto-oxidation for 1 year without addition of anti-oxidant.

Air Pollutants↗
PubMed1994

Allergenicity of rosin (colophony) esters (II). Glyceryl monoabietate identified as contact allergen.

In the esterification of rosin with glycerol, the main compound formed, glyceryl triabietate, shows low allergenic activity. In this study, compounds formed in smaller amounts, when abietic acid (main component in rosin) was esterified with glycerol, were identified as glyceryl-1-monoabietate (GMA), glyceryl-1,2-diabietate (GDA1,2) and glyceryl-1,3-diabietate (GDA1,3), using nuclear magnetic resonance (NMR), infra-red (IR) and mass spectrometry (MS) analyses. According to animal experiments, GMA was a contact allergen. No cross-reactivity was seen to allergens in unmodified rosin. Some patients allergic to unmodified rosin reacted when tested with GMA. No reactions were seen to the 2 diabietates. Some patients also reacted to commercial glycerol-modified rosins. GMA together with unmodified abietic acid were identified in these rosin samples. The reactions seen in rosin-sensitive patients to commercial glycerol-esterified rosins probably derive from the unmodified material still present in the product, but could also be the result of GMA obtained from the glycerol derivatization.

Abietanes↗
PubMed1994

Quantification of tissue damage in the feline small intestine during ischaemia-reperfusion: the importance of free radicals.

Intestinal ischaemia is accompanied by characteristic mucosal lesions, which can be graded according to a six-grade system proposed by Chiu et al. (1970). This report describes a continuous grading system which makes it possible to quantify the intestinal damage in connection with ischaemia-reperfusion. The present morphometric method is based on quantitative histological analysis of intestinal biopsies performed on 200 histological sections from 44 cat experiments. Radical formation was quantified by infusing close i.a. a spin trap, OXANOH, which produces a secondary stable radical, OXANO., after reacting with radicals in the tissue. OXANO. concentration was determined in venous blood samples with electron spin resonance. We demonstrate a highly significant correlation between the grading system of Chiu et al. (1970) and the morphometric analysis of this study. The tissue damage was located exclusively in the intestinal villi. Comparing the mucosal damage that occurs during 60 min of intestinal ischaemia (superior mesenteric artery pressure 15-25 mmHg) with that seen during the first 30 min reperfusion this study shows that the villus damage occurring during ischaemia is at least twice as large as the aggravation seen upon reperfusion. Furthermore, the authors demonstrate a significant correlation between rate of radical formation and villus tissue damage particularly during the first 30 min after ischaemia. It is concluded that the proposed quantitative morphological method represents a non-discrete grading system for evaluating tissue damage in connection with ischaemia-reperfusion in the small intestine. The ischaemia itself inflicted a more severe damage to the intestine than reperfusion. A significant correlation between damage and radical formation was demonstrated during the reperfusion. However, the results suggest that factors other than radical formation are of importance in explaining the tissue damage upon reperfusion. The nature of these factors is presently unknown.

Animals↗
PubMed1994

Adsorption of fibrinogen and some other proteins from blood plasma at a variety of solid surfaces.

Enzyme linked immunosorbent assay (ELISA) was used for the estimation of protein adsorption from blood plasma at some model solid surfaces. The majority of those surfaces were made in the wells of microtiterplates of polystyrene commonly used for ELISA purposes. Three of the model surfaces were made by radio frequency plasma discharge polymerization (RFPD) of the microtiterplates of polystyrene. The monomers we used were diaminocyclohexane, hexamethylenedisiloxane, and acrylic acid. Other surfaces investigated were: unmodified polystyrene, oxidized polystyrene, hydrophilic silicon oxide, and methylized silicon oxide. Two substances, Tween and bovine serum albumin (BSA), for the prevention of unintended adsorption of ELISA conjugate were also tested and the BSA method was found to be superior for this kind of investigation. Human blood plasma at different dilutions was incubated in the surface-modified microtiterplates followed by incubation of rabbit antibodies against fibrinogen (FG), fibronectin (FN), human serum albumin (HSA), complement factor 3 (C3), and immunoglobulin G (IgG). Visualization of bound antibodies was then made by standard ELISA procedure. At low blood plasma concentrations (plasma dil 1/1000), anti-IgG and anti-HSA were detected at high levels at the majority of surfaces. At high blood plasma concentration (plasma dil 1/10), anti-FG dominated at most surfaces. ELISA activity of FN and C3 were low at most of the surfaces at both plasma concentrations. An 'optimum' plasma dilution for the detection of surface bound FG (the Vroman effect) was not found with the use of the ELISA on any of the surfaces except for the silicon oxide surface. This is in contrast to findings by others who had used isotope-labelled fibrinogen diluted in plasma. However, 'false' Vroman effects occurred if nonionic surfactant was used for the prevention of unspecific binding in the ELISA.

Adsorption↗
PubMed1994

Synthesis of the 2"-hydroxy, 4"-deoxy and 4"-epi analogues of beta-D-GalNAc-(1-->3)-alpha-D-Gal-(1-->4)-beta-D-Gal, the terminal trisaccharide of globotetraose.

Radical deoxygenation of methyl 3,6-di-O-benzoyl-2-deoxy-4-O-imidazol-1-yl-thiocarbonyloxy-2-phtha limido-beta-D - glucopyranoside 5 gave methyl 3,6-di-O-benzoyl-2,4-dideoxy-2-phthalimido-beta-D-glucopyranoside 6, which was converted into the corresponding methyl thioglycoside donor 9. Methylsulfenyl trifluoromethane-sulfonate-promoted glycosylation of 2-(trimethylsilyl)ethyl 2,3,6-tri-O-benzyl-4-O-(2,4,6-tri-O-benzyl-alpha-D-galactopyranosyl)-bet a-D- galactopyranoside 10, followed by removal of protecting groups gave the 4"-deoxy analogue 12 of the terminal trisaccharide of globotetraose. Silver trifluoromethanesulfonate-promoted glycosylation of the same disaccharide alcohol 10 with 3,4,6-tri-O-acetyl-2-deoxy-2- phthalimido-alpha/beta-D-glucopyranosyl bromide 13 and 2,3,4,6-tetra-O-acetyl-alpha-D-galactopyranosyl bromide 16, followed by deblocking, gave the 2"-hydroxy and 4"-epi analogues 15 and 18, respectively.

Carbohydrate Conformation↗
PubMed1993

Crystal structure of transketolase in complex with thiamine thiazolone diphosphate, an analogue of the reaction intermediate, at 2.3 A resolution.

The crystal structure of the complex of transketolase and thiamine thiazolone diphosphate has been determined at 2.3 A resolution. The complex has a structure which closely resembles that of this enzyme with the cofactor ThDP. This is consistent with the observation that the binding of the analogue to transketolase involves ground state rather than transition state interactions. Since thiamine thiazolone diphosphate resembles an expected intermediate in the catalytic pathway, the structure of the intermediate was modelled from the crystal structure. Based on this model, enzymic groups responsible for binding of the intermediate and proton transfer during catalysis are suggested.

Binding Sites↗
PubMed1993

Complement activation during tryptophan immunoadsorption treatment.

Antibodies against human lymphocyte antigens (HLA) are frequently seen among patients undergoing repeated renal transplantations. Graft survival can be improved by eliminating these antibodies by plasmapheresis before transplantation. In this study, we have tried a new extracorporeal procedure to remove the anti-HLA antibodies. An immunoadsorption column (IM-TR) with a matrix of polyvinyl alcohol (PVA) gel conjugated with a hydrophobic amino acid tryptophan was utilized. Previous results have shown that repeated IM-TR treatments are at least equally effective as plasmapheresis in reducing levels of specific immunoglobulins in treated patients. In this study, 7 HLA-immunized patients were treated before renal transplantation. Each patient was subjected to a total of 12 treatment sessions divided into 3 sessions per week. After each treatment session, the reduction of the immunoglobulins was less than what has been reported for plasmapheresis. This suggests that mechanisms other than immunoglobulin depletion are involved in the reduction of the total immunoglobulin levels. The IM-TR treatment resulted in a strong complement activation triggered by the alternative pathway. Since the adsorbed plasma was returned to the patient, exceedingly high levels of the activation fragment C3d (C3dg) were found in plasma during and after the treatment. We conclude that the extensive generation of C3dg may be one of the factors that plays a role in the reduction of the antibody levels since the C3dg fragment has been shown to down-regulate the immune response.

Adult↗
PubMed1993

The allergenicity of glycerol esters and other esters of rosin (colophony).

To investigate whether esterification of rosin with polyalcohols changes its allergenic potential, abietic acid, the main component of rosin, was esterified with glycerol at high temperature. The major product formed was isolated and identified, using nuclear magnetic resonance (NMR), infra-red (IR) and mass spectrometry (MS) analyses, as glyceryl triabietate (GTA), an ester between one glycerol molecule and 3 abietic acid molecules. According to animal experiments GTA was not allergenic and no cross-reactivity was seen to allergens in unmodified rosin. When testing patients allergic to unmodified rosin, no reactions were found to GTA. Some of the patients reacted to glycerol- and pentaerythritol-esterified rosins. According to HPLC analyses, these esterified rosins still contained unmodified material to which the patients may have reacted. It seems that the esterification of rosin with polyalcohols such as glycerol reduces its allergenic activity, possibly because of the formation of much larger molecules with reduced bioavailability. However, making methyl esters of rosin causes little alteration in the molecular weights of the components and, when unmodified and methylated rosin were tested in patients, we saw no difference between the 2 forms.

Abietanes↗
PubMed1993

In vivo x-ray fluorescence measurements of cadmium and lead.

Information about the body burden of a metal is important in the evaluation of both exposure and risk. In vivo methods for determining cadmium and lead have become available in the last 20 years. Thus cadmium in kidney can be assessed by neutron activation or X-ray fluorescence analysis. The X-ray fluorescence technique allows kidney cadmium determinations even in subjects without occupational exposure. The level reflects the burden, long-term exposure, and the risk of toxic effects. Furthermore, in vivo determination of lead in finger bone, tibia, or calcaneus by X-ray fluorescence indicates long-term exposure and should become a valuable tool in epidemiologic studies, especially of chronic effects.

Bone and Bones↗
PubMed1993

Biological monitoring of inorganic lead.

In exposure and risk evaluation, monitoring lead biologically has several advantages over technical exposure assessment. Traditionally, the concentration in blood (B-Pb) has been widely used. However, the erythrocytes tend to become saturated, and this phenomenon causes a nonlinear relationship between B-Pb and uptake and between metabolic and toxic effects and B-Pb. Recently, several techniques for determining lead in finger bone, tibia, or calcaneus in vivo by X-ray fluorescence have become available. Bone lead reflects long-term exposure and should prove valuable in epidemiologic studies. Mobilization tests have been widely used to monitor lead biologically. They mainly seem to reflect the lead in soft tissues and may not be an index of total body burden, most of which is in the skeleton. It thus seems that, at least in adults, mobilization tests do not provide more information than traditional lead determinations in blood and urine. A metabolic model for lead in humans is presented.

Adult↗